ADVANCES IN
Applied Microbiology VOLUME 8
CONTRIBUTORS
TO THIS VOLUME
Emanuel Borker
S. G. Bradley Thomas D. Broc...
21 downloads
935 Views
19MB Size
Report
This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!
Report copyright / DMCA form
ADVANCES IN
Applied Microbiology VOLUME 8
CONTRIBUTORS
TO THIS VOLUME
Emanuel Borker
S. G. Bradley Thomas D. Brock Cecil W. Chambers Norman A. Clarke Arnold L. Demain Paul A. Hartman Heiner Hoffman Nino F. Insalata Stephen Alan Kollins Colette P. Levi Wesley 0. Pipes George W. Reinbold Martin
H. Rogoff
Devi S. Saraswat John
S. Witzeman
ADVANCES IN
Applied Microbiology Edifed by WAYNE W. UMBREIT Department of Bacteriology Rutgers, The State University New Brunswick, New Jersey
VOLUME 8
ACADEMIC PRESS, New York and London
COPYRIGHT @ 1966, BY ACADEMICPmss INC. ALL RIGHTS RESERVED.
NO PART OF THIS BOOK MAY BE REPRODUCED I N ANY FORM, BY PHOTOSTAT, MICROFILM, OR ANY OTHER MEANS, WITHOUT WRITTEN PERMISSION FROM THE PUBLISHERS.
ACADEMIC PRESS INC. 111 Fifth Avenue, New York, New York 10003
United Kingdom Edition published by ACADEMIC PRESS INC. (LONDON) LTD. Berkeley Square House, London W.l
LIBHAHY O F CONGRESS CATALOG C A R D
NUMBER:59-13823
PRINTED I N THE UNITED STATES OF AMERICA
CONTRIBUTORS Numbers in parentheses indicate the pages on which the authors’ contributions begin.
EMANUELBORKER, General Foods Technical Center, White Plains, New York (315) S. G. BRADLEY, Department of Microbiology, University of Minnesota, Minneapolis, Minnesota (29)
THOMAS D. BROCK,Department Bloomington, Zndianu ( 61 )
of
Microbiology, Indiana University,
CECILW. CHAMBERS, U. S. Department of Health, Education, and Welfare, Federal Water Pollution Control Administration, Robert A. Tap, Sanitary Engineering Center, Cincinnati, Ohio ( 105) NORMAN A. CLARKE, U. S. Department of Health, Education, and Welfare, Federal Water Pollution Control Administration, Robert A. Tuft Sanitary Engineering Center, Cincinnati, Ohio (105) ARNOLD L. DEMAIN, Merck Sharp G Dohme Laboratories, Merck G CO., Inc., Rahway, New Jersey ( 1 ) PAULA. HARTMAN,Departments uf Bacteriology and Dairy Food Zndustry, Iowa State University, Ames, Iowa ( 2 5 3 ) HEINERHOFFMAN,Department of Microbiology, New York University, College of Dentistry, New York, New York (195) NINOF. INSALATA, General Foods Technical Center, White Plains, New York (315) STEPHEN ALANKOLLINS,Department of Environmental Sciences, College of Agriculture and Environmental Science, Rutgers University, New Brunswick, New Jersey (145)l COLETTEP. LEVI, General Foods Technical Center, White Plains, New York (315) WESLEY0. PIPES,Department of Civil Engineering, Northwestern University, Evanston, Zllinois (77) GEORGEW. REINBOLD,Departments of Bacteriology and Dairy Food Industry, Iowa State University, Ames, Iowa ( 2 5 3 ) 1
Present Address: University of Cincinnati College of Medicine, Cincinnati, Ohio. V
vi
CONTRIBUTORS
MARTIN H. ROGOFF, Znternational Minerals G Chemical Corporation, Wasco, California (291)
DEVIS. SARASWAT, Departments of Bacteriology and Dairy Food Industry, Iowa State Uniuersity, Ames, Zowa (253)' S. WITZEMAN, General Foods Technical Center, W h i t e Plains, New York (315)
JOHN
2
Present Address: Rajasthan College of Agriculture, Udaipur, India.
PREFACE This volume, the eighth - of this serial -publication, continues its relatively wide coverage, which is characteristic of the essay type of approach, of the problems of transmission of information, ideas, and evaluations, an approach which appears to be more useful as the range of knowledge expands and the erudition required of the applied microbiologist increases. It is curious how a group of essays assembled somewhat at random, and prepared independently, tend to coalesce about a theme. Thus, since this does occur without plan or previous design, we take it to mean that such subjects are uppermost in the minds of the applied microbiologist, and that this type of publication provides a prime source in which the thinking in the field is summarized. Two such themes can easily be discerned in the present volume. One is the importance of the application of the latest knowledge of genetic information to microbiology which is discussed in two chapters. The other is the general importance of microbial ecology which is specifically elaborated in another chapter bearing this title. It is also clear that ecology is of importance in activated sIudge, in the control of bacteria in water, and even in oral microbiology. Related to this central theme are reviews on the removal of viruses in sewage treatment, on methods for work with enterococci, and on the curious crystal insect toxins produced by bacteria. A newly developing area, which is undoubtedly to grow in importance, is represented for the first time in this serial publication by a chapter on mycotoxins. There is, as we see it, another problem faced by the applied microbiologist, not necessarily faced by his contemporary in the university or research institute. The demands on the time of the applied microbiologist are such that he rarely is able to keep up with developments other than in his own restricted field. By providing interesting essays in related but perhaps impinging areas, this serial publication affords a means of broadening the basic knowledge of the practicing applied microbiologist. We do not wish to single out any special article, but perhaps the reader involved in antibiotic production would find relaxation and stimulation in reading about aflatoxin, or the crystalline protein of certain bacilli, or oral bacteriology, or ecology. It is the enrichment of mind which leads to new concepts and to fresh approaches to immediate problems.
W.W. UMBREIT Rutgers University September, 1966 vii
This Page Intentionally Left Blank
CONTENTS CONTRIBUTORS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . PREFACE ........................................................... CONTENTS OF PREVIOUS VOLUMES. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
ii Vii
xiii
Industrial Fermentations and Their Relation to Regulatory Mechanisms ARNOLDL . DEMAIN I . Introduction ................................................. I1 Metabolic Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11 Accumulation of Normal Intermediates and End Products Due to Alteration of Feedback Controls ................................ IV. Regulatory Mechanisms and the Biosynthesis of Antibiotics . . . . . . . . . . V Synthesis of Macromolecules .................................... VI . Summary .................................................... References ...................................................
. .
.
1 2 6 17 23 24 25
Genetics in Applied Microbiology S . G. BFLADLEY
I. I1. I11. IV . V. VI . VII . VIII .
Introduction .................... Mutation and Selection . . . . . Recombinational Mechanisms ................................... Phage-Host Interactions . . . . . . . . . . . .......... Genetic Control of Biosynthesis . . . . . . . . . Cell-Free Syntheses . . . . . . . . . . . . . . . . . . .......... Future Applications ........................................... Conclusions .................................................. References ...................................................
29 30 36 43 46 50 53 55 56
Microbial Ecology and Applied Microbiology THOMAS D. BROCK
I . Introduction ................................................. I1. The Search for a New Antibiotic: A Problem in Microbial Ecology? 111. Summary .................................................... References ...................................................
61 63 74 75
The Ecological Approach to the Study of Activated Sludge WESLEY0. PIPES
I. 11. I11. IV. V.
Introduction . . . . . . ........................................ Organisms Present . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ecological Factors .................... ... Ecological Relationships . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary ....................................... References ...................................................
ix
77 81
101
X
CONTENTS
Control of Bacteria in Nondomestic Water Supplies CECIL w . CHAMBEHS AND NORMAN A . CLARKE
I. I1. I11. IV. V. VI . VII . VIII . IX. X.
............................... Introduction . . . . . . . . . . . . . Water as a Source of Bacte ntamination . . Water as a Bacteriological Medium . . . Areas Where Contaminants Multiply . Biological Factors Affecting Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physical Methods of Control ...................... ......... Chemical Methods of Control . . . . . .......................... Economics of Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Evaluation of Effectiveness of Control ethods .................... General Comments and Conclusions .............................. References ......................
105
115 116 121 135 136 138
The Presence of Human Enteric Viruses in Sewage and Their Removal by Conventional Sewage Treatment Methods STEPHENALAN KOLLINS I. I1. I11. IV. V. VI . VII .
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Nature of Enteric Viruses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enteric Viruses in Feces ... ................................ Transmission of Viruses Throu Water . . . . . . . . . . . . . . . . . . . . . . . . . . Presence of Viruses in Sewage . . . . . . . . . . . . . . . . . . Removal of Viruses by Sewage Treatment Methods . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
145 146 159 162 170 175 189 191
Oral Microbiology HEINEHHOFFMAN
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. History of Oral Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Present State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
195 195 199 243
Media and Methods for Isolation and Enumeration of the Enterococci
PAULA . HARTMAN. GEORGE W . REINBOLD. AND DEW S. SARASWAT
I.
............................
I1. Media Available . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I11. Comparative Studies on Media and Methods . . . . . . . . . . . . . . . . . . . . . . References ..................................
253 254 279 283
Crystal-Forming Bacteria as Insect Pathogens MARTINH . ROGOFF
I . Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I1. Toxic Materials Produced by the Crystal-Forming Bacilli . . . . . . . . . . . . I11. Host Susceptibility and the Toxic Factors Produced by Bacillus thuringiensis
.................................................
291 294
306
xi
CONTENTS
IV. Some Industrial Considerations .................................. V. Future Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
308 312 312
Mycotoxins in Feeds and Foods EMANUELBORKER, NINOF. INSALATA, COLETTEP. LEVI,AND JOHN S. WITZEMAN I. 11. 111. IV.
Introduction . . . . . . . . . . . ................................ 315 Aflatoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316 Other Mycotoxicoses . . . . . . . . . . . . . ........... . . . 336 Control Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346
AUTHOR INDEX . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SUBJECTINDEX . . . . .
..........
. . . . . . . . . . . . . . 353 ..
373
This Page Intentionally Left Blank
CONTENTS OF PREVIOUS VOLUMES Volume 1
Protected Fermentation MiloB Herold and Jan NeEhsek The Mechanism of Penicillin Biosynthesis
Arnold L. Demain Preservation of Foods and Drugs by Ionizing Radiations W . Dexter Belhmy The State of Antibiotics in Plant Disease Control
David Pramer Microbial Synthesis of Cobamides
D. Perlman Factors Affecting the Antimicrobial Activity of Phenols
E . 0. Bennett Germfree Animal Techniques and Their Applications Arthur W. Phillips and James E . Smith Insect Microbiology S. R. Dutky The Production of Amino Acids by Fermentation Processes
Shukuo Kinoshita
Continuous Industrial Fermentations Philip Gmhardt and M . C . Bartlett The Large-Scale Growth of Higher Fungi Radcliffe F . Robinson and R. S .
Microbial Control Brewery Gerhard J . H a m
in
the
Newer Development in Vinegar Manufactures Rudolph J . Allgeier and Frank M .
Hildebranclt The Microbiological Transformation of Steroids
7'. H . Stoudt Biological Transformation of Solar Energy Willium J. Oswald and Clurence G.
Golueke SYMPOSIUMON ENGINEEHING ADVANCES IN FERMENTATION PRACTICE Rheological Propcrties of Fermentation Broths
Fred H . Deindoerfer and John M . West Fluid Mixing in Fermentation Processes J.
y. OUshue
Scale-up of Submerged Fermentations W . H . Bartholemew ~ i , sterilization .
Arthur E . Humphrey Sterilization of Media for Biochemical Processes
Lloyd L. Kempe Fermentation Kinetics and Model Processes
Davidson AUTHOR INDEX-SUB
Methods
Fred H. Deindoerfer JECT INDEX
Volume 2
Continuous Fermentation W . D. Maxon
Newer Aspects of Waste Treatment
Control Applications in Fermentation
Aerosol Samplers
Volume 3
George J . Fuld
Nandor Porges
Harold W . Batchelor A Commentary on Microbiological Assaying F. Kavanagh Application of Membrane Filters Richard Ehrlich
Preservation of Bacteria by Lyophilization
Robert J. Heckly Sphaerotilus, Its Nature and Econoniic Significance
Norman C . Dondero xiii
xiv
CONTENTS OF PREVIOUS VOLUMES
Large-Scale Use of Animal Cell Cultures Donah! J . Merchant and C . Richard Eidam Protection against Infection in the Microbiological Laboratory: Devices and Procedures Mark A. Chatigny Oxidation of Aromatic Compounds by Bacteria Martin H . Rogoff Screening for and Biological Characterizations of Antitumor Agents Using Microorganisms Frank M . Schabel, Jr., and Robert F . Pittillo The Classification of Actinomycetes in Relation to Their Antibiotic Activity Elio Baldacci The Metabolism of Cardiac Lactones by Microorganisms Elwood Titus Intermediary Metabolism and Antibiotic Synthesis J. D. Bu'Lock Methods for the Determination of Organic Acids A. C . Hulme AUTHOR INDEX-SUB
JECT INDEX
Volume 4
Induced Mutagenesis in the Selection of Microorganisms S. I . Alikhanian The Importance of Bacterial Viruses in Industrial Processes, Especially in the Dairy Industry F . J . Babel Applied Microbiology in Animal Nutrition Harlow H . Hall Biological Aspects of Continuous Cultivation of Microorganisms T . HoEme Maintenance and Loss in Tissue Culture of Specific Cell Characteristics Charles C . Morris
Submerged Growth of Plant Cells L. G . Nickell AUTHOR INDEX-SUB
JECT INDEX
Volume 5
Correlations between Microbiological Morphology and the Chemistry of Biocides Adrien Albert Generation of Electricity by Microbial Action J. B . Davis Microorganisms and the Molecular Biology of Cancer G . F . Gause Rapid Microbiological Determinations with Radioisotopes Gilbert V. Levin The Present Status of the 2,S-Butylene Glycol Fermentation Sterling I<. Long and Roger Patrick Aeration in the Laboratory W. R. Lockhart and R. W. Squires Stability and Degeneration of Microbial Cultures on Repeated Transfer Fritz Reusser Microbiology of Paint Films Richard T . Ross The Actinomycetes and Their Antibiotics Selnian A, Waksman Fuse1 Oil A . D ~ ~ S ~ OWOe bTb nnd John L. Ingraham AUTHOR INDEX-SUB
JECT INDEX
Volume 6
Global Impacts of Applied Microbiology: An Appraisal Carl-Goran Heddn and Mortimer P . Starr Microbial Processes for Preparation of Radioactive Compounds D. Perlman, Arb P . Bayan, and Nancy A. Giuffre Secondary Factors in Fermentation Processes P . Margalith
xv
CONTENTS OF PREVIOUS VOLUMES
Nonmedical Uses of Antibiotics Herbert S . Goldberg
Cold Sterilization Techniques John B . Opfell and Curtis E . Miller
Microbial Aspects of Water Pollution Control K . Wuhrmann
Microbial Production of Metal-Organic Compounds and Complexes D . Perlman Development of Coding Schemes for Microbial Taxonomy S. T . Cowan Effects of Microbes on Germfree Animals Thomas D . Luckey
Microbial Formation and Degradation of Minerals Melvin P . Silverman and Henry L . Ehrlich Enzymes and Their Applications Irwin W . Sizer
A Discussion of the Training of Applied Microbiologists B . W. Koft and Wayne W . Umbreit AUTHOR INDEX-SUB
JECT INDEX
Volume 7 Microbial Carotenogenesis Alex Ciegler
Biodegradation : Problems of Molecular Recalcitrance and Microbial Fallibility M . Alexander
Uses and Products of Yeasts and Yeastlike Fungi Walter J . Nickerson and Rohert G . Brown
Microbial Amylases Walter W. Windish and Nagesh S . Mhatre The Microbiology of Freeze-Dried Foods Gerald J . Silverman and Samuel A. Goldblith Low-Temperature Microbiology Judith Farrell and A. H . Rose AUTHOR INDEX-SUB
JECT INDEX
This Page Intentionally Left Blank
ADVANCES IN
Applied Microbiology VOLUME 8
This Page Intentionally Left Blank
Industrial Fermentations and Their Relation to Regulatory Mechanisms ARNOLDL. DEMAIN Merck Sharp CL. Dohme Research Laboratories, Merck G Co., Inc., Rahway, New Jersey
I. Introduction ..................................... 11. Metabolic Regulation ............................. 111. Accumulation of Normal Intermediates and End Products due to Alteration of Feedback Controls . . . . . . . . . . A. Decreasing the Concentration of End Product .... B. Alteration of Enzymes or Enzyme-Forming Systems IV. Regulatory Mechanisms and the Biosynthesis of Antibiotics .......................................... A. Inhibition of Penicillin Biosynthesis by Lysine . . . . B. Stimulation of Cephalosporin C Formation by Methionine .................................. V. Synthesis of Macromolecules ....................... A. Microbial Enzymes ........................... B. Microbial Nucleic Acids ....................... VI. Summary ....................................... Acknowledgment .................................. References ......................................
1.
1 2 6 8
14 17 18 20 23 23
24 24 25 25
Introduction
At one time it was thought that the success of an industrial fermentation depended on the addition of the correct precursor compound to the medium. For example, the marked stimulation of the biosynthesis of benzylpenicillin by the addition of phenylacetate was one of the first major breakthroughs in penicillin production during World War 11. The structural similarity of the precursor to its product is obvious. However, c&.-CH2-
H
H s C' '?( I I C-N-CH-COOH 0
CO-NH-C-
CH,),
Benz ylpenicillin
C&-
C&-
COOH
Phenylacetic acid
SH I bN-CH-CH, I COOH
Cysteine 1
YH(CIZh &N-CH-COOH
Valine
2
ARNOLD L. DEMAIN
in recent years, it has become evident that precursor addition fails more often than it succeeds. For example, the demonstration that L-valine and, L-cysteine were precursors of the penicillin ring system required refined techniques; stimulation was not apparent when the amino acids were merely added to fermentations. The reason was that the organism made these readily from sugar and ammonia and they were not in limiting supply. In many fermentations, compounds other than precursors are very important and control production by virtue of their stimulatory or inhibitory properties. Consider the control of the glutamic acid fermentation by biotin, the stimulation of cephalosporin C production by methionine or norleucine, the inhibition of the biosynthesis of benzylpenicillin by lysine, and the inhibition of the lysine fermentation by threonine. It has become apparent that in many fermentations inhibition by end products is more important than stimulation by precursors. We have learned in recent years that most modern-day microorganisms tend to avoid overproduction of natural compounds and that the fermentation microbiologist must find ways to break down controls that have evolved to prevent waste of organic materials by oversynthesis. By learning all we can about the controls operating in living cells, we increase our ability to circumvent or alter these fundamental regulatory mechanisms and to allow overproduction and excretion of desired compounds without destroying the metabolic activities of the cell.
II. Metabolic Regulation' It has been known for many years that the genotype of a microbial cell dictates the constitution of its enzymatic machinery. Yet, as the environment changes, microbes have an amazing ability to change their composition and metabolic activities. The genotype is not affected by these alterations in environment-only the phenotypic expression of the genes. Despite profound changes in the environment, microbial cells generally do not oversynthesize metabolites. For example, consider the usual wild-type microorganism. When fed glucose, ammonia, and salts, the cell synthesizes its own histidine and all of its other building blocks needed for formation of protein and nucleic acids. Now, if histidine is added to the medium, the endogenous synthesis of this amino acid is 1 In all of the diagrams depicting biosynthetic pathways and metabolic controls, solid arrows represent enzymatic steps; dashed arrows represent control mechanisms; I is inhibition; R is repression; a double line intersecting a dashed arrow indicates breakdown of the control mechanism; compounds enclosed in rectangles are those accumulated by the organism. A solid arrow replaced by a series of dots indicates absence of the active enzyme.
3
INDUSTRIAL FERMENTATIONS
suddenly blocked and the exogenous histidine is taken into the cell and used for protein synthesis. This ability of the cell to recognize the presence of an end product, to stop wasteful synthesis until no more end product remains in the medium, and then to resume production, is a function of feedback regulation. The two main mechanisms involved are inhibition and repression (Fig. 1). Inhibition is the phenomenon by which the final metabolite of a biochemical sequence inhibits the action of an early enzyme (usually the first) of that sequence. Repression refers to the inhibition of the formation of the enzymes in a biochemical path-
i
A
End -ro&ct
FIG. 1. Feedback inhibition and repression (see footnote 1 ).
way by a derivative of the end product. Both are economy measures adjusting the rate of production of low-molecular-weight end products to the rate of synthesis of macromolecules. In feedback inhibition (also called end-product inhibition or allosteric inhibition), the inhibitor is the end product and not a derivative. In contrast to classical competitive inhibition, the end-product inhibitor and the substrate do not necessarily resemble each other in size, shape, and charge. It has been shown that the enzyme has two separate binding sites-one for the substrate and one for the inhibitor. However, occupancy of the inhibitor site by the end product interferes with the attachment of the substrate at its binding site by altering the conformation of the enzyme. By treatment of the purified enzyme with heat, urea, or heavy metal ions, it is possible to eliminate the sensitivity of the enzyme to the inhibitor without decreasing the enzymatic activity (Gerhart and Pardee, 1962). Whereas end-product inhibition is a rapid mechanism for maintaining
4
ARNOLD L. DEMAIN
a constant concentration of low-molecular-weight metabolites, repression would be a sluggish mechanism for controlling the flow of metabolites. Instead, repression appears to control the capacity for synthesis of certain proteins. Repression is thought to operate by combination of the end product with a high-molecular-weight “aporepressor” (probably a protein) to produce an active repressor. Such a repressor appears to stop the production of messenger RNA by the structural genes that
FIG. 2. Multiple enzymes as protective devices in branched pathways. Isoenzymes one, two, and three carry out the same reaction but are individually regulated by end products P,, Ps, and P,, respectively.
dictate formation of the enzymes in the biosynthetic pathway. The phenomenon of enzyme induction, in which enzymes that normally are barely detectable are formed rapidly after addition of substrates or substrate analogs, is thought to be a reversal of repression. Some workers suggest that enzyme inducers inhibit the combination of an endogenous product with aporepressor. Although research on the mechanisms of repression and induction is of the utmost importance and interest, details of the current hypotheses will not be presented here. For those readers who are interested in the development of current thought in this area, the following reviews are recommended: Vogel ( 1961), Riley and Pardee (1962), Moyed and Umbarger (1962), Jacob and Monod (1963), Monod et al. (1963), Umbarger (1964), Ames and Martin (1964), Maas and McFall (1964), Changeux (1965).
INDUSTRIAL FERMENTATIONS
5
Although the concepts of feedback regulation were not developed until recently, microbial geneticists had known that the end product of a biosynthetic pathway exercised strict control over the amount of an intermediate accumulated by a mutant blocked in that pathway. Only at growth-limiting concentrations of the required end product would large accumulations of the substrate of the deleted enzyme occur. Today we know that the high levels of the end product inhibited an early enzyme and repressed the enzymes of the pathway, thus limiting the accumulation of the intermediate.
H
FIG.3. Multivalent ( o r concerted) feedback as a protective device in branched pathways. All three products are required for feedback to occur.
Since most end products arise from branched pathways, the cell must have more involved mechanisms to prevent one end product from interfering with the production of others derived from the common metabolic sequence. For example, in a pathway leading to three end products, if any one of them could completely inhibit the first enzyme, an excess of this product would cause a bacteriostatic deficiency of the other two end products. Means of control that prevent this type of regulatory suicide have been detected only in the last 5 years. To date, three main mechanisms have been demonstrated and each one can apply to inhibition as well as to repression. First, multiple enzymes (isoenzymes) can be produced each of which carries out the same reaction but is controlled by a different end product (Stadtman, 1963). This type of protective device is diagrammatically shown in Figure 2; compound A is
6
ARNOLD L. DEMAIN
converted to B and then to C, where branching occurs, leading to end products PI, PB, and P3. Three isoenzymes are present, each of which converts A to B. Isoenzyme one is controlled only by PI, isoenzyme two is specifically regulated by P2, while P3 controls only isoenzyme three. The second type of protective device is that of rnultivalent (or concerted) feedback (Freundlich et al., 1963) in which all end products must be present in excess to inhibit or repress the enzyme (Fig. 3 ) . An idealized version of multivalent feedback is shown in Table I. Also shown in the table is the third mechanism, cumulatioe feedback (WoolTABLE I IDEALIZED EFFECTS OF PRODUCTS ON AN ENZYME IN A BRANCHED PATHWAY Inhibition or repression Multivalent feedback
Additiona
0 0 0 0 0 0 100
Cumulative feedback 50 50 50 75
75
75 88
a Each additive in excess.
folk and Stadtman, 1964). Here each end product is capable of a limited degree of inhibition or repression by itself; increasing the concentration over a certain level does not lead to further effects. Combinations of end products result in increased inhibitory activities. However, all the end products are required for complete inhibition or repression. Thus, in all three cases, the excess of one end product does not cause a drastic inhibitory effect on the production of the others. It should be mentioned here that after the branchpoint, the individual pathways act like simple biosynthetic routes; i.e., the end product usually inhibits the first enzyme and represses all of the enzymes after the branchpoint (Yugari and Gilvarg, 1962).
Ill. Accumulation of Normal Intermediates and End Products Due to Alteration of Feedback Controls
A large number of microbial fermentations in which high concentrations of valuable organic compounds are produced by the cells from inexpensive carbon and nitrogen sources and then excreted into the medium are results of alterations in feedback controls. This is not to
INDUSTRIAL FERMENTATIONS
7
say that all have been designed as such. In many cases, the fermentations were first empirically developed by screening techniques and only later found to be due to modifications of regulatory mechanisms. In others, more rational steps were used. The extreme importance of bypassing feedback controls in order to accumulate products is illustrated in Figure 4 by the conversion of DL-"hreonine
a-Threonine
aLKetobu tyrate I
@I
a-Acetohydroxybutyrate
a, 3-Dihydroxy-P-methylvalerate I
c
a-Keto-P-methylvalerate I
FIG.4. Conversion of D-threonine to L-isoleucine by Serratiu marcescens.
threonine to L-isoleucine using Serratia marcescens ( Kisumi et al., 1964). When L-threonine is the substrate, no isoleucine accumulates because of feedback inhibition of enzyme one by isoleucine. However, D-threonine is deaminated by a different enzyme not subject to control by isoleucine. Thus, an efficient conversion of D-threonine to L-isoleucine is obtained. Using various fermentations as examples, the following paragraphs illustrate various methods of breaking down control mechanisms which result in excretions of organic compounds produced from sugar. Not all of the illustrations have been commercially exploited, and some involve only small accumulations noted in laboratory experiments. However,
8
ARNOLD L. DEMAIN
they all demonstrate principles that can be used in developing successful fermentations on an industrial scale. In general, there are two means available to the microbiologist to modify feedback regulation. The simplest is to decrease the concentration of the end product and the other is to alter the enzyme or the enzyme-forming mechanism. A. DECHEASINC THE CONCENTRATION OF END PRODUCT 1. Ornithine Fermentation
In a simple unbranched pathway, this method can be used to accumulate high concentrations of intermediates. This is done by obtaining a mutant which lacks the enzyme which would ordinarily act on the desired intermediate. By feeding growth-limiting amounts of the end product in the presence of high levels of sugar and ammonium salts, high concentrations of intermediates are excreted. An example is the ornithine fermentation (Fig. 5) carried out by a citrullineless mutant of Corynebacterium glutamicum' ( Udaka and Kinoshita, 1958). The absence of enzyme six allows the microbiologist to control the concentration of arginine. By feeding low levels of arginine, the feedback inhibition of enzyme two by arginine is broken and high levels of ornithine are excreted. 2. Inosinic Acid and Xanthylic Acid Fermentations Accumulation of nucleotide intermediates in a branched pathway has been achieved by decreasing the concentration of the end products, adenylic acid (AMP) and guanylic acid (GMP) (Nakayama et al., 1964; Demain et al., 196513). Figure 6 shows that AMP and GMP inhibit the first enzyme of de nouo purine nucleotide synthesis. Certain adenineless mutants of C . glutamicum blocked at eleven, when grown with low levels of adenine, can accumulate high concentrations of inosinic acid (IMP), further conversion of IMP to GMP being inhibited by GMP at thirteen. By further mutation of the adenineless strain to guanine dependency, strains lacking enzyme fourteen are obtained ( Misawa et al., 1964; Demain et al., 1965b). In such strains, feedback inhibition at all three sites shown in Figure 6 are broken down and xanthylic acid (XMP) is accumulated rather than IMP. 3. Lysine Fermentation It is obvious that decreasing the concentration of an end product would be a useless method of accumulating the end product itself. 2
previously called Micrococcus glutamicus.
INDUSTRIAL FERMENTATIONS
9
However, certain end products can be accumulated in branched pathways. Thus, in a branched pathway which normally leads to products PI and Pa, decreasing the concentration of PI often leads to overproduction and excretion of Pz. Even before feedback regulation was recognized as an important biological phenomenon, Davis (1950) noted that phenylalanineless mutants excrete tyrosine whereas tyrosine auxotrophs excrete phenylalanine. An example which illustrates that the decrease in concentration of
Glutamic acid
I
N-Acetyl-glutamic acid I
c-----
I I
II
I
I
I I I I
I
I I I
4
------_-
-1-43
1
N-Acetyl-y-glutamyl phosphate
N-Acetyl glutamic semialdehyde
1.
N-Acetyl-ornithine
I
I
I I I I I I
I
_L T
I I I
Citrulline
lo I
Arginino-succinic acid
I
I
I
I
L - __-_
__ ---- - Arghine
FIG.5. Ornithine fermentation by a citrullineless mutant of Corynebacterium glutamicum.
10
ARNOLD L. DEMAIN
--__
----__-
Phosphoribosyl pyrophosphate
_ _ - ------I--
_ _ _ _ _ _ _ _ _ _ _ _-______ ~
Phosphoribos ylamine
Glycinamide ribotide I
"1
@I
Formylglycinamide ribotide
Formylglycinamidine ribotide
4
Aminoimidazole ribotide
Aminoimidazole carboxylic acid ribotide 1
I
I
I
6
Aminoimidazole-N-succil;o-carboxamide ribotide
II
I I _L T
I
I
II I 1
I I I
I I
@I
f
Aminoimidazole carboxamide ribotide
Formamido-imidazole carboxamide ribotide
I
FIG. 6. Inosinic acid fermentation by an adenineless mutant of Corynebacterium glutamicum.
11
INDUSTRIAL FERMENTATIONS
one end product of a branched pathway results in the accumulation of another is the commercial lysine fermentation (Nakayama et al., 1961b). Figure 7 shows the probable mechanism by which a homoserineless
i
Aspartate
Aspartyl bhosphate
I
Aspartic semi-aldehyde . . . . . . . . . . .69 .......................
I
a-Amino-€ - ketopimelate
Homos e r h e
@I
t -ke topimelate
N-Succinyl-a-amino-€
N-SUCcinyl- diaminopimelate
L-
Diaminopimelate I
meso -Diaminopimelate
T I I
I
I I
Threonine
Methionine
I I
\ *Inhibition is assumed to be multivalent
FIG. 7. Lysine fermentation by a homoserineless mutant of Corynebacterium glutamicum.
12
ARNOLD L. DEMAIN
auxotroph of C. glutamicum excretes over 50gm. of lysine per liter. Although the true mechanism is not known, it has been established that excess threonine inhibits lysine formation whereas methionine, lysine, and other amino acids normally found in protein have no marked effect. Feedback inhibition rather than repression appears to be important
1
Aspartate
10
I
I
II I
I I I
I
I I I
I
I
I I I
I
I
I
_L I
I
T
I I
I I
I
I
I
0
I
I
I I
II
I I
I
I
@
/I
Lysine
I
1 Threonine
Methioiiine
I
/
-2
*Exact position of block in lysine path is not known
FIG.8. Threonine fermentation by cherichia coli.
R
lysinelcss, methionineless mutant of Es-
here. These results of fermentation studies are in agreement with the concept of multivalent inhibition of enzyme one ( fl-aspartokinase) by Iysine plus threonine, a situation recently discovered in Bacillus species by Paulus and Gray (1964) and in Rhodopseudomonas by Datta and Gest (1964). By virtue of the block at ten (homoserine dehydrogenase), the concentration of threonine can be kept low and inhibition of enzyme one would not occur even in the presence of high levels of lysine. Daoust and Stoudt (1966) reported that the inhibition of lysine ac-
13
INDUSTRIAL FERMENTATIONS
cumulation by threonine can be completely reversed by methionine. Apparently methionine competes with threonine at its binding site on enzyme one. The principle of reversal of feedback inhibition by other end products of the same pathway was originally discovered by Sturani
t
Threonine
lo -I---
+
!
a-Acedlactate
[
a, P-Dihydroxy
isovalerate
I
t
1
a-Ketoisovalerate
a-Acetohydroxybutyrate
a, 0-Dihydroxyp -methylvalerate
t
cY-Keto-P-methylvalerate
Isoleucine I I
1
I
'_-----___--__----j
'*_- -_ -__-___ -- --------__ --
I
-1
*Multivalent repression FIG.9. Valine fermentation by an isoleucineless mutant of Corynebacteriurn glutamicum.
and co-workers ( 1963). They found that in Rhodopseudomonas threonine inhibition of homoserine dehydrogenase can be reversed by methionine. 4. Threonine Fermentation
In the threonine fermentation, the creation of Iysine and methionine deficiencies by auxotrophic mutations leads to threonine accumulation
14
ARNOLD L. DEMAIN
by Escherichia coli (Huang, 1961). This is shown in Figure 8. The exact site of the lysine mutation is unknown. Excess lysine or methionine interferes with accumulation of threonine. The work of Stadtman et al. (1961) revealed that E. coli possesses multiple forms of enzyme one (P-aspartokinase). One of the three isoenzymes is specifically repressed and inhibited by lysine. Creation of the lysine deficiency could result in a derepressed and uninhibited enzyme leading to threonine accumulation despite the feedback effects on a second P-aspartokinase by threonine. Excess lysine would then be expected to interfere with the fermentation. The interference by excess methionine could be due to repression of enzyme three ( homoserine dehydrogenase ) as recently found in C . glutamicum (Nara et al., 1961). 5. Valine Fermentation
The pathway depicted in Figure 9 is normally responsible for the formation of isoleucine, valine, leucine, and pantothenate. Enzymes two, three, four, and five are common enzymes for both the valine and the isoleucine segments. It has been found that isoleucine auxotrophy in C . glutamicum leads to excretion of high concentrations of valine (Nakayama et al., 1961a). This could be explained by the occurrence of multivalent repression by all four end products as had been found in E. coli and in Salmonella typhimurium (Freundlich et al., 1963). Creation of an isoleucine deficiency would be expected to derepress the pathway completely. Although the exact mechanism in the isoleucineless strain of C. glutamicum is not known, the postulated importance of multivalent repression in the fermentation is consistent with the known fact that excess isoleucine interferes with valine accumulation and that this occurs by repression rather than by feedback inhibition. Furthermore, imposition of a mutational block in the leucine branch (instead of the isoleucine mutation) also leads to valine accumulation (Nakayama et al., 1961a).
B. ALTERATIONOF ENZYMES OR ENZYME-FORMING SYSTEMS 1. Mutation to Resistance to Antimetabolites It is obvious that one could not use the previously described technique of decreasing end-product concentration to accumulate an end product of an unbranched pathway. The way to accomplish such a feat is to modify the sensitive enzyme or the enzyme-forming mechanism so that it is less sensitive to feedback inhibition or repression respectively. Although this is easily done with isolated enzymes (Gerhart and Pardee, 1962), the trick is to modify enzymes in the living cell. One such technique involves the production of mutants resistant to a growth-
15
INDUSTRIAL FERMENTATIONS
inhibitory analog of the desired compound. Before the development of the feedback concept, it had been noted that such mutants excrete the natural compound and it was felt that the accumulation itself was responsible for the reversal of growth inhibition. However, upon detailed analysis, it turns out that the accumulation is a result, rather than the cause, of the resistance (Moyed, 1964). Apparently, the analog mimics the natural compound in its feedback effects, and the major TABLE I1 EXCRETION BY ANALOGRESISTANT MUTANTS Analog
Excreted compound
p-Fluorophenylalanine
Amino acids tyrosine
Ethionine
Organism
Escherichia coli
methionine methionine
E. coli
Ethionine Ethionine
methionine
Neurospora crassa
6-Methyl tryptophan
tryptophan
Salmonellu typhimurium
Sulfonamide Isoniazid 3-Acetylpyridine
2,6-Diaminopurine
Vitamins p-ABA pyridoxine nicotinic acid Purines adenine
Candida utilis
Reference Cohen and Adelberg (1958) Adelberg (1958) Musilkova and Fend (1964) Metzenberg et al. (1964) Lingens et al. (1964)
Staphylococcus aureus Saccharomyces microsporus Chlamydomonas eugametos
Oakberg and Luria (1947) Scherr and Rafekon (1962)
S. typhimurium
Kalle and Gots ( 1962)
Nakamura and Gowans (1984)
part of a population exposed to such an antimetabolite cannot grow because of the resulting deficiency of the end product. However, rare mutants with modified enzymes or enzyme-forming systems that are insensitive to feedback effects of both the analog and the natural compound are selected by the inhibitory analog. Because of the genetic insensitivity to feedback, some of these mutants overproduce and excrete the end product. Examples of such excretions of amino acids, vitamins, and purines are shown in Table 11. Further examples in which the mechanisms responsible for the overproduction are known are described below. Maas ( 1961) showed that canavanine-resistant mutants of E . coli
16
ARNOLD L. DEMAIN
possess an enzyme-forming system for the arginine pathway which is not repressible by arginine and thus the culture excretes arginine. Calvo and Umbarger ( 1964) found that trifluoroleucine-resistant mutants of S. typhirnurium excrete leucine as a result of a similar mutation con-
Threonine
1
Pyruvate
lo
t a-acetolactate
k
a, P-Dihydroxy isovalerate
__
I a-Ketobutyrate I
c
a-Acetohydroxybutyrate I
a,0-Dihydroxy-0methylvalerate
I
---- --_ -
f-
a-Keto-P-methylvale rate
FIG. 10. Isoleucine excretion by a valine-resistant mutant of Escherichia coli.
trolling repression of the leucine pathway. Methionine excretion by norleucine-resistant mutants of E . coli is due to insensitivity of the pathway enzymes to repression ( Howbury, 1965). An interesting variation in a branched pathway is shown in Figure 10. In certain strains of E . coli, exogenous valine is a growth inhibitor, the effects of which can be reversed by isoleucine. Using vaIine as an inhibitor, certain valine-resistant mutants were obtained which excreted isoleucine. Analysis showed
INDUSTRIAL FERMENTATIONS
17
that, in these strains, enzymes one, four, and five were no longer subject to multivalent repression ( Ramakrishnan and Adelberg, 1964). In the histidine pathway, both histidine and 2-thiazolealanine inhibit the first enzyme, phosphoribosyl-ATP-pyrophosphorylase.Thiazolealanine-resistant mutants of both E . coli (Moyed, 1961) and S. typiiimurium (Sheppard, 1964) which are found to excrete histidine possess initial enzymes which are insensitive to feedback inhibition. A similar mechanism is involved in the excretion of tryptophan by E . coli mutants resistant to 5-methyl tryptophan ( Moyed, 1960). The observation that thienylalanine-resistant mutants of E. coli excrete phenylalanine and tyrosine (Adelberg, 1958) can be explained by the recent finding by Ezekiel (1965) that such mutants possess an altered initial enzyme of the pathway leading to aromatic amino acids. The mutant enzyme, in contrast to that in the wild-type, is not inhibited by phenylalanine or thienylalanine. Proline excretion by a mutant of E. coli resistant to 3,4-dehydroproline is due to insensitivity of the initial reaction of the pathway to feedback inhibition. The reaction involves the conversion of glutamic acid to its semialdehyde (Baich and Pierson, 1965). 2. Reverse Mutations Resistance to antimetabolites is not the only way of selecting mutants which excrete end products by virtue of derepressed enzyme systems or desensitized enzymes. Reverse auxotrophic mutations (reversion ) in structural genes have been known to result in enzymes which differ in structure from the original enzyme but are active nevertheless (Riley and Pardee, 1962). There is no reason to expect that such alterations in structure would not affect feedback characteristics. Umbarger ( 1963) reported that reversion of auxotrophic mutants lacking the first enzyme in a metabolic pathway often leads to revertants which excrete the end product of that pathway. Obviously the enzyme in the revertant is active but differs from the original enzyme in that it is insensitive to feedback inhibition.
IV. Regulatory Mechanisms and the Biosynthesis of Antibiotics
The two antibiotics to be discussed in this section are benzylpenicillin (penicillin G ) and cephalosporin C, shown below. Both have a side-chain, a 4-membered (3-lactam ring and a sulfur-containing ring, Penicillin, the first commercially produced antibiotic, is still the most potent and one of the least toxic drugs in use today for chemotherapy of bacterial infections. However, over the years, the staphylococcic population was
18
ARNOLD L. DEMAIN
C~H~--CH,-CO+NH-C-C/ I
I
I
H
H
s
YH3
\c-cH, l
I l C-N-CH
I
0
COOH
I
I
Be& ylpenicillin I
I HWC-CHW-
(CH,h,--
i
CO+NHI
I
H C-c
HS,,
I
C-N, 0
I
FH, ,C-CH,O-CO-CH, C’
I
COOH Cephalosporin C
building up resistance to penicillin via selection of penicilIin 0-lactamase ( penicillinase ) -producing strains. New drugs were clearly needed to combat these resistant forms, and cephalosporin C was a start in this direction. Although it contains the p-lactam ring, cephalosporin C is not attacked by penicillin p-lactamase, being protected by its 6-membered dihydrothiazine ring. The disadvantage of cephalosporin C lies in its weak activity. However, derivatives with other side-chains have been produced which are about as active as penicillin G. A. INHIBITION OF PENICILLIN BIOSYNTHESIS BY LYSINE The earliest studies on the penicillin fermentation showed that addition of phenylacetic acid or its derivatives markedly stimulated formation of the antibiotic. This represented a simple precursor-product relationship where synthesis of side-chain precursor by the mold was the rate-limiting reaction. Supplementation of the medium with side-chain precursor became an established practice. The precursors of the penicillin nucleus, 6-aminopenicillanic acid, were not detectable by such supplementation tests. However, by the use of starved resting cells, amino acid analogs, and tracer studies, it became obvious by the late 1950’s that L-cysteine and L-valine were the precursors of the remainder of the molecule (for a review of these early studies, see Demain, 1959). A peculiar observation made during studies on resting cell synthesis of penicillin in 1957 was that lysine inhibited production ( Demain, 1957). This was reminiscent of some earlier results obtained by Bonner (1947) while studying the relationship between biochemical deficiencies and penicillin production in Penicillium notatum; it was noted that about 25% of the lysineless strains faiIed to produce the antibiotic. During those 10 years, it had become known that lysine synthesis in fungi in-
INDUSTRIAL FERMENTATIONS
19
volves a-aminoadipic acid as a precursor. Soon thereafter it was found that a-aminoadipic acid couId reverse the inhibitory effects of Iysine on penicillin biosynthesis (Somerson et al., 1961). At about the same time, Arnstein and Morris (1960) isolated from P. chrysogenum a most interesting tripeptide believed to be a precursor of the penicillin nucleus; HOOC-CHN?i2-CH2-
C&-
0 SH II I C-NH-CH-CCH, CH--(CH& I I CO-NH-CHCOOH
C&-
-
6-(wAminoadipy1) cysteinylvaline
i.e., 6- ( a-aminoadipyl ) -cysteinylvaline. The work of Arnstein and Morris suggests that benzylpenicillin is formed as shown in Figure 11. a-Aminoadipic acid would combine first with cysteine and then with valine in
t
L-LY-AAA-L-CYS
I.-
i-
L-Val
a-AA A- L- Cys - L- Val (Tripeptide)
~-(~-AAA-6APA~Isopenicillin N)
1
C6H5- C%-
C6H5- CHa-CO-
COOH
6 APA(Benzylpenicil1in)
FIG.11. Possible scheme of benzylpenicillin (penicillin G ) biosynthesis. Abbreviations are as follows: AAA, aminoadipic acid; CYS, cysteine; VAL, valine; 6-APA, 6-aminopenicillanic acid.
a manner similar to that of glutathione formation. The cysteine-valine moiety of the tripeptide would then undergo a series of reactions to form the two-ring penicillin nucleus to which the side-chain of L-a-aminoadipic acid is still attached. Actually, this compound was recently isolated (Flynn et al., 1962; Cole and Batchelor, 1963) from P . chrysogenum broths and was named “isopenicillin N.” Penicillin G could
20
ARNOLD L. DEMAIN
then be formed by side-chain exchange with phenylacetic acid. It is clear that a-aminoadipic acid is involved in the initial reactions of penicillin biosynthesis. Bonner's lysineless nonproducers of penicillin were probably blocked before a-aminoadipic acid and thus could not form the tripeptide. Penicillin and lysine can be considered as end products of a branched pathway. The inhibition of penicillin synthesis
sugT-NH3 I
I
II I
II
1 t
Aminoadipic acid
LyS ine
FIG. 12. Possi'ble mechanism of inhibition of penicillin biosynthesis by lysine.
by lysine is most probably due to feedback inhibition and/or repression of the enzymes of the lysine pathway resulting in a deficiency of a-aminoadipic acid and consequent reduction of penicillin formation ( Fig. 12). The recent report of Trupin and Broquist (1965) that growth in excess lysine completely inhibited accumulation of a-aminoadipic acid by Neurospora crassa strain 4545, a mutant blocked in the conversion of aminoadipic acid to its semialdehyde, contributes to the credibility of a feedback regulation of penicillin synthesis by lysine.
B. STIMULATION OF CEPHALOSPORXN C FORMATION BY METHIONINE The stimulation of biosynthesis of cephalosporin C by methionine (for review, see Demain, 1963a) is of considerable interest since the sidechain of the antibiotic is u-a-aminoadipic acid and a metabolic relation-
21
INDUSTRIAL FERMENTATIONS
ship between the two amino acids is unknown. However, they both have similar structures from carbon atom 1 to atom 4.Nevertheless aminoadipic acid failed to duplicate the stimulatory effect of methionine; neither did other compounds with that same four-carbon structure: a-amino-n-butyric acid, lysine, or arginine ( Table I11 ). Additional compounds related to aminoadipic acid such as ketoadipic acid, pipecolic acid, diaminopimelic acid, and ornithine were also inactive. Apparently, methionine did not act as a precursor of a-aminoadipic acid. Its role as a sulfur source was TABLE 111 STIMULATION O F CEPHALOSPORIN c
FORMATION
Compound
Structure
Methionine a-Aminoadipic acid a-Aminobutyric acid Lysine
H:,C-S-CH,-CH,-CHNH,-COOH HOOC-CH2-CH,-CH,-CHNH,COOH CH:3-CH2-CHNHy--COOH
Activity
H2N-CH2-CH,-CH,-CH2-CHNH2-COOH
+
-
HN Arginine H2N
/
C--NH--CH,--CH,--CH,--CHNH,--COOH
-
considered by surveying a large number of sulfur compounds as methionine replacements. One group of compounds showed no activity at all. A second group showed a mild enhancement of activity which failed to approach the activity of methionine; this group included cysteine. A third group, which could replace methionine, was composed of methionine sulfoxide and s-methylcysteine. Both were thought to act after conversion to methionine. Subsequently the nonsulfur methionine antagonist, norleucine, was surprisingly found to be fully active. As with methionine, both isomers of norleucine were active, the D-form being the more potent. The activity of norleucine eliminated the possibility that methionine was acting primarily as a source of sulfur for the cephalosporin C molecule. Methionine
H,C-S-CH,-CH,-
Norleucine
H&-
CH2--C&-
C H m - COOH
Cq-
CHN&-
COOH
Further evidence against a precursor role for methionine was the finding that it had to be added early in the growth phase of the fermentation for maximal effect, rather than during the phase of antibiotic production (Demain, 1963b). The above data indicate that the major role of methionine is an indirect one which takes effect during growth of the fungus; this suggests repression. In E. coli, methionine represses cystathionine synthetase,
22
AANOLD L. DEMAIN
cystathionase, and homocysteine methylase, the three enzymes which convert cysteine to methionine (Rowbury, 1965). Cystathionase is identical to cysteine desulfhydrase (Rowbury and Woods, 1964) and, in addition to acting on cystathionine, converts cysteine to pyruvate, ammonia, and hydrogen sulfide. Thus, methionine represses cysteine breakdown. If cysteine is the true precursor of cephalosporin C sulfur, the addition of Sugar
NH,
so:-
FIG. 13. Possible mechanism of stimulation of cephalosporin C synthesis by mcthionine.
methionine might be expected to spare cysteine and stimulate antibiotic formation ( Fig. 13). Although exogenously added cysteine is only a weak stimulator of cephalosporin C biosynthesis, its low activity could be due to its oxidation to insoluble cystine during shaking. In agreement with the hypothesis depicted in Figure 13, Trown et al. (1963) showed that labeled cystine was incorporated into cephalosporin C at the site where one would expect it to enter. Some of the radioactivity from C14-cystine was distributed in other parts of the molecule as if there had been a partial degradation to pyruvate by cysteine desulfhydrase. This enzyme has been found in Cephalosporium sp. by Wixom ( 1963).
INDUSTRIAL FERMENTATIONS
23
The enhancement of cephalosporin C formation by norleucine is also consistent with regulatory prevention of cysteine breakdown. This analog has been shown to repress homocysteine methylase in E. coli (Rowbury and Woods, 1961) and there is no reason to expect that such would not be the case with cystathionase. Norleucine has also been found to inhibit the production of H2S from cystine in Proteus morganii (Porter and Meyers, 1945).
V. Synthesis of Macromolecules A. MICROBIALENZYMES Although this review emphasizes the production of small molecules, mention should be made of the possible application of knowledge of control mechanisms to industrial production of enzymes. Prior to the discovery of feedback regulation, the existence of inducible enzymes was known and substrates were generally tested for ability to increase enzyme production. In certain cases, it is easy to understand that such a procedure would be too expensive to use commercially. However, the realization that enzyme induction is related to repression-the inducer behaving in a manner opposite to that of an end-product “co-repressor”-allows us to expand considerably our abilities to produce enzymes in high yield. Since inducibility (and also repressibility ) is a genetically determined characteristic, it is possible to obtain constitutive mutants of an organism which normally produces an inducible enzyme (Pardee and Beckwith, 1963). Such constitutive or derepressed mutants produce the enzyme in the absence of inducer. Amazingly enough, by proper selection methods, mutants have been obtained which produce 25% of their protein as catalase (Clayton, 1962) or as @-galactosidase ( Novick and Horiuchi, 1961) . The importance of feedback control in the formation of extracellular enzymes is revealed by recent studies on protease. It had been known that formation of proteolytic enzymes by bacilli and actinomycetes was inhibited by protein hydrolysates or mixtures of amino acids. Chaloupka et al. (1963a) showed that addition of a single amino acid, isoleucine or threonine, caused a specific repression of protease synthesis by Bacillus megaterium; i.e., general protein synthesis was not inhibited. Furthermore, it was found (Chaloupka et al., 1963b) that the repression was related to the concentration of the amino acid in the medium and not to the concentration inside the cell. Thus, not only is evidence mounting that many extracellular enzymes are produced at the cell surface (Lampen, 1965) but now it appears that control mechanisms may be also acting at the exterior of the cell. An alternative explanation could be that the true
24
ARNOLD L. DEMAIN
“co-repressor” is an internal activated form of isoleucine or threonine, rather than the free amino acid.
13. MICROBIAL NUCLEIC ACIDS A control mechanism not yet mentioned is that concerned with the regulation of ribonucleic acid (RNA) synthesis by amino acids. As an example, consider a histidine-requiring microorganism growing in the presence of the amino acid. As long as histidine is added, the cell makes protein, RNA, etc.; it grows and multiplies. When the supply of histidine is exhausted, not only does synthesis of protein cease but also that of RNA. This regulation of RNA synthesis by the concentration of intracellular amino acids is under control of a special genetic locus. Certain E. coli mutants called “relaxed mutants (in contrast to the usual strains, which show “stringent”’ dependence of RNA synthesis on adequate supply of amino acids), are capable of continuing RNA synthesis in the absence of a required amino acid ( Neidhardt, 1964). Although this intracellular accumulation of RNA is not striking from the viewpoint of an industrial fermentation, it may be related to the RNA excretion phenomenon recently discovered in B. subtilis MB-1480 (Demain et al., 1965a). Thib strain was found to excrete large amounts of intact RNA during growth in a medium containing sugar, citrate, and inorganic salts. The excretion paralleled growth and ceased when the cells stopped growing. At the point of maximum cell yield, the weight of excreted RNA and its breakdown products was equal to over half the weight of the cells present. One could conceive B . subtilis MB-1480 to be a “relaxed” prototroph in which RNA is produced from glucose and ammonia faster than is protein, the “excess” RNA being excreted from the cells. This concept is supported by the observation that addition of amino acids to the medium stimulates growth and decreases excretion.
VI. Summary More often than not, the success of an industrial fermentation does not depend on addition of precursors. Rather, the effects of regulatory mechanisms such as feedback inhibition and repression are of extreme importance. Modification of feedback effects without destroying the metabolic activities of the cell has become a major activity of the ferments. tion microbiologist. Such alteration in living cells can be done by (1) decreasing the concentration of the end product and ( 2 ) modifying the sensitive enzyme or enzyme-forming system. The first principle has been used to accumulate intermediates of simple pathways (ornithine), and of branched pathways (inosinic acid, xanthylic acid) and also end products
INDUSTRIAL FERMENTATIONS
25
of branched pathways (lysine, threonine, valine). Use of the second principle has resulted in accumulation of end products of both simple and branched pathways ( tyrosine, methionine, tryptophan, arginine, leucine, isoleucine, histidine, phenlyalanine, proline, adenine, p-aminobenzoic acid, pyridoxine, nicotinic acid). Regulatory controls probably affect antibiotic production as well. The inhibition of penicillin formation by lysine and the stimulation of biosynthesis of cephalosporin C by methionine are apparently the results of normal feedback effects of these amino acids on their own biosynthesis. Knowledge of control mechanisms can be expected to stimulate much effort toward the industrial production of microbial enzymes and nucleic acids. Constitutive mutants have already been obtained which produce 25% of their protein as a single enzyme. Production of extracellular protease is controlled through repression by exogenous amino acids. Excretion of high concentrations of intact ribonucleic acid is another process controlled by amino acids added to the growth medium. AS more information is obtained, industrial fermentation will move from art to science and success in the fermentation field will no longer depend on “cookbook microbiology. Instead, the rewards will be reaped by those who appreciate the microbiological, biochemical, and, most important, the genetic relationship between the microorganism and its manmade environment. ACKNO WLEDCM ENT I wish to acknowledge the enthusiasm and stimulation provided by David Hendlin who was responsible for pointing out to me the implications and significance of regulatory mechanisms in fermentation research. I also thank Barbara Maling for reviewing the manuscript.
KEFERENCES Adelberg, E. A. (1958). J. Bacteriol. 76, 326-327. Ames, B. N., and Martin, R. G. (1964). Ann. Reo. Biochem. 33, 235-258. Arnstein, H. R. V., and Morris, D. (1960). Biochem. J. 76, 357-361. Baich, A., and Pierson, D. J. (1965). Biochim. Biophys. Acta 104, 397-404. Bonner, D. (1947). Arch. Biochem. 13, 1-9. Calvo, J. M., and Umbarger, H. E. (1964). Federation Pmc. 23, 377. Chaloupka, J., Kreckova, P., and Rihova, L. ( 1963a). Biochem. Biophys. Res. Commun. 12, 380-382. Chaloupka, J., Kreckova, P., and Rihova, L. ( 1963b). Nature 200, 885-886. Changeux, J. P. (1965). Sci. Am. 214, No. 4, 36-45. Clayton, R. K. (1962). Biochim. Biophys. Acta 58, 360-362. Cohen, G. N., and Adelberg, E. A. (1958). J. Bacteriol. 76, 328-330. Cole, M., and Batchelor, F. R. (1963). Nature 198, 383-384. Daoust, D. R., and Stoudt, T. H. (1966). Ueuelop. Ind. Microbiol. 7, 22-34. Datta, P., and Gest, H. (1964). Proc. Natl. Acad. Sci. US‘. 52, 1004-1009. Davis, €5. D. (1950). Experientia 6, 41-50.
26
ARNOLD L. DEMAIN
Demain, A. L. (1957). Arch. Biochem. Biophys. 67, 244-246. Demain, A. L. (1959). Advan. Appl. Microbiol. 1, 23-47. Demain, A. L. (1963a). Trans. N . Y. Acad. Sci. [2] 25, 731-740. Demain, A. L. (1936b). Clin. Med. 70, 2045-2051. Demain, A. L., Burg, R. W., and Hendlin, D. (1965a). J. Bacteriol. 89, 640-646. Demain, A. L., Jackson, M., Vitali, R. A., Hendlin, D., and Jacob, T. A. (196513). Appl. Microbiol. 13, 757-761. Ezekiel, H. D. (1965). Biochim. Biophys. Actu 95, 54-62. Flynn, E. H., McCormick, M. H., Stamper, M. C., DeValeria, H., and Godzeski, C. W. (1962). J . Am. Chem. Soc. 84, 4594. Freundlich, M., Bums, R. O., and Umbarger, H. E. (1963). In “Informational Macromolecules” (H. J. Vogel, V. Bryson, and J. 0. Lampen, eds.), pp. 287-391. Academic Press, New York. Gerhart, J. C., and Pardee, A. B. (1962). J. Biol. Chem. 237, 891-896. Huang, H. T. (1961). Appl. Microbiol. 9, 419-424. Jacob, F., and Monod, J. (1963). In “Biological Organization at the Cellular and Supercellular Level” (€3. J. C. Harris, ed.), pp. 1-24. Academic Press, New Yo&. Kalle, G. P., and Gots, J. S. (1962). Proc. SOC. Exptl. Biol. Med. 109, 277-281. Kisumi, M., Kato, J., and Chibata, I. (1964). J. Biochem. (Tokyo) 56, 450-456. Lampen, J. 0. ( 1965). In “Function and Structure in Microorganisms” (M. R. Pollak and M. H. Richmond, eds.), pp, 115-133. Cambridge Univ. Press, London and New York. Lingens, F., Kraus, H., and Lingens, S. (1964). Z. Physiol. Chem. 339, 1-8. Maas, W. K. (1961). Cold Spring Harbor Symp. Quunt. Biol. 26, 183-191. Maas, W. K., and McFall, E. (1964). Ann. Rev. Microhiol. 18, 95-110. Metzenberg, R. L., Kappy, M. S., and Parson, J. W. (1964). Science 145, 1434-1435. Misawa, M., Nara, T., Udagawa, K., Abe, S., and Kinoshita, S. (1964). Agr. Biol. Chem. (Tokyo) 28, 690-693. Monod, J., Changcux, J. P., and Jacob, F. (1963). J. Mol. BioZ. 6, 306-329. Moyed, H. S. (1960). J. Biol. Chem. 235, 1098-1102. Moyed, H. S. (1961). J. Biol. Chem. 236, 2261-2267. Moyed, H. S. (1964). Ann. Reu. Microbiol. 18, 347-366. Moyed, H. S., and Umbargcr, H. E. (1962). Physiol. Reo. 42, 444-466. Musilkova, M., and Fencl, Z. ( 1964). Folk Microbiol. (Prague) 9, 374-379. Nakamura, K., and Gowans, C. S. (1964). Nature 202, 826-827. Nakayama, K., Kitada, S., and Kinoshita, S. (1961a). J. Gen. Appl. Microbiol. (Tokyo) 7, 52-69. Nakayama, K., Kitada, S., and Kinoshita, S. (1961b). J. Gen. Appl. Microbiol. (Tokyo) 7, 145-154. Nakayama, K., Suzuki, T., Sato, Z., and Kinoshita, S. ( 1964). J. Gen. Appl. Microbiol. (Tokyo) 10, 133-142. Nara, T., Samejima, H . , Fujita, C., Ito, M., Nakayama, K., and Kinoshita, S. (1961). Agr. Biol. Chem. (Tokyo) 25, 532-541. Neidhardt, F. C. (1964). Progr. Nucleic Acid Res. 3. 145-181. Novick, A., and Horiuchi, T- ( 1961). Cold Spring .Harbor Symp. Quant. Biol. 26, 239-245. Oakberg, E. F., and Luria, S. E. (1947). Genetics 32, 249-261. Pardee, A. B., and Beckwith, J. R. (1963). In “Informational Macromolecules” (H. J. Vogcl, V. Bryson, and J. 0. Lampen, eds.), pp. 255-269. Academic Press, New York.
INDUSTRIAL FERMENTATIONS
27
Paulus, Ii.,and Gray, E. (1964). J. Biol. C h m . 239, PC4008-PC4009. Porter, J. R., and Meyers, F. P. (1945). Arch. Biochem. 8, 169-176. Ramakrishnan, T., and Adelherg, E. A. (1964). J. Bacteriol. 87, 566-573. Riley, M., and Pardee, A. B. (1962). Ann. Reu. Microbio2. 16, 1-34. Rowbury, R. J. (1965). Nature 206, 962-963. Rowbury, R. J., and Woods, D. D. (1961). J. Gen. Microbiol. 24, 129-144. Rowbury, R. J., and Woods, D. D. (1964). J. Gen. Microbiol. 35, 145-158. Scherr, G. H., and Rafelson, M. E. (1962). J. Appl. Bacteriol. 25, 187-194. Sheppard, D. E. (1964). Genetics 50, 611-623. Somerson, N. L., Demain, A. L., and Nunheimer, T. D. (1961). Arch. Biochem. Biophys. 93, 238-241. Stadtman, E. R. (1963). Bacteriol. Rev. 27, 170-181. Stadtman, E. R., Cohen, G. N., LeBras, G., and DeRohichon-Szulmajster, H. (1961). J . Biol. Chem. 236, 2033-2038. Sturani, E., Datta, P., Hughes, M., and Gest, H. (1963). Science 141, 1053-1054. Trown, P. W., Smith, B., and Abraham, E. P. (1963). Biochem. J. 86, 284-291. Trupin, J. S., and Broquist, H. P. (1965). J. Biol. Chem. 240, 2524-2530. Udaka, S., and Kinoshita, S. (1958). J . Gen. Appl. Mierobiol. (Tokyo) 4, 283-288. Umbarger, H. E. (1963). Ann. Reu. Plant Physiol. 14, 19-42. Umbarger, H. E. (1964). Science 14, 674-679. Vogel, H. J. ( 1961). In “Control Mechanisms in Cellular Processes” (D. M. Bonner, ed.), pp. 23-65. Ronald Press, New York. Wixom, R. L. (1963). Personal communication. Woolfolk, C. A,, and Stadtman, E. R. (1964). Biochem. Biophys. Res. Commun. 17, 313-319. Yugari, Y., and Gilvarg, C. ( 1962). Biochim. Biophys. Actu 62, 612-614.
This Page Intentionally Left Blank
Genetics in Applied Microbiology S. G. BRADLEY Wepartment of Microbiology, Uniuersity of Minnesota, Minneapolis, Minnesota
I. Introduction A. B. C. D.
................
.................
Direct Selection .................. Mutation Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Rational Screening . . . . . . . . . . . . . . ....... Mutagens . . . . . . . . . . .
C. Syncytic Recombination D. Transformation . . . . . . . IV. Phage-Host Interactions . . . .
................
29
31 32
42
C. Transduction . . . . . . . . . . . . . . . . . . . A. Induction and Repression . . . . . . . . . . . . . . . . . . . . . 13. End-Product Inhibition . . . . . . . . . . . . . . C. Protein Synthesis . . . . . . . . . . . . . . . . . . . VI. Cell-Free Syntheses . . . . . . . . A. Polypeptides ......... B. Chemical Transformatio VII. Future Applications . . . . . . ................. A. General Significance . . B. Antibody Synthesis . . . VIII. Conclusions . . . . . . . . . . . . . References ...................... ............
46
53
56
I. Introduction A search for new products initially centers on discovering the desired activities, for example, production of a novel antibiotic or alteration of a steroid. To date, the principal source of microbes able to synthesize a desired substance or to modify chemically a particular compound is nature (Calam, 1964). Immediately, the organism able to mediate a significant process is purified by serial subculture from well-separated single colonies, or by isolation of single cells or spores by micromanipulation. Numerous clones are examined on a variety of nutritional concoctions and subjected to diverse cultural conditions in order to detect strains possessing desirable features and to develop improved operating procedures ( Krasilnikov and Skriabin, 1960). Concurrently, new cultures 29
30
S. G. BRADLEY
which resemble the original organism are independently isolated from nature (Raper, 1946). Ultimately, the increase in yield that can be obtained by manual picking of spontaneous variants and by empirical studies on cultural conditions is limited, and additional approaches must be employed ( Backus and Stauffer, 1955). Spontaneous variants must also be picked and characterized in order to delineate the range of variation within the particular taxon because the identity of the microbe has import in the process patent.
II. Mutation and Selection A. D~RECT SELEC~ION Strains may be improved by direct selection; for example, phageresistant cultures can be derived from phage-sensitive populations by serial subculture of the survivors of viral lysis (Bradley and Jones, 1966). Technically, this can be accomplished by infecting a broth culture of a given microbe with a bacterial virus, incubating the mixture for several days, and then inoculating fresh medium with phage and the organisms that survived phage infection. Alternatively, the phage-resistant variants can be selected on solid medium. It should be noted that the survivors of phage infection may be either genetically resistant or physiologically resistant ( Welsch, 1957). The latter will prove virus sensitive upon reexamination; therefore, selection must be repeated until the bacteria grow equally well whether phage is present or not. Direct selection may also be used to develop resistance to the antibiotics produced by an organism. Many antibiotic-producing streptomycetes, as originally isolated, are remarkably susceptible to their own metabolic products. This restriction is not as severe as it first seems because most antibiotic biosynthesis occurs during the stationary phase of growth ( Bradley, 1961) ; however, streptomycete populations are physiologically heterogeneous and some hyphal elements enter the stationary phase of growth while other hyphae are still in the rapid phase of growth. Self-inhibition applies not only to antibiotic-producing microbes but to vitamin-producing cultures as well. Although the desired end-product is a growth factor, many organisms concurrently synthesize inhibitory metabolites. Drug resistance is of two types, frequently referred to as one-step resistance, or the streptomycin-pattern, and multi-step resistance, or the penicillin-pattern (Pollock, 1960). These designations are partially misleading because the pattern of resistance is a function of the medium and microorganism, as we11 as of the antibiotic. In general, drug-resistant variants can be selected directly by the following procedure: a sensitive
GENETICS IN APPLIED MICROBIOLOGY
31
population is inoculated into a minimally inhibiting concentration of antibiotic; after a few days’ incubation, the survivors are transferred to fresh medium with a doubled antibiotic concentration. Repeated subcultures, each with an augmented amount of drug, continue until the desired resistance is achieved, or until surviving microbes cannot be found.
B. MUTATIONRATE It is common practice to describe development of resistance by a bacterium to an antibiotic in terms of the number of subcultures required for it to become insensitive to a given drug concentration and to state whether acquisition of insensitivity occurs in one step or many steps. In my opinion, actual mutation rates should be determined for the most common pathogens treated with particular antibiotics. Mutation rate is often incorrectly used in the sense of frequency of mutants in a population. There are several strict, acceptable definitions of mutation rate; one is the probability that a cell division will yield one mutant offspring, expressed symbolically a = E / D , where a is the mutation rate, E is the number of mutational events, and D is the number of cell divisions. It must be emphasized that the number of mutational events ( E ) is not the same as the number of mutants ( M ) because a mutant, once formed, can grow. The number of mutants in a population, therefore, increases by new mutations and by growth of existing mutants. Because the number of cell divisions equals the cell increment, the number of divisions is approximately the same as the number of cells when the inoculum is small; therefore, a = E / N or E = uN. The Poisson distribution can be applied to the problem if the experiment is properly designed (Luria and Delbruck, 1943). It is necessary to set up the experiment such that in a large series of tubes, about one-third of the cultures contain no mutants. Generally, a trial run is required in order to get within the right range. The Po, that is, the probability that a tube will have no mutants, is used for two reasons: firstly, it is easy to score; secondly, the Poisson distribution for Po simplifies from P, = e-mmx/xlto Po = r m where , m is the mean value and x is a particular value. With respect to calculation of mutation rate, rn in the Poisson distribution refers to the average number of mutants per tube. The number of mutants per tube, however, is not known since only the presence or absence of mutants is scored. Because of the experimental design, that is, because m is approximately 1, we can assume that all of the mutational events occur in the last generation. Accordingly, the average number of mutants per tube is equivalent to the average number of mutational events per tube. Therefore a w can be substituted for
32
S . G . BRADLEY
-
m to give P , == e-aY. Solving for a, we obtain a = (2.3 log l / P ~ ) / where ~, N is the average number of cells per tube. The units of a are mutational events per cell division, It should be noted that many workers express mutation rates as the probability that a mutation will occur per cell per division. To convert the above equation into this unit, it is necessary to substitute N/ln2 for ??. There are various criticisms of the Poisson formulation; one is that the investigator must be able to set up the experiment such that Po is about %3 (actually as close to l / e as possible). Nevertheless, the null fraction of the Poisson is satisfactory for most purposes. Mutation rates are useful not only for characterizing drug-pathogen interactions but also for studies on mutagenesis and antimutagenesis (Novick, 1956). A search for substances that suppress development of drug resistance now seems feasible (Sevag and Drabble, 1962).
C. RATIONALSCREENING Because desirable variants cannot be directly selected, the success of the strain improvement program rests more on the ability to detect a superior strain than on the ability to produce one. Many thousand clones can be reliably screened manually. By use of replica plating, a few million clones can be examined cursorily. If 100-fold more organisms are to be surveyed, alternative methods must be devised. Unfortunately, mass screening techniques are confronted with two serious limitations : one is the lack of correlation between performance of a culture under survey conditions and under production conditions; the other is the decreasing ability of the system to recognize superior variants. In the early stages of a strain development program, an absolute yield increment of 500 units may well represent a 10-fold gain in activity. This difference is easily recognized. A few years later, the developed culture may produce 5000 units; then an absolute increment of 500 units represents only a 10% increase. Strains differing by 10% in productivity usually cannot be differentiated by a screening system; therefore, detection of superior variants becomes progressively more difficult. Several methods have been used differentially to recognize desirable variants; for example, antibiotic production can be estimated by measuring the diameter of the zone of inhibition around colonies overlaid with sensitive bacteria. Similarly, growth-factor production can be estimated by measuring the diameter of the ring of growth around colonies overlaid with microbes that specifically require the nutrient in question. The principal reservation about this approach is whether apparently increased activities reff ect superiority of a genetic strain or physiological variation such as colony size or stage of growth. It should be noted that optimal
GENETICS IN APPLIED MICROBIOLOGY
33
resolution is not necessarily achieved by using indicator strains that respond to very low concentrations of drug or growth factor. Rather than rely on differential techniques, selective techniques should be sought, that is, the growth or survival of superior strains is favored and the growth of inferior strains is arrested (Jones, 1966). Conceivably, an antibiotic-producing culture can be inoculated into a nutritionally limiting semisolid medium along with an organism that is sensitive to the antibiotic elaborated by the first microbe. Hopefully, superior strains will inhibit the competitor in their own microcosmos, whereas inferior strains will be starved. After several cycles of selection, the favored antibiotic-producing culture can be freed of the competitor, perhaps by adding high concentrations of drug to the plating medium. Superior growth-factor-producing variants may be selected by addition of analogs, which function as competitive inhibitors, to minimal medium (Adelberg, 1958; Scherr and Rafelson, 1962). Optimistically, variants with increased biosynthetic capacity will be able to grow whereas the parental types will be inhibited. Alternative approaches for selecting improved strains are needed, Obviously, no universally applicable method is known. Antiserum directed against an antibiotic (as the combining group) may be able to agglutinate preferentially viable organisms with greater synthetic capability than the parental culture. Organisms with increased biosynthetic potential may, at some critical stage of their life cycle, have altered buoyant densities or electrophoretic properties. The desired product, while held internally or at the cellular surface, may protect the microbes from noxious agents such as ultraviolet radiation or metallic salts. In closing this discussion on selection, it merits reemphasis that the success of a strain-development program is very much dependent upon the power of the selection system.
D. MUTAGENS The incidence of variants in a population can be increased by mutagens (Dulaney et al., 1949). Accordingly, a mutation project is an important part of every strain-improvement program ( Alikhanian, 1962). All of the known mutagens act by producing alterations in deoxyribonucleic acid (DNA) (Orgel, 1965). Because DNA is found in autonomous plasmids, as well as integrated into the intact genophore, treatment of an organism with a mutagen may cause alteration of episomes and plasmids (Gregory and Huang, 1964) as well as of conventional genes. Different mutagens have varied mechanisms of action; therefore, they produce diverse results and must be used under particular conditions ( Kihlman, 1961) .
34
S . G . BRADLEY
1 . Radiation The first truly effective means discovered for regularly inducing mutations was ionizing radiation (Stadler, 1928). However, the mechanism of action of ionizing radiation is not yet understood. Because the number of mutations produced by X-rays is directly proportional to the dose and independent of the rate of dosage, the mutagenicity of ionizing radiation seems to be attributable to direct hits on the genetic material, resulting in alteration or breakage of the DNA molecules (Kimball, 1963). It is obvious, though, that radiation also induces mutations indirectly, by active radicals formed in the milieu surrounding the genetic material. Anaerobiosis and sulfhydryl-containing compounds reduce the mutagenicity of X-rays (Kimball, 1955). Because X-rays in general act directly on DNA, the actual mutation is immediate and may affect both strands of the DNA double helix. The time required for the new phenotype to be expressed is determined by the number of unaffected genomes present, by the nature of the mutation (dominant or recessive), and by the time needed to replenish a significant proportion of the physiological machinery concerned with that expression. Perhaps the most widely used mutagen is ultraviolet radiation. The most effective wave lengths for producing mutations in microbes are between 253 mp and 265 mp (J. K. Setlow, 1963). Irradiation of media with ultraviolet light in the region of 200 mp is also mutagenic. The temperature of the substrate at the time of treatment affects the yield of mutants; cooling reduces the incidence of mutation. Moreover, ultraviolet radiation is most effective as a mutagen if the microorganisms are placed in an environment favoring protein synthesis immediately after exposure. For example, a population of bacteria treated with ultraviolet radiation and placed in chloramphenicol, a recognized inhibitor of protein synthesis, yields fewer mutants than cells not exposed to the antibiotic. In addition, bright light restores viability and causes a loss of mutations induced by ultraviolet radiation (Rupert et d.,1958). It should be noted that ultraviolet light is absorbed by glass and medium components; therefore, microbes should be exposed as a film on the surface of solid medium or in a thin layer of an aqueous suspension, Pyrimidines are photochemically altered by ultraviolet radiation, but purines are not affected. The pyrimidines become hydrated at their C-4 and C-5 positions; cytosine is more vulnerable than thymine. In addition, ultraviolet radiation induces thymine rings to coalesce in pairs to form thymine dimers (R. B. SetIow and Setlow, 1962). D'imers are probably first formed by adjacent thymines on the same DNA strand.
GENETICS I N APPLIED MICROBIOLOGY
35
This causes distortions in the double helix which allow dimers to form between thymines OR opposite strands. These bridges will prevent DNA strand separation. Although thymine dimer formation is a major cause of cell death by ultraviolet radiation, hydration of cytosine seems to be the alteration responsible for mutation. Because ultraviolet radiation usually affects only one strand of DNA, replication is necessary to fix the mutant genotype, as well as to permit development of the phenotype. 2. Chemicals Analogs of purines and pyrimidines are mutagenic (Freese, 1959); examples are 5-bromouracil, which is an analog of thymine, and 2aminopurine, which is an analog of adenine. Bromouracil is in fact extensively incorporated into the DNA of bacteria. The mere substitution of bromouracil for thymine does not itself result in mutation. The mutation arises as a result of mispairing. There are two possible errors: one starts with adenine ( A ) aligning with bromouracil, thereby leading to the incorporation of bromouracil into the DNA instead of thymine ( T ) . Later, bromouracil will accidentally mispair with guanine ( G) resulting in the change A-T to G-C.Alternatively, guanine may align with bromouracil, thereby incorporating it into the DNA instead of cytosine ( C ) . Later bromouracil will pair with adenine, resulting in the change G-C to A-T. The majority of the bromouracil-induced mutations seem to be G-Cto A-T transitions (Lawley and Brookes, 1962). Aminopurine, unlike bromouracil, is not extensively incorporated into DNA; however, it is an effective mutagen. When incorporated into DNA, 2-aminopurine substitutes for adenine, pairing with thymine. Aminopurine, in the imino-form, can pair with cytosine. As with bromouracil, there are two possible errors: thymine may align with 2-aminopurine, which subsequently will pair with cytosine, resulting in a change from A-T to G-C; or cytosine may align with 2-aminopurine, ultimately resulting in a change from G-Cto A-T. In order for an analog to induce a mutation, it must be incorporated into the DNA; moreover, DNA replication is necessary to provide opportunity for mispairing to occur. To induce variants, the microbes must be cultivated in the presence of the analog for many generations. To obtain sufficient incorporation of analog, it may be necessary to use mutants unable to synthesize the parent compound, or to inhibit its synthesis with another drug. Sulfanilamide or fluorouracil, for example, can be used to increase incorporation of bromouracil into DNA. Like radiation, some chemicals alter the structure of DNA directly. Nitrous acid, for example, oxidatively deaminates adenine to form hypoxanthine, guanine to form xanthine, and cytosine to form uracil. Thy-
36
S . G . BRADLEY
mine is not affected. Xanthine and guanine pair alike in the DNA double helix, so this alteration is not mutagenic. Hypoxanthine, however, aligns with cytosine, thereby resulting in a change from A-T to G-C, and uracil aligns with adenine, resulting in a change from G-C to A-T. Hydroxylamine apparently reacts only with cytosine, altering it in such a way that it can pair with adenine. Hydroxylamine, therefore, results in the change from G-C to A-T (Freest: et al., 1961). The alkylating agents such as ethyl ethanesulfonate and ethyl methanesulfonate also act directly on DNA ( Lawley and Brookes, 1961; Bautz and Freese, 1960). They attack the N-7 position of guanine, possibly forcing guanine to align with thymine, thereby producing the change G-C to A-T. More commonly, the alkylated guanine is released from the sugar-phosphate chain. The missing guanine may subsequently be replaced by any of the four DNA bases. The direct-acting chemical mutagens affect resting cells, and only limited DNA replication is needed in order to fix the mutation. Several divisions, however, may be required for expression of the new phenotype. Mutagens should be chosen for diversity in mechanism of action. A mutational program employing one base analog and one alkylating agent is more likely to produce novel variants than one using only base analogs. Moreover, the mechanisms of action must be considered in the experimental design; some mutagens act directly on DNA, whereas others act only in replicating systems. All experiments must allow for phenotypic lag. Conceivably, it may be profitable to treat synchronous cultures with mutagens for short intervals during the replicative cycle (Ryan, 1963). This approach, fraught with technical problems, may give some specificity to the mutational alterations. It must be emphasized that mutants with desirable attributes will constitute a minority of the population, and an intense, critical selective system must be imposed upon the treated cultures ( Davies, 1964). As noted earlier, spontaneous variants should be picked and characterized in order to delineate the range of variation within the taxon. In addition, it is prudent to determine the range of variation encountered after treatment with mutagens.
Ill. Recombinational Mechanisms A. SEXUALRECOMBINATION
Many eukaryotic microbes possess true sexual cycles involving meiosis ( Beadle, 1959; Lewis, 1961; Papazian, 1958); industrially important examples include the genera Saccharomyces, Penicillium, and Phycomyces. In Saccharomyces cereuisiae, both the haploid and diploid phases may
GENETICS IN APPLIED MICROBIOLOGY
37
be prolonged indefinitely. Under appropriate conditions, the diploid cells will develop into asci. The asci regularly contain four ascospores in an unordered fashion. The ascospores germinate to form homothallic or heterothallic populations which subsequently clump, undergo cell fusion and zygote formation. The diploid zygote, which is readily propagated, is usually more hardy and vigorous than the parental haploid strains. Most characteristics in yeasts segregate according to the rules of classical genetics. Expressions controlled by nuclear genes include fermentative capabilities, sensitivity to drugs and cations, clumping, frothing, lipid production, and alcohol tolerance (Erebo et al., 1961). In addition, the genetic determination of cephalosporin production by the homothallic ascomycete Emericellopsis has been partially analyzed by meiotic recombination (Fantini, 1962). Antibiotic production seems to be controlled by both multiple alleles and multiple loci. Sexual recombination provides a means for introducing new attributes into established strains and for increasing strain variability (Nelson, 1963). Sexuality cannot be expected to be the panacea for strain improvement programs; however, it should not be relegated to the academicians because it was tried once and failed!
B. PARASEXUALITY Detection of a cryptic and rare recombinational event in a microbe necessitates conditions that select for the unique phenotype in a vast population of parental organisms. Two types of selective markers are available: those in which desired forms can grow but all others are inhibited; and those which are readily recognizable even in crowded cultures (Bradley, 1962). The first class of selective characteristics is exemplified by nutritional sufficiency in contrast to requirement for exogenous growth factor, and resistance to toxic substances in contrast to susceptibility. Morphological characteristics are of the second type. Although appropriately marked strains can be obtained as a result of direct selection and by use of mutagens, this approach takes time and may concurrently induce undesirable alterations in the stock cultures. Naturally existing variants, for example, organisms differing in their ability to utilize particular carbohydrates or organic nitrogen sources, will provide adequate suitable selective markers. Appropriate strains are grown together on an unselective medium, and the resulting mycelium subcultured to a selective medium. Hyphal fragments from nonparental growth are serially plated on a selective medium to establish the intracellular nature of the interaction. Next, spores are tested; if the spores yield predominantly parental types, the nonparental growth is heterokaryotic. From the heterokaryon, a heterozygous diploid
38
S. G. BRADLEY
must be derived, by plating uninucleate conidiospores, if available, onto a selective medium. The heterozygous diploid nuclei will ultimately give rise to recombinants, either by vegetative haploidization or by mitotic crossing over. In this way, the parasexual cycle (Pontecorvo, 1953) has been demonstrated in the following fungi: Aspergillus nidulans; A. niger (Pontecorvo et al., 1953), which produces citric acid; the koji molds A. oryzae and A. sojae (Ikeda et al., 1957); Penicillium chrysogenum ( Pontecorvo and Sermonti, 1954), which produces penicillin; Fusarium ( Buxton, 1956) ; and Ustilago ( Holliday, 1961). Numerous strain improvement programs have used parasexual recombination ( Macdonald et al., 1964). Although recombinants producing more penicillin than the parental P . chrysogenum cultures have occasionally been found (Alikhanian and Kameneva, 1961), the segregants are generally the same as or inferior to the parents. Sermonti (1961) has expressed reservations about the utility of parasexuality in a strain development program; however, Alikhanian (1962) has advocated its use as an important adjunct to the mutation and selection project.
C. SYNCYTICRECOMBINATION I . Recombination in Actinomycetes Formation of nonparental types from mixed growth of genetically marked actinomycetes has been demonstrated for Streptomyces uiolaceoTuber (Bradley, 1959; Hopwood and Sermonti, 1962), S. aureofaciens ( Alikhanian and Borisova, 1961 ), S. rimosus ( Alikhanian and Mindlin, 1957), S. griseus (Bradley and Lederberg, 1956), and Nocurdia opaca ( Adams and Bradley, 1963). The fundamental recombinational processes seem to be identical in each of these microbes and in enterobacteria (Bradley, 1963). To detect syncytic recombination, stocks having dissimilar auxotrophic characteristics are grown together, and prototrophs are selected on media that will not support growth of the parental types individually. A heterogenomic mycelium is established by hyphal fusion, which in these coenocytic prokaryotic organisms is tantamount to nuclear fusion. Because there are no nuclear membranes to restrict interactions between different genomes ( Fuhs, 1965), classical heterokaryosis and diploidy do not exist in the prokaryotes. Hyphal anastomosis as determined cytologically does not invariably lead to formation of a functional heterogenomic mycelium (Bradley and Anderson, 1960). Genetic interaction among actinomycetes is restricted by compatibility systems (Bradley and Anderson, 1958; Adams, 1964). The exuberant growth that develops from a mixture of auxotrophs can be maintained indefinitely by subculture of long hyphal strands, although selective conditions may be needed to prevent dissociation. Spores borne
GENETICS IN APPLIED MICROBIOLOGY
39
by heterogenomic mycelia of S. griseus rarely perpetuate the prototrophic character; a chain of spores usually contains conidiospores of each parental type. S. violaceoruber spores are primarily recombinant types. Usually the recombinants initially display the dominant characteristics : drug sensitivity over resistance; prototrophy over auxotrophy, and actinophage sensitivity over resistance. From primary recombinants of S . violaceoruber, both parental types can be recovered as clones derived by serial subculture of spores from single colonies. Nonparental types are also found; some of these are stable with respect to one characteristic but remain heterogeneous with respect to other characteristics. Another nonparental class is stable for all expressions studied. Antibiotic production by S. violaceoruber is genetically controlled (Bradley, 1965a). Recombinants formed by the interaction of a productive strain and a nonproductive strain are able to synthesize antibiotic. Yields by recombinants resulting from a cross between a low-producing mutant and a high-producing one are usually intermediate between the parents; however, clones with increased and decreased productivity have been encountered. Infrequently, recombinants from a cross between two nonproducing mutants give yields comparable to the wild type. In addition to antibiotic yields, other industrially important attributes, such as foaming in S. rimosus, have been shown to be genetically controlled and manipulatable in breeding programs ( Mindlin et al., 1961). Applied studies with S. aureofaciens, the chlortetracycline producer ( Borisova et al., 1962), and S. erythreus, the erythromycin culture (Huang-Lo, 1962), have given promising results. Syncytic recombination has been demonstrated not only between independently isolated members of the same strain but also between different species. Interspecific recombination, involving pairwise mixtures of S. aureofaciens, S. violaceoruber, and S. rimosus, has been reported ( Alacevic, 1963). Interspecific hybridization, like intraspecific hybridization, can be detected by selecting for nutritionally nonexacting recombinants from mixtures of nutritionally exacting parents. Organisms for which auxotrophic variants are not available can be tested for ability to recombine with auxotrophic, drug-resistant tester strains. The organisms to be tested may be prototrophic and must be sensitive to the particular antibiotic, for example, streptomycin or chlortetracycline. The recombinants are selected from mixed cultures on a minimal medium containing drug. In the nocardiae and streptomycetes, natural markers can be used; these include carbohydrate utilization, drug susceptibility, and actinophage host range. It should be noted that these screening methods are also useful for detecting heterothallic, intraspecific recombination.
40
S . C . BRADLEY
2. Genetic Homology among Actinomycetes Although recombinants have been obtained from pairs of distinctly different streptomycetes, the base compositions of their deoxyribonucleic acids are essentially identical. In fact, the base compositions of the DNAs of the streptomycetes and nocardia are all approximately the BASE
TABLE I COMPObll lONS OF DEOXYHlBONUCLEIC ACIDS FROM ACTINOMYCETES BASEDON THEHMAL DENAlUHATlON TEMPEHATURES
Organism Streptomyces aureofaciens Cellulomoms biazoteu Orskov’s motile Nocardia ATCC 12288 S. viridochromogenes S. a2bus Actinoplanes philippinensis S . fradiae Micrococcus lysodeikticus S. violaceoruber S. griseus S. venezuelae
Actinophage MSP8 Actinophage MSP2 Myxococcus zanthus Nocardh farcinica Streptosporangium roseum S . cinnamomeus S. niveus N . asteroides 92 N . brasiliensis N . asteroides 96 Mycobacterium phlei N . rubra M. stercoicles Mycohacterium sp. ( wotochromogenic) Arthrobacter glohiformis N . erythropolis N . canicniria
-
% Guanine + Cytosine _..___ ~
~
76 76 75 74 74 74 73 72 72 71
71 70 70 70 70 70 69 69 68
67 67 67 66 66 66 64 62 62,
same (Table I ) . Therefore, sufficient genetic homology may exist within the actinomycetes to permit extensive interspecific hybridization. Syncytic recombinational studies are technically tedious and are biologically restricted by the experimental conditions and by compatibility systems. Accordingly, genetic homology can be better assessed by measuring renaturation between single-stranded DNAs of diverse origin ( Bradley, 1965b). The actinomycetes are grown in a chemically defined medium with N15-ammonium salts or Nl5-nitrate salts, or in minimal medium supple-
41
GENETICS IN APPLIED MICROBIOLOGY
mented with peptone and either uridine-CI4 or thymidine-C1*. The DNA is prepared as follows: the mycelium is disrupted with a combination of lysozyme and sodium dodecyl sulfate; protein is denatured with chloroform; nucleic acids are precipitated with ethanol; the ribonucleic acid (RNA) is digested with ribonuclease; the DNA is precipitated with isopropanol. Nl"1abeled DNA of the tester strain, and unlabeled DNA from another strain, are denatured by heating, then mixed, incubated at 60°C., and allowed to cool slowly to permit renaturation. Deoxyribonuclease active on single-stranded DNA is used to degrade the remaining denatured DNA. The hybrid DNA molecule is detected in CsCl density gradient-equilibrium centrifugation ( Schildkraut et al., 1961) . Alternatively, genetic homology is determined with denatured DNA trapped in agar. The DNA-agar is forced through a sieve, washed, and put into test tubes with denatured, sheared DNA-C14 of the tester strain; the mixture is incubated at 60°C. for 15 hours. The DNA-agar DNAC14 is washed to remove unbound DNA. The hybridized DNA is removed with 0.01 M saline-citrate at 75°C. The hybrid DNA carrier DNA is precipitated with trichloroacetic acid and collected on a membrane filter and the radioactivity is measured. The extent of renaturation is the index of genetic homology between the two organisms ( McCarthy and Bolton, 1963). Preliminary studies have shown that the members of the genus Streptomyces possess nearly complete genetic homology ( Tewfik and Bradley, 1966). These results give credence to the reports of biological interspecific recombination, and suggest that breeding programs may well create offspring with novel and useful biosynthetic properties.
+
+
3. Unified Viezcj of Bacterial Recombination
In closing this discussion on syncytic recombination, it should be noted that the recombinational process in the actinomycetes resembles the parasexual cycle of Aspergillus in all its essential features except that the actinomycetes lack an organized nucleus surrounded by a double membrane ( Hopwood and Glauert, 1960). Moreover, the fundamental genetic processes in the true bacteria and the actinomycetes seem to be the same. For example, Escherichia coli cells and S. violaceoruher hyphae each contain several to many sets of genetic information. In neither organism is the germ plasm separated from the cytoplasm by a well-defined nuclear envelope. In both organisms, cellular or hyphal fusion is tantamount to nuclear fusion. The resulting heterogenomic state is maintained during several cell divisions in E. coli and indefinitely in the mycelia of S. violaceoruber. Homogenomy is established in S. violaceoruber by serial plating of spores. In both microbes, parental types are recovered frequently from primary recombinants, and many recombinant phenotypes may be derived from a single heterogenomic clone. Different sublines of a hetero-
42
S. G. BRADLEY
genomic clone often show disparate patterns of segregation for one or more characters. Recombinant clones may appear homogeneous for some expressions but heterogeneous for others. These results are best explained in terms of repeated opportunity for recombination during vegetative growth. Streptomyces violaceoruber differs from E. coli in that the mycelial growth habit of the former stabilizes the heterogenomic state, whereas the relatively regular cell division of the latter establishes homogeneity rather quickly. If pedigrees based on single cells of E. coli (Lederberg, 1957) are compared with ones based on serial spore analysis in S . violaceoruber (Bradley et al., 1959), the differences disappear. D. TRANSFORMATION Genetic transformation is the transfer of a limited amount of genetic information as free DNA from a disrupted cell to an intact cell. Although transformation may be obtained with DNA directly and spontaneously liberated by the donor, extracted and purified DNA is more often used. Obviously, DNA extracts from a single clone contain more than one genetic factor; in fact, as many transforming activities can be detected in a suitable recipient as there are powerful selective systems to detect them. In other words, all characters that can be recognized can also be transferred. However, genetic transformation is well established for only a limited number of species; for example, pneumococcus (Avery et al., 1944), Hemophilus influenzae (Alexander and Leidy, 1951), Bacillus subtilis ( Spizizen, 1958), Neisseria catarrhalis ( Catlin and Cunningham, 1961), Rhizobium ( Balassa, 1963), and Streptococcus ( Bracco et al., 1957). Among these organisms, not all strains of a species can be transformed. The general protocol for a transformation experiment can be summarized as follows: the donor cells are lysed, preferably with deoxycholate or lysozyme and sodium dodecyl sulfate. Mechanical or sonic disruption of the cell is to be avoided; even pipetting of the solutions should be performed gently and kept to a minimum so that the DNA is not excessively sheared. Protein is then removed from the crude DNA preparation by treatment with chloroform, detergents, or phenol. The purified DNA is sterilized during the last ethanol precipitation. Filtration is not effective and is deleterious. The stock DNA is stable in 0.15 M to 2 M NaCl at 5°C. for years. The DNA is added to a suitable recipient at a concentration of about 1pg./ml.; after 10 to 30 minutes, deoxyribonuclease is added. Claims for transformation based upon experiments using 100-fold more DNA and exposure periods of several days should be viewed with skepticism because selection may well account for the emergence of new phenotypes. The bacteria, after exposure to DNA, must be
GENETICS
IN
APPLIED MICROBIOLOGY
43
given adequate time for integration and expression of the new character. The principal factor limiting detection and application of transformation is the genetic and physiological competence of the recipient to incorporate DNA. competence develops only under narrow cultural conditions which must be empirically determined; determinative factors include pH, ionic content, temperature of incubation, and agitation. Experience gained with one species is useless with another; for example, competence in pneumococcus or Hemophilus is obtained in complex media but for Bacillus a minimal medium is better. The point on the growth curve at which competence develops varies with the species; it is the early exponential phase for Rhizobium and the late exponential phase for Bacillus and Hemophilus. A search for transformation in a particular organism can best begin with a study of the cultural conditions that will induce competence. A reasonable index of competence is the ability of the microbe to take up DNA labeled with P33 or C14 (Lerman and Tolmach, 1957). The DNA used must be homologous because heterologous DNA may be, but is not invariably, taken up by competent cells. Escherichiu coli DNA, for example, is readily taken up by pneumococcus but Bacillus DNA is not taken up by Hemophilus. Although there are a few reports of genetic transformation in the actinomycetes (Jarai, 1961; Matselyukh, 1964)) these are not yet well enough documented to accept without reservation. Conditions for DNA uptake, however, have been worked out for S. griseus (Germaine and Anderson, 1966) and for S. uureofuciens (Kuroda and Bradley, 1966). In both systems, a complex medium is used, and DNA is extensively taken up during the late exponential phase of growth. These studies by independent groups strongly suggest that reproducible transformation systems for actinomycetes will be established in the near future.
IV. Phage-Host Interactions A. RESISTANCE AND LYSOGENIZATION When a population of sensitive bacteria is exposed to a large number of bacteriophage, the vast majority of the host viable units is infected and killed. Upon continued incubation, a secondary growth develops; it is often phage sensitive even though the medium contains millions of bacterial virus particles. This is possible because bacterial metabolites and depletion of the nutrients suppress additional phage multiplication. However, truly resistant mutants do arise; as many as one spontaneous resistant variant may be found per million organisms. The resistant variants remain susceptible to unrelated phages but simultaneously acquire resistance to phages related to the challenging virus. Resistance may be
44
S. G . BRADLEY
due to inability of the phage to infect, grow vegetatively, or mature, or to alteration of the attachment sites, or to lysogenization. The resistant variants often deviate markedly from the parental bacterium with respect to morphological, cultural, and biochemical characteristics. Alikhanian and Iljina (1958) have reported that 99% of the Streptomyces olioaceus organisms that survived phage lysis are markedly altered phenotypically. Some of these variants have been found to produce more antibiotic and more vitamin than the parental strains. These workers suggest that the actinophage is acting as a mutagen. Cultures of actinomycetes once exposed to actinophages often liberate virus during subsequent subcultures. Such cultures are frequently referred to as lysogenic, but most of the surviving mycelia are actually pseudolysogenic, that is, are mixtures of externally carried phage and a partially resistant actinomycete population. Partial phage resistance may be a genetically determined cellular phenotype, may be due to the presence of truly resistant variants in a population of sensitive organisms, or may be due to physiological heterogeneity of mycelial fragments. In general, young hyphae are more vulnerable to phage infection than are spores and older hyphal strands. Pseudolysogenic cultures are readily freed of phage by serial subculture from single colonies and by treatment of the culture with antiserum directed against the phage. Small volumes of specific antiserum can be prepared by injecting mice twice intraperitoneally with lo1(’ phage. To make the antiserum phage specific, it is mixed with washed host mycelium and incubated at 30°C. for 1 hour. The mycelium is removed by centrifugation. True lysogeny is widespread among the actinomycetes; in fact about 3570 of streptomycete cultures are reputed to be lysogenic. To detect iemperate phage in culture filtrates, it is necessary to test them against a variety of possibly sensitive indicator strains. Other criteria which suggest lysogeny are sporadic, spontaneous plaque formation and the observation of phage-like elements in electron micrographs of filtered culture fluid. Induction of temperate actinophage formation by lysogenic cultures has not been reproducibly achieved. Lysates prepared by infection with a virulent phage often contain temperate phage. It has been equally difficult to free lysogenic actinomycetes of their prophages ( Welsch, 1959).
B. LYSOCENIC CONVERSION Cultures of virulent Corynebacterium diphtheriae invariably contain free phage, indicating that some fraction of the population is lysogenic and has been induced, or that the culture is pseudolysogenic (Freeman, 1951). Lysogenization of a nontoxigenic strain with a phage from a toxi-
GENETICS I N APPLIED MICROBIOLOGY
45
genic culture may, but does not invariably, confer toxigenicity upon the recipient bacterium. Significantly, a nontoxigenic culture which is not converted by one phage may be converted by another phage from a toxigenic organism. Generally, lysogenic conversion can be demonstrated only with phage derived from toxin-producing cultures of C . diphtheriae; however, there are a few examples in which nontoxigenic strains that cannot themselves be converted to toxigenicity liberate temperate phage which can confer the power to make toxin upon other nontoxigenic cultures. Undoubtedly toxin production is the result of an interaction between the phage and host genomes (Barksdale et al., 1961). In lysogenic conversion there is a one-to-one relationship between lysogenicity and toxigenicity ( Groman, 1955). Clearly, certain phages of Corynebacterium carry genetic determinants governing toxin production. The expression of the toxigenic characteristic occurs as a result of infection with an appropriate lytic phage or by induction of the proper temperate phage. In either instance, toxin production is correlated with vegetative proliferation of phage. Phage proliferation, however, does not in itself ensure that toxin will be produced ( Barksdale et al., 1960). Addition of iron to an induced toxigenic culture will allow phage to replicate but will not permit toxin production. Lysogenic conversion is not restricted to the genus Corynebacterium. The synthesis of polysaccharide moieties of salmonella 0 antigens is determined by the presence of the suitable phage genome.
C. TRANSDUCTION Lysogeny is widespread in the genus Bacillus; many temperate phages of Bacillus can be induced to initiate lytic growth by ultraviolet radiation, mitomycin C, or hydrogen peroxide. Moreover, phages isolated from the soil may be able to lysogenize appropriate strains. In addition, virulent phages can mutate to temperancy. Thorne (1962) isolated from soil a bacillus-phage that has the power to carry a limited amount of genetic information from a lysed cell to a suitable recipient. The phage, virulently propagated on a streptomycin-resistant, prototrophic bacterium, is able to transfer wild-type alleles individually to mutants requiring exogenous indole, arginine, histidine, adenine, guanine, thiamine, leucine, or methionine. This general transduction occurs at a frequency of once per lo6 plaque-forming units. Deoxyribonuclease does not affect the incidence of transduction, but phage-specific antiserum prevents genetic transfer. In general, treatment of the phage lysate with ultraviolet light increases the frequency of transduction. General transduction is well established within the enterobacteria and the genera Pseudomonas and Staphylococcus ( Campbell, 1964).
46
S. G. BRADLEY
Analysis of results from syncytic crosses in Escherichia coli have established that the temperate phage h is integrated into the bacterial genome per se and is closely linked to the loci controlling galactose fermentation. Morse et al. ( 1956) have induced galactose-fermenting, lysogenic cultures and have added the purified phage to galactose nonfermenters. Galactose-fermenting cells are recovered when lysogenic or h-sensitive cultures are used as the recipient but not when h-resistant cultures are the recipient. In general, anything which inactivates h phage also destroys transductive capacity. The h system differs from general transduction in two significant respects: induced h phage has the power to transduce genes but lytically propagated phage is not effective; h phage serves as a vector for only those loci which are adjacent to its position within the bacterial genome. The transfer of genetic information by actinophages has been reported. Alikhanian and Teteryatnik (1962) have claimed that the ability to produce streptomycin can be transferred to nonproducing variants by means of phage. Some of the strains derived in this manner synthesize more antibiotic than the parental cultures. Similar results have been described for S. erythreus. It is impossible to decide whether these variants arose by mutation and selection or whether they are true recombinants.
V. Genetic Control of Biosynthesis A stock culture of a bacterium, yeast, or mold kept by serial transfer in the laboratory retains its characteristics for years. To a large extent, this is a reflection of the stability of the genes of microbes. When the microbe is subcultured into a different environment, however, its morphological and physiological expressions may be altered. Certain enzymes are absent unless their substrates are available, other enzymes are present only if their products are kept scarce. Pigments may be formed at one temperature but not at another. These changes in outward manifestations can be attributed to alterations of the genetic material itself; alternatively, all of the genic composition of an organism may not be expressed at any one time. Dominance and epistasis are examples wherein some of the genetic potentiality is not expressed.
A. INDUCTION AND REPRESSION In microbes, there are many examples of nonhereditary enzymic variations which take place against a constant genetic background (Maas and McFall, 1964). Induced enzymes, which are formed only when the substrate is present in the medium, are readily demonstrable in yeasts and bacteria. Certain strains of E. coli, for example, possess no lactose-
GENETICS IN APPLIED MICROBIOLOGY
47
splitting activity ( (3-galactosidase) when grown in glucose medium but develop it soon after transfer to lactose medium. When these cells are returned to glucose medium, (3-galactosidase activity is lost. It must be emphasized that although an expression is variable, the potential to adapt is definitely a regularly inherited factor (Jacob and Monod, 1961). In yeasts, adaptive enzymes can be produced by nongrowing populations. This clearly indicates that the process involves conversion of enzymically deficient cells into active cells, and not the emergence of a new population of capable cells. This observation also suggests that replication of the genetic material itself is not required for induction to occur. Metabolic inhibitors which affect protein synthesis and ribonucleic acid syntheses do interfere with adaptation. Indeed, it has been shown that induced enzyme results from new synthetic activity and not by release of preformed, intact enzyme molecules.
1. (3-Galactosidase Synthesis Because bacteria have short generation times, it is not possible usually to demonstrate adaptation in the absence of growth. This makes it difficult to distinguish between direct inductive effects and indirect effects, such as availability of energy, particularly when a utilizable inducer is used. However, inducers which are not substrates and substrates which are not inducers have been found. It is possible, therefore, to induce cells in such a way that they are not dependent upon utilization of the inducing substrate, and to measure induced enzyme activity without the problem of continuing induction. Nitrophenyl-(3-D-galactosideis one example of a noninducing substrate for (3-galactosidase. When E. coZi is grown in media containing succinate as the carbon and energy source, no lactose-splitting (3-galactosidaseis formed. When nonutilizable methyl(3-D-galactoside is added to the succinate medium, the induced enzyme develops. Enzyme synthesis begins almost immediately upon addition of inducer and increases directly proportional to the increase in mass of the growing population. This suggests that each induced cell makes enzyme maximally and that there is no noticeable transition from the fully negative to the fully positive state. When inducer is removed, further (3galactosidase synthesis stops promptly. The observations described above are for cells confronted with a large concentration of non-substrate inducer. When E. coli cells are grown in succinate broth with a low concentration of non-substrate inducer, the amount of enzyme per unit of bacterial protoplasm is intermediate between that of a fully induced culture and that of an uninduced culture. The question arises: does this intermediate enzymic activity come about because the individual cells contain less enzyme, or because some of the
48
S. G . BRADLEY
cells are fully induced whereas others are unaltered? When these populations are examined on an indicator medium, some of the cells are found to be enzymically active whereas others are inactive. The adapted cells can be isolated and cultivated in the presence of an even lower inducer concentration; these cells will retain their ability to produce (3-galactosidase. The unadapted cells grown in the medium containing minute amounts of inducer will remain unable to make (3-galactosidase. The same end result can be obtained by taking adapted and unadapted cells and placing them in a medium containing inducer and a suitable concentration of glucose. Glucose inhibits the formation of induced (3-galactosidase by E. coli. The induced cells remain induced and the uninduced cells remain uninduced, even though they are cultivated in the same medium! A nonhereditary alteration in phenotype has been overtly stabilized. These observations should forewarn you that differentiation between mutation and induction is not always easy. In addition to the structural genes concerned with the synthesis of p-galactosidase and the permeation system, there are regulatory genes. According to one popular theory, regulatory genes direct the production of substances called repressors, which control the activities of specific structural genes or a coordinated group of structural genes. A repressor presumably acts by combining with a genetic locus, designated the operator, which is responsible for the transcription of the genetic information contained in a group of genes. The regulator and operator loci themselves are subject to mutation and are amenable to genetic manipulation by transduction, transformation, and syncytic recombination. An alteiiiative explanation is that repressors act at the level of the ribosome, affecting release of nascent proteins, rather than at the level of transcription of the DNA itself. Both of these proposed mechanisms may be operative. It should be noted that induction and repression are merely different aspects of the same phenomenon ( Hayes, 1964). In general, repression is associated with anabolic systems and induction with catabolic systems.
2. Antibiotic Synthesis The biosynthesis of antibiotic by S. uiolaceoruber has most of the attributes of an adaptive or derepressed system (Bradley, 1961). Washed mycelia of S. zjiolaceoruher grown in a complex medium that does not support antibiotic production begin to produce antibiotic after several hours’ incubation in aqueous glucose. If growth medium or chloramphenicol is added prior to detectable antibiotic synthesis, no antibiotic is made. Between the sixth and twentieth hours of incubation of washed mycelium in aqueous glucose, the yield and rate of antibiotic production increase. If growth medium or chloramphenicol is added between the
GENETICS IN APPLIED MICROBIOLOGY
49
twelfth and eighteenth hours of incubation, antibiotic synthesis continues, but at a linear rate. These observations suggest that the enzyme system for antibiotic synthesis is developed as a nonhereditary response to the internal or external environment of the cell. The adaptive system may be induced specifically by substances generated within starving or nutritionally imbalanced cells, or metabolites repressing fabrication of the needed enzymes may be present in growing hyphae but be depleted in nonproliferating systems.
B. END-PRODUCX INHIBITION In most biosynthetic pathways leading to the formation of essential metabolites, the end-product of the pathway will inhibit the activity of the first enzyme of the sequence or, where branched pathways are concerned, of the enzyme at the point of branching (Gorini, 1963). Accordingly, the activity of an anabolic system is controlled by the rate at which its end-product is utilized or removed. End-product inhibition has several notable characteristics: it is specific in that only the end-product, or an analog of it, is effective; a single enzyme is inhibited; and the action is immediate. Another significant feature of this type of control is that the inhibitory end-product has little steric relationship to the substrate of the affected enzyme. The enzymes susceptible to end-product inhibition, therefore, bear two sites of specificity, one for its substrate and the other for control. The two sites are independently vulnerable to alteration by mutation. Mutations affecting the sites for attachment of the inhibitor may release the enzyme from end-product control and lead to the excretion of large amounts of the metabolite into the medium. This is the rationale which led Adelberg (1958) to select for mutants which overproduced metabolites. Karlstrom ( 1965) has applied these principles to development of superior strains for amino acid production. C. PROTEIN SYNTHESIS I t has been recognized for many years that the chemical basis of heredity is deoxyribonucleic acid (DNA) and that the metabolic attributes of an organism are due to the catalytic action of specific proteins (Burton, 1955). If, as is presently believed, the specificity of an enzyme is determined by its amino acid sequence, the problem becomes: how does information stored in DNA direct protein synthesis? DNA does not intervene directly, but instead relies on three species of ribonucleic acid (RNA), all of which are made as complementary copies of a strand of DNA, with uracil substituting for thymine (Commoner, 1964). The site of protein synthesis is the ribosome, an entity composed of ribosomalRNA and protein. The nucleotide composition of ribosornal-RNA is con-
50
S . G . BRADLEY
stant, even between diverse genera. The ribosomes are nonspecialized structures which receive specific information in the form of messengerRNA. Messenger-RNA is complementary to the DNA of the structural genes. The information in the messenger-RNA is stored in the following manner: ( a ) three bases specify one amino acid, ( b ) the code is not overlapping, ( c ) the sequence of bases is read from a fixed starting point, ( d ) there are no commas to protect a given triplet “reading frame,” and ( e ) several triplets code for one amino acid. The third class of RNA is the soluble- or transfer-RNA. The first step in protein synthesis is activation of an amino acid by adenosine triphosphate. The activated amino acid is transferred to the appropriate soluble-RNA ( s-RNA) . In prokaryotes, activated amino acids may be transferred to nonmethylated s-RNA, but in eukaryotic systems, activated amino acids are transferred only to methylated s-RNA. The charged s-RNA carries its amino acid to the proper site on the ribosome. As noted earlier, the specificity of the ribosome is due to the attached messenger-RNA (m-RNA ). Actually several ribosomes are usually attached to a single m-RNA molecule. This aggregate is called a polysome. In bacterial systems, m-RNA reacts directly with intact, preformed ribosomes, but in eukaryotes, m-RNA may first bind with a ribosomal precursor. The amino acid-charged s-RNA molecules, in order to align themselves according to the base sequence of the m-RNA, must become affixed to the ribosome. Next, adjacent amino acids are joined, thereby adding to the polypeptide chain; the uncharged s-RNA molecule is released from the ribosome and a charged s-RNA molecule takes its place. With the addition of each amino acid to the polypeptide chain, the point of contact between the ribosome and the m-RNA changes; the m-RNA advances, or is advanced, one “reading frame” or triplet toward the 3’ end of the m-RNA molecule. Ultimately, the entire message is translated, and the m-RNA and polypeptide are individually released from the ribosome.
VI. Cell-Free Syntheses A. POLYPEPTIDES Several cell-free systems for protein synthesis have been described for microorganisms; these include Saccharomyces ( So and Davie, 1963), Neurospora (Wainwright, 1959), Bacillus brevis (Uemura et al., 1963), Bacillus subtilis (Frontali et al., 1964), Salmonella typhimurium ( Martin and Ames, 1962), Escherichia coli (Tissieres et al., 1960), and Streptomyces aureofuciens (Mizukami and Bradley, 1966). Determinative factors in the S. aureofaciens system for incorporation of C1*-amino acids
51
GENETICS IN APPLIED MICROBIOLOGY
into the matter insoluble in hot 10% trichloroacetic acid are the concentrations of adenosine triphosphate ( ATP), the components of the ATPgenerating system, guanosine triphosphate, mercaptoethanol, K+ and Mg++, and the pH of the buffer. NUCLEOT~DE SEQUENCES OF Amino Acid Alanine Arginine Asparagine Aspartic acid Cystein'e Glutamine Glutamic acid Glycine Histidine Loleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan Tyrosine Valine
THE
TABLE I1 RNA-TRIPLETS SPECIFYING EACHAMINO Acma Ordered T ~ i p l e t GpCpA CpGpA ApGpA CpUpA
; ; ; ;
CpApA ; GpApA ; GpGpA ; UpUpA ; ApApA ; UpApA ; ( ApUpA 1e; CpCpA UpCpA ApCpA UpCpA
; ; ; ;
GpUpA ;
GpCpC ; GpCpU CpGpG ; CpCpU (ApGpC) ( C P U ~ G//) ~ApApU GpApU UpGpU CpApG GpApG //ApCpU GpGpC ; GpGpU CpApU ApUpU UpUpG / / C p U p u ApApC (UpApGId ApUpG upupu CpCpG ; CpCpU U p C p G ; UpCpU ApCpC ; ApCpU UpGpG UpApU GpUpG ; GpUpU
; ;
GpCpC CpGpC
; ;
ApApC GpApC upcpc
;
; ApGpCC ; GpCpC ; CpApC ; ApUpC ; cpupc
;
upupc
; CpCpC ; UpCpC ; ApCpC ; ;
UpApC GpUpC
a Based primarily on the work of Khorana (1965) and Nirenberg et al. (1965). Italicized triplets are assigned on the basis of both trinucleotide-stimulated binding of charged s-RNA and polynucleotide-stimulated polypeptide synthesis. Triplets in brackets represent the author's predictions. All other triplets are assigned on the basis of binding or synthesis, but not both. b Based upon trinucleotide-stimulated binding of s-RNA, this pair of triplets should be assigned to leucine. c Based upon trinucleotide-stimulated binding of s-RNA, this pair of triplets should be assigned to serine. 6 Nirenberg et at. (1965) have designated this pair of triplets as nonsense codons. e Jukes (1965) believes that the triplet ApUpG solely codes for methionine and that ApUpA codes for isoleucine.
The discovery by Nirenberg and Matthaei (1961) and Lengyel et d. ( 1961) that synthetic polynucleotides stimulate incorporation of C14amino acids into protein by cell-free extracts has made it possible to determine the actual nucleotide triplet code for each amino acid. For example, when synthetic polyuridylate is added to the E. coli cell-free
52
S. G . BHADLEY
system for protein synthesis, only phenylalanine-C14 is incorporated. Polycytidylate stimulates incorporation of proline. By addition of different synthetic copolymers containing nucleotides in different ratios, coding triplets for the 20 amino acids have been determined. Each amino acid has more than one set of code words. It is significant that no matter what organism is used to prepare a cell-free extract for protein synthesis, the code word UU U invariably causes phenylalanine incorporation. The identification of the sequence of bases in the coding triplet has been facilitated by the discovery by Leder and Nirenberg (1964) that ribosomes combined with synthetic trinucleotides will bind s-RNA molecules charged with their specific CI4-amino acids. Accordingly, only valinecharged s-RNA will adhere to ribosomes bearing the trinucleotide GpUpU, cysteine-charged s-RNA to ribosomes bearing UpGpU, and leucine-charged s-RNA to ribosomes bearing UpUpG (Table 11). Biologically active polypeptides can be synthesized by cell-free systems; for example, gramicidin S is made by extracts of Bacillus brevis (Hall and Sedat, 1965). Presently the role of m-RNA in directing the synthesis of polypeptide antibiotics is not resolved. In fact, several cyclic polypeptides, and peptides containing unusual linkages, unusual components, or D-amino acids, seem to be synthesized without the direct intervention of ribosomes and m-RNA (Mach and Tatum, 1964; Adiga et al., 1965). B. CHEMICAL TRANSFORMATIONS
It is sometimes desirable to separate in time and place the growth of microbes from the actual manufacturing process. Although the living organisms may be harvested and stored as such, frozen or dried preparations are more convenient and reliable. Presently, crude enzyme preparations, which are in reality dried microorganisms, are used to increase the nutritional value of livestock feed by converting indigestible fiber into utilizable materials, for removing hair from hide, and to produce a finer grain and greater pliability in leather. A more important potential application of these microbial reagents is for chemical transformations, in particular, hydroxylation, hydrogenation, and dehydrogenation. It should be pointed out that many of these enzymes are adaptive; therefore, crude enzyme powders may have to be prepared from mycelia that have been exposed to a suitable inducer. Ultimately, purified enzymes, affixed to a resin ( Bar-Eli and Katchalski, 1960), will be used extensively to perform simple chemical transformations and will be used in sequence to produce derivatives modified in several sites.
GENETICS I N APPLIED MICROBIOLOGY
53
VII. Future Applications A. GENERAL SIGNIFICANCE Microbial genetics constitutes the rationale for strain improvement programs (Elander, 1966). More important, it provides an approach for developing new products; for example, it is reasonable to expect that recombinants will produce unique antibiotics. A continuing, perplexing problem in clinical medicine is the emergence of drug-resistant organisms during chemotherapy ( Finland, 1955) . Presently, drug combinations are used so that mutants resistant to one antibiotic will be killed by the second agent. Alternatively, the incidence of the mutation from drug sensitivity to resistance can conceivably be reduced. In fact, numerous substances which decrease the mutation rate are known. Some of these show promise in preclinical studies (Johnson and Bach, 1965). Microbial genetics, as a tool for applied microbiologists, is primarily limited by the goals set and not by inability to meet them. Applied microbiologists must include in their domain all problems which can be studied by microbiological techniques. Microbiologists should be developing continuous cultures of diploid cell lines which have lost the antigens responsible for homograft rejection ( Merrill, 1959). Conceivably, organ culture using economical substrates can produce hormones on a commercial scale. Critical experiments evaluating the contributions of autoimmunity and accumulation of deleterious mutations to the aging process must be devised ( Szilard, 1959). Genetic transformation must be explored as a means of correcting hereditary metabolic defects. The regulatory systems of mammalian tissues ought to be understood (Cox and MacLeod, 1964): Do the steroid hormones control transcription of the DNA? Can antimetabolites be used to select in vivo for derepressed variants which will excrete needed substances? It is reasonable to suppose that obesity and hypercholesterolemia can be overcome by inhibiting the appropriate synthetic enzymes by adding suitable repressors. Alternatively, anabolic activity may be increased by stimulating transcription of the proper structural genes. Memory may be stored as stable informational RNA (Agranoff et ak., 1965). Learning ability or retention may be improved by use of inhibitors of ribonuclease; conversely, mental illness can conceivably respond to drugs affecting stability and translation of m-RNA. B. ANTIBODY SYNTHESIS Control of antibody synthesis is one of the most challenging problems in applied microbiology ( Billingham et al., 1956). Classical humoral
54
S. G . BRADLEY
antibody constitutes an important part of the body’s defense against disease (Jerne, 1960). Contrarily, most of the symptomatology associated with viral diseases is due to immune reactions. Other deleterious aspects of immunity are allergy, autoimmune disease, and rejection of needed homografts. Can we use our understanding of microbial genetics to gain insight into the mechanism of antibody synthesis and thereby learn how to control and exploit it? Antibodies are elicited specifically by antigens. Antigens are foreign substances which must be partially degradable by the reticuloendothelial system. A foreign substance which is not degradable, or one which is quickly and completely degraded, is not immunogenic. Complex antigens are apparently split into several fragments, each containing a reactive site or hapten. The hapten fragment is probably coupled with a transport molecule, Recent work suggests that the transport molecule is RNA. The hapten-RNA is produced by phagocytes and subsequently transferred to plasma cell precursors. Based upon these suppositions, a primary immune response in uitro requires the interaction of two cell-types. Antibodies are proteins of high specificity; yet all y-globulin molecules have a common component, The specificity of the antibody molecule resides in combining sites which are formed by polypeptide chains arranged in a prescribed order (Fudenberg, 1965). If four polypeptides make up the combining site, it is possible for each immature plasma cell to contain a finite number of antibody precursors which will allow it to respond to any antigen. Logically, the amino acid sequence of each of these peptides and of the common component of y-globulin molecules is determined by the DNA in structural genes (Dreyer and Bennett, 1965). It is reasonable to propose that each structural gene makes a molecule of m-RNA which becomes attached to a ribosome. The m-RNA may direct the synthesis of a single molecule of polypeptide, which remains attached to the m-RNA-ribosome. A region of this polypeptide chain will become a part of the combining site of the completed antibody molecule. The antigenic site of the hapten-RNA, which has been transferred from a phagocyte to an immature plasma cell, reacts with the individual, incomplete combining sites of the polypeptides which are still attached to their m-RNA-ribosome. In this way, the ribosomes directing the synthesis of all of the elements of the combining site of a specific antibody become bound together. The RNA portion of the hapten-RNA may serve as the m-RNA for the common component of the y-globulin molecule. According to this concept, antigen actually plays the determinative role in organizing the polysome that produces a specific antibody. In immature plasma cells, antibody is accumulated within the cells and released only by cell death, usually provoked by the interaction of cell-bound antibody,
GENETICS IN APPLIED MICROBIOLOGY
55
antigen, and complement. A vast excess of antigen will destroy all of the cells producing a specific antibody, resulting in immunological unresponsiveness. This proposal predicts that immunological paralysis and tolerance require continuous presence of excess antigen ( Burnet, 1961). The antibody released by immature plasma cells is a macroglobulin. As the plasma cells mature, they develop a secretory system which fixes the molecules so that they have a constant molecular weight and acquire resistance to mercaptoethanol. The primary structure of euglobulin, that is, classical antibody, is essentially the same as that of corresponding macroglobulin. In the absence of antigen, and therefore in the absence of hapten-RNA, no new antibody polysomes will be organized. Euglobulin production, therefore, in the absence of antigen is inherited semiclonally. During continued exposure to antigen or upon secondary presentation of antigen, some new induction of antibody synthesis occurs, but the principal response is proliferation of the differentiating plasma cells which are already dedicated to the production of a given specific antibody. All antibody cells, however, are presumed to require hapten-RNA in order to organize their antibody-producing polysomes. This model for antibody synthesis indicates that in vitro production of specific euglobulin will require continued presence of either antigen or a degraded antigen coupled with RNA. Moreover, the model leads to the prediction that inhibitors of nucleic acid and protein syntheses will suppress both macroglobulin and euglobulin production. In order to affect euglobulin synthesis differentially, substances which alter cellular morphogenesis are apt to be more useful than are analogs of metabolites.
VIII. Conclusions Mutation and selection have gained universal acceptance as the principal tools for strain development. More effort, however, needs to be made to increase the discrimination of the selective systems because rational screening is an integral part of any program to improve an existing fermentation or to discover a new product (James et al., 1956; Ostroukhov and Kuznetsov, 1963). Proposed projects employing genetic recombination as an approach to strain improvement have been received reluctantly. This pessimism is largely based upon a misunderstanding of the probable results of a breeding program. Recombination is a means of introducing new genetic information into an organism and increasing its variability. Crosses should not be expected to yield a high proportion of desirable progeny; stringent selection must be imposed upon vast populations in order to recover desired types (Alikhanian et al., 1962). Undoubtedly our understanding of biological systems will continue to
56
S. G . BRADLEY
advance at a rapid rate. The new information and more precise techniques will have to be considered in the design of strain improvement programs and in the search for new products. The success of an applied microbiologist will depend upon his ability to assimilate new ideas and to adapt existing methods in order to accommodate a particular microbe or product. The greatest challenge lies in the recognition of new goals. The philosophy of microbial genetics should exert a determinative role in the development of cell-free syntheses on a commercial scale (Merrifield, 1965), and in the forced and directed evolution of microbes to serve our needs here and extraterrestrially ( Abelson, 1961) .
REFERENCES Abelson, P. H. (1961). Proc. Natl. Acad. Sci. U.S. 47, 575-581. Adams, J. N. (1964). 1. Bacteriol. 88, 856-876. Adams, J. N.,and Bradley, S. G. (1963). Science 140, 1392-1394. Adelberg, E. A. (1958). J. Bacteriol. 76, 326-328. Adiga, P. R., Ucmura, I., Yukioka, M., and Winnick, T. (1965). Federation Proc. 24, Pt. I, 283. Agranoff, B. W., Davis, R. E., and Brink, J. J. ( 1965). Proc. Natl. Acad. Sci. U.S. 54, 788-793. Alacevic, M. (1963). Nature 197, 1323. Alexander, H., and Leidy, G. (1951 ). 3. Exptl. Med. 93, 345-359. Alikhanian, S. I. (1962). Aduan. Appl. Microbiol. 4, 1-50. Alikhanian, S. I., and Borisova, I. N. ( 1961). J. Gen. Microbiol. 26, 19-28. Alikhanian, S. I., and Iljina, T. S. (1958). Nature 181, 1476-1477. Alikhanian, S. I., and Kameneva, S. V. (1961). Sci. R e p . Ist. Super. Sanita 1, 454457. Alikhanian, S. I., and Mindlin, S. Z. (1957). Nature 180, 1208-1209. Alikhanian, S. I., and Teteryatnik, A. F. (1962). Mikrobiologiya 31, 54-60. Alikhanian, S. I., Chernosvitova, V. I., and Lubinskaya, S. I. (1962). Antibiotiki 7, 491. Avery, 0. T., MacLeod, C. M., and McCarty, M. (1944). J. ExptZ. Med. 79, 137-158. Backus, M. P., and Stauffer, J. F. (1955). Mycologia 47, 429-463. Balassa, G. (1963). Bacteriol. Reu. 27, 228-241. Bar-Eli, A., and Katchalski, E. (1960). Nature 188, 856-857. Barksdale, W . L., Garmise, L., and Horibata, K. (1960). Ann. N.Y. Acad. Sci. 88, 1093-1108. Barksdale, W. L., Garmise, L., and Rivera, R. (1961). J . Bacteriol. 81, 527-540. Bautz, E., and Freese, E. (1960). Proc. Natl. Acad. Sci. U.S. 46, 1585-1594. Beadle, G. W. (1959). Science 129, 1715-1719. Billingham, R. E., Brent, L., and Medawar, P. B. (1956). Nature 178, 514-519. Borisova, L. N., Konyoukhova, M. V., and Ivakina, N. S. (1962). Antibiotiki 7, 685. Bracco, R. M., Krauss, M. R., Roe, A. S., and MacLeod, C. M. (1957). J. Exptl. Med. 106, 247-259. Bradley, S. G. (1959). Ann. N.Y. Acad. Sci. 81, 899-906. Bradley, S. C. (1961). Develop. Ind. Microbiol. 3, 362-369. Bradley, S. G. (1962). Ann. Reu. MiCTObiol. 16, 35-52.
GENETICS IN APPLIED
MICROBIOLOGY
57
Bradley, S. G. (1963). Proc. 11th Intern. Congr. Genet., The Hague, 1963 Vol. 1, p. 14. Pergamon Press, Oxford. Bradley, S. G. (1965a). Deuelop. Ind. Microhiol. 6, 296-301. Bradley, S. G. (1965b). Intern. Bull. Bacteriol. Nomenclat. Taxonomy 15, 239-241. Bradley, S. G., and Anderson, D. L. (1958). Proc. SOC.Exptl. Biol. Med. 99, 476-478. Bradley, S. G., and Anderson, D. L. (1960). J. Gen. Microbiol. 23, 231-241. Bradley, S. G., and Jones, L. A. (1966). Progr. Ind. Microhiol. 7 (in press). Bradley, S. G., and Lederberg, J. (1956). J. Bacteriol. 72, 219-225. Bradley, S. G . , Anderson, D. L., and Jones, L. A. (1959). Ann. N.Y. Acacl. Sci. 81, 811-823. Burnet, F. M. (1961). Science 133, 307-311. Burton, K. (1955). Biochem. J. 61, 473-483. Buxton, E. W. (1956). 1. Gen. Microbiol. 15, 133-137. Cnlam, C. T. (1964). Progr. Id.Microbiol. 5, 1-53. Campbell, A. M. (1964). In “The Bacteria” ( I . C. Gunsalus and R. Y. Stanier, eds.), Vol. 5, pp. 49-85. Academic Press, New York. Catlin, B. W., and Cunningham, L. S. (1961). J. Gen. Microbiol. 26, 303-312. Commoner, B. ( 1964). Am. Scientist 52, 365-388. Cox, R. R., and MacLeod, C. M. (1964). Cold Spring Harbor Symp. Quunt. Biol. 29, 233-251. Davies, 0. L. (1964). Biometrics 20, 576-591. Dreyer, W. J., and Bennett, J. C. (1965). Proc. Natl. Acad. Sci. U.S. 54, 864-869. Dulaney, E. L., Ruger, M., and Hlavac, C. (1949). Mycologia 41, 388-397. Elander, R. P. (1966). Deuelop. Ind. Microbiol. 7, 61-73. Erebo, L., Johnsson, H., Nordstrom, K., and Moller, A. (1961). J. Inst. Brewing 67, 76. Fantini, A. A. (1962). Genetics 47, 161-177. Finland, M. (1955). New Engl. J. Med. 252, 570-580. Freeman, V. J. ( 1951). J. Bacteriol. 61, 675-688. Freese, E. (1959). J. Mol. Biol. 1, 87-105. Freese, E., Bautz, E., and Freese, E. B. (1961). Proc. Natl. Acad. Sci. U.S. 47, 845855. Frontali, L., Leoni, L., and Tecce, G. (1964). Nature 203, 84-85. Fudenberg, H. H. ( 1965). Ann. Reti. Microhiol. 19, 301-338. Fuhs, G. W. (1965). Bacteriol. Reti. 29, 277-293. Germaine, G. R., and Anderson, D. L. (1966). Bacteriol. Proc. p. 34. Gorini, L. (1963). Bacteriol. Rev. 27, 182-190. Gregory, K. F., and Huang, J. C. C. (1964). J. Bacteriol. 87, 1287-1294. Groman, N. B. (1955). J. Bacteriol. 69, 9-15. Hall, J. B., and Sedat, J, W. (1965). Federation Proc. 24, Pt. I, 282. Hayes, W. (1964). “The Genetics of Bacteria and Their Viruses,” pp. 594-627. Wiley, New York. Holliday, R. (1961). Genet. Res. 2, 231-248. Hopwood, D. A., and Glauert, A. M. (1960). J. Biophys. Biochem. Cytol. 8, 267-278. Hopwood, D. A., and Sermonti, G. (1962). Aduan. Genet. 11, 273-342. Huang-Lo, L. (1962). Mikrobiologiya 31, 61-65. Ikeda, Y., Nakamura, K., Uchida, K., and Ishitani, C. (1957). J. Gen. Appl. Microbiol. ( T o k y o ) 3, 93-101. Jacob, F., and Monod, J. (1961). J. Mol. Biol. 3, 318-356.
58
S. G . BRADLEY
James, L. V., Rubbo, S. D., and Gardener, J. F. (1956). J. Gen. Microhiol. 14, 223227. Jarai, M. (1961). Acta Microbiol. Acad. Sci. Hung. 8, 81-87. Jeme, N. K. (1960). Ann. Rev. Microbiol. 13, 341-358. Johnson, H. G., and Bach, M. K. (1965). Bacten'ol. Proc. p. 17. Jones, L. A. (1966). Deuelop. Ind. Microbiol. 7 , 124-128. Jukes, T. €1. (1965). Am. Scientist 53, 477-487. Karlstrom, 0. (1965). Biotech. Bioeng. 7, 245-268. Khorana, H. G. ( 1965). Federation Proc. 24, Pt. I , 1473-1487. Kihlman, B. A. (1961). Aduan. Genet. 10, 1-59. Kimball, R. F. (1955). Ann. N.Y. Acad. Sci. 59, 638-648. Kimball, R. F. (1963). Genetics 48, 581-595. Krasilnikov, N. A., and Skriabin, G. K. (1960). Antibiotiki 5, 121. Kuroda, S., and Bradley, S. G. (1966). Unpublished results. Lawley, P. D., and Brookes, P. (1961). Nature 192, 1081-1082. Lawley, P. D., and Brookes, P. (1962). J. Mol. Biol. 4, 216-219. Leder, P., and Nirenberg, M. W. (1964). Proc. Natl. Acad. Sci. U.S. 52, 1521-1529. Lederberg, J. (1957). Proc. Natl. Acad. Sci. U S . 43, 1060-1065. Lengyel, P., Speyer, J. F., and Ochoa, S. (1961). PTOC. Natl. Acad. Sci. U.S. 47, 1936-1942. Lerman, L. S., and Tolmach, L. J. (1957). Biochim. Biophys. Acta 26, 68-82. Lewis, D. (1961). Nature 190, 399-400. Luria, S. E., and Delbruck, M. (1943). Genetics 28, 491-511. Maas, W. K., and McFall, E. (1964). Ann. Rev. Microbiol. 18, 95-110. Macdonald, K. D., Hutchinson, J. M., and Gillett, W. A. (1964). Antonie van Leeuwenhoek, J . Microbiol. Serol. 30, 209-224. Mach, B., and Tatum, E. L. (1964). Proc. Natl. Acud. Sci. U S . 52, 876-884. Martin, R. G., and Ames, B. N. (1962). Proc. NatE. Acad. Sci. U S . 48, 2171-2178. Matselyukh, B. P. (1964). Dokl. Akad. Nauk S S S R 154, 710-715. McCarthy, B. J., and Bolton, E. T. (1963). Proc. Natl. Acad. Sci. U S . 50, 156-164. Merrifield, R. B. (1965). Science 150, 178-185. Merrill, J. P. (1959). Physiol. Rev. 39, 860-884. Mindlin, S. Z., Alikhanian, S. I., Vladimirov, A. V., and Mikhailova, C. R. (1961). Appl. Microbiol. 9, 349-353. Mizukami, I., and Bradley, S. G. (1966). Develop. Ind. Microbiol. 7, 326-334. Morse, M. L., Lederberg, E. M., and Lederberg, J. (1956). Genetics 41, 142-156. Nelson, R. R. (1963). Ann. Reu. Microbiol. 17, 31-48. Nirenberg, M. W., and Matthaei, J. 13. (1961). Proc. Natl. A d . Sci. U S . 47, 1588-1602. Nirenberg, M., Leder, P., Bemfield, M., Brimacombe, R., Trupin, J., Rottman, F., and ONeal, C. (1965). Proc. Natl. Acad. Sci. U.S. 53, 1161-1168. Novick, A. (1956). Brookhaven S y m p . Biol. 8, 201-215. Orgel, L. E. (1965). Aduan. Enzymol. 27, 289-346. Ostroukhov, A. A., and Kuznetsov, V. D. (1963). Antihiotiki 8, 33-35. Papazian, H. P. (1958). Advan. Genet. 9, 41-69. Pollock, M. R. (1960). Brit. Med. Bull. 16, 16-22. Pontecorvo, G. (1953). Aduan. Genet. 5, 141-238. Pontecorvo, G., and Sermonti, G. (1954). 1. Gen. Microbiol. 11, 94-101. Pontecorvo, G., Roper, J., and Forbes, E. (1953). J. Gen. Microbiol. 8, 198-210. Raper, K. B. (1946). Ann. N.Y. Acad. Sci. 48, 41-57.
GENETICS IN APPLIED MICROBIOLOGY
59
Rupert, C . S., Goodgal, S . H., and Herriot, R. M. (1958). J. Gen. Physiol. 41, 451471. Ryan, F. J. (1963). 8th Symp. Intern. Congr. Microbiol., 1962 pp. 297-300. Scherr, G. A., and Rafelson, M. E., Jr. (1962). J. Appl. Bacteriol. 25, 187-194. Schildkraut, C. L., Marmur, J., and Doty, P. (1961). J. MoZ. Biol. 3, 595-617. Sermonti, G. (1961). Sci. Rept. 1st. Super. Sanita 1, 449-454. Setlow, J. K. (1963). Photochem. Photobiol. 2, 393-399. Setlow, R. B., and Setlow, J. K. (1962). Proc. Natl. Acad. Sci. U S . 48, 1250-1257. Sevag, M. G., and Drabble, W. T. (1962). Biochem. Biophys. Res. Commun. 8, 446-452. So, A. G., and Davie, E. W. (1963). Biochemistry 2, 132-136. Spizizen, J. (1958). Proc. Natl. Acad. Sci. U.S. 44, 1072-1078. Stadler, L. J. (1928). Science 68, 186. Szilard, L. (1959). Proc. Natl. Acad. Sci. U S . 45, 30-45. Tewfik, E. M., and Bradley, S. G. (1966). Unpublished results. Thome, C. B. (1962). J. Bacteriol. 83, 106-111. Tissieres, A,, Schlessinger, D., and Gros, F. (1960). Proc. Natl. Acad. Sci. U.S. 46, 1450-1462. Uemura, I., Okuda, K., and Winnick, T. (1963). Biochemistry 2 , 719-725. Wainwright, S. D. (1959). Can. I. Biochem. Physiol. 37, 1417-1430. Welsch, M. (1957). Antonie wan Leeuwenhoek, 1. Microbiol. Serol. 23, 59-80. Welsch, M. (1959). Ann. N.Y. Acad. Sci. 81, 974-993.
This Page Intentionally Left Blank
Microbial Ecology and Applied Microbiology THOMAS D. BROCK Department of Microbiology, Indiana University, Bloomington, Indianu
I. Introduction ........................ ......... 11. The Search for a New Antibiotic: A Problem in Microbial Ecology? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. What Are Antibiotics? ........................ B. What Role Do Antibiotics Play for the Organism Producing Them? ............................ C. What Is the Role of the Antibiotic in Nature? . . . D. Are There Any More New Antibiotics? . . . . . . . . . . E. What Kinds of Organisms Produce Antibiotics? . . . F. Factors Influencing the Detection of Antibiotics . . G. The Screening Method of Looking for New Antibiotics ...................................... H. The Ecological Approach ...................... I. Another Ecological Approach .................. J. After Discovery, Then What? . . . . . . . . . . . . . . . . . . 111. Summarv ....................................... References ....................................
61 63 64
64 66 66 67 69 71 72 73 73 74 75
..
“, there were many who believed that the state of mind required for the successful prosecution of pure science was almost incompatible with the attitude imposed by applied science. But this was not Pasteur’s opinion. Immediately he formulated the view, and taught it with passion to his students, that in fact there was no incompatibility. I n his words, ‘There are not two different kinds of science; there is science and there are the applications of science.’ The two types constantly interplay, and one type cannot progress far without contact with the other” Dubos (1960).
1.
Introduction
The thesis of the present article is that to a great extent applied microbiology represents the application of the concepts, principles, and facts of microbial ecology. Microbial ecology attempts to understand the relationships between microorganisms and their environments. It is a completely fundamental discipline which derives from microbial physiology, biochemistry, and genetics but which represents a higher level of integration than these other disciplines. Microbial ecology has developed slowly in recent years, partly because of experimental difficulties and partly because it has become out of fashion with microbiologists. Yet microbial ecology can become an experimental science of considerable sophistication and can provide the fundamental principles which are needed so that we can better understand the role of microorganisms in nature. 61
62
THOMAS D. BROCK
The goals of microbial ecology include the explanation at the physiological, biochemical, and genetic levels of the roles of microorganisms: ( 1) as biogeochemical agents in weathering, soil formation, decomposition of organic materials, and transformation of inorganic elements, as primary producers of organic matter, and in the deposition of minerals; ( 2 ) as specific pathogens; ( 3 ) as producers of specific antagonistic or stimulatory agents; (4) as specific symbionts, e.g., as essential agents in digestive processes of herbivores, as producers of specific growth factors for higher organisms, or as agents of biological nitrogen fixation.
In no environment in which higher organisms are present are microorganisms absent. In many environments devoid of or inimical to higher organisms, microorganisms exist and even flourish. Microbial ecology can be studied at a number of levels, depending on our interest. 1. At the cellular level, we can investigate the relationships between individual cells and their environments in the kind of study best called physiological ecology. The response of organisms to environmental variables such as temperature, salinity, or light, the nutrition of organisms, the relation of cell structure and morphogenetic behavior to environment, the relation of microorganisms to surfaces, and the alteration of the environment by the organism, are all areas of study. 2. At the population level, investigations can be made of the interactions between cells which lead to new properties, and studies can be made of the role of the environment in population growth and control. Another large area of study is population genetics and the evolution of microorganisms. 3. At the next level, studies can be made of interactions between diverse organisms, as represented by the ideas of commensalism, neutralism, mutualism, competition, antagonism, parasitism, and predation. 4. At the next level, we can study microbial ecosystems such as in the rumen, soil, or a sewage treatment plant. Problems of energy flow, biogeochemical cycle, productivity, and population fluctuations and adaptations can be studied best at this level. 5. At the next leveI, we can study how microorganisms interact with higher organisms, either as pathogens or as symbionts. 6. At the global level, we can study the macrocosmic roles of microorganisms. At all of these levels, the goal is the same: to study fundamental probIcms, to uncover new concepts, and to devise an ever more sophisticated
MICROBIAL ECOLOGY AND APPLIED MICROBIOLOGY
63
predictive science. Thus habitats and organisms are studied not for their human significance but as model systems which provide suitable experimental tools for uncovering basic principles. In this way, microbial ecology differs from applied microbiology. The applied microbiologist is committed to a specific organism, process, or habitat, and his goal is to achieve the best possible control over it. To do this, he uses the concepts and techniques provided by the microbial ecologist. However, information flow is not one way. The applied microbiologist can provide for the microbial ecologist experimental systems which may be adaptable to his own ends. Thus an activated sludge sewage treatment plant provides one of the best examples of a steady-state microbial ecosystem, and the ecologist can study it as such. Furthermore, the applied microbiologist will uncover new puzzles or problems which the ecologist can take up and solve, and thus further develop new concepts. For instance, nitrification was discovered by Schloesing and Muntz in 1877 during the course of experiments on sewage treatment. The best training for any scientist is research training, and research can be done most effectively on clearly defined well-controlled systems. Thus the applied microbiologist should be trained first as a microbial ecologist. The present author believes that training in ecology, with strong emphasis on physiological and biochemical ecology, provides a rigor and an attitude of mind which will carry the applied microbioIogist far. He further believes that this training should be given not in an applied department but in a microbiology department. To illustrate the ways in which microbial ecology and applied microbiology interact, I have chosen the problem of how to find a new antibiotic, and I have attempted to analyze this problem briefly from the points of view of both the ecologist and the applied microbiologist. It is hoped that this analysis will illuminate, better than any special pleading, the thesis of this article. For a detailed analysis of microbial ecology at all levels, the reader is referred to a forthcoming book by the author: Principles of Microbial Ecology ( Brock, 1966).
II. The Search for a New Antibiotic: A Problem in Microbial Ecology?
The exciting days of the antibiotic era are over. No longer do we read that yet another new wonder drug has been discovered. Within a few years the patents will have expired on most of the important medically useful antibiotics. With the highly profitable exclusives gone, what will happen next?
64
THOMAS D. BROCK
The need for new antibiotics has not passed. There are infectious diseases yet to be conquered; people do still die from bacterial infections; antibiotic-resistant mutants do arise; some of the antibiotics currently used are toxic. But the search for new antibiotics must receive a new outlook if it is to succeed. I hope the present article will show that an ecological viewpoint may be valuable. A. WHAT ARE ANTIBIOTICS? Antibiotics are chemical substances produced by living organisms which are able to inhibit the growth of or kill other living organisms. Usually the living organisms in question are microorganisms, but this need not be so. The key word in our definition is “chemical.” Antibiotics are not mysterious marvelous substances of unknown nature. They are like any other chemical substances, but instead of being produced in the laboratory by organic chemists they are produced in the laboratory or in nature by microorganisms. We can look on the producing creatures as little organic chemists. An important distinction is that the microorganism is usually doing a job which the organic chemist cannot do, or cannot do well. (However this need not always be so; the antibiotic chloramphenicol is produced today by chemical synthesis even though it was discovered initially as a product of a microorganism.) An important distinction here is that the microorganism is producing a substance that the organic chemist would not dream of producing if left to his own devices. He is using the microorganism to show him the way. Most antibiotics, however, are too complicated for chemical synthesis, containing many asymmetrical carbon atoms or complicated rings, so that the chemist usually relies on the organism for commercial production as well as for initial demonstration.
B. WHAT ROLE Do ANTIBIOTICS PLAYFOR PRODUCING THEM?
THE
ORGANISM
This question, which would seem reasonable and pertinent, is unfortunately mixed up in legal matters. A patent cannot be obtained on a natural product, such as an oak tree or a rose, unless this is a hybrid that does not exist in nature. Therefore, if an antibiotic were considered a natural product, it would not be patentable as a new composition in the way nylon, for instance, is patentable. Only the process for producing the antibiotic, say by a microorganism in a fermenter, would be patentable. However, a process patent is less desirable commercially than a composition patent, since the latter keeps a rival from making the material by any means for the life of the patent. Thus when an antibiotic
MICROBIAL ECOLOGY AND APPLIED MICROBIOLOGY
65
is patented as a new composition it is argued that the antibiotic is not produced in the soil from which the organism producing it was isolated, but only in the laboratory. It is argued that the scientist, using his ingenuity, imagination, and genius, has devised unique culture media and environmental conditions which “force” the organism to produce a new substance which it would not otherwise have manufactured. This patent position, which seems to me very tenuous, has not been contested through the years, presumably because drug companies have felt that it is to their advantage not to change the status quo. Unfortunately, once such a position is taken, it is impossible to do any significant work on the question of whether antibiotics are produced in nature, since it would be fatuous to look for something which must not be there. Thus, the legalistic position inhibits the prosecution of either fundamental or applied research on this question. Is it true that antibiotics are merely laboratory creations rather than natural products? I believe that antibiotics are natural products, and my reasoning follows. It is very easy to isolate antibiotic-producing organisms from almost any soil. It is unreasonable to believe that the genotype of an organism is changed when it is isolated into pure culture, and thus the antibioticproducing organism in the soil probably possesses the genes to make the enzymes needed to make the antibiotic. Competition in the microbial world is fierce, and it is hard to imagine an organism containing genes and producing enzymes which did not aid in its survival. If a gene were unessential, the loss of this gene would result in the loss of an enzyme, and this would be one less protein that the organism would have to make with the possibly limited nutrients at its disposal. It is well known from bacterial physiology that the fewer enzymes an organism has to make the faster the organism is able to grow. This evolutionary argument strongly suggests, but does not prove, that antibiotics are not merely laboratory creations. In addition, there is some direct evidence that antibiotics are produced in nature. Waksman and Woodruff ( 1942) extracted antibacterial materials directly from natural soil, and Casas Campillo (1947) found substances inhibitory to rhizobia in soil. Recently, Stephen Allen in my laboratory (unpublished observations ) has extracted antibacterial activities from grassland and woodland soils and has shown by paper chromatography that different soils yield different antibiotics and that more than one antibiotic may be extracted from a single soil. Of course, such direct extraction methods reveal only the most common of the antibiotics. What is urgently needed is a method capable of measuring antibiotic activity in extremely tiny microenviron-
66
THOMAS D. BROCK
ments of soil and root surfaces. Perhaps micromanipulation could be coupled with microbiological assay in microcapillaries to achieve this end. There is considerably more evidence (reviewed by Brian, 1957) that antibiotics are produced in natural soils supplemented with organic materials, or in sterile supplemented or unsupplemented soils which have been inoculated with known antibiotic-producing organisms. It thus seems safe to assume that antibiotics are produced in nature.
C. WHATIs THE ROLE OF THE ANTIBIOTICIN NATURE? First, it need not function as an antibiotic. It might be an intermediate or end product produced for some other reason that just incidentally happens to be an antibiotic. The polypeptide antibiotic bacitracin seems to be part of the bacterial cell wall and is released during the sporulation process. Other antibiotics might be coenzymes or repressors or might function in some other way. However, it is fairly safe to assume that some of these substances do act in nature as antibiotic agents and play roles in competition or defense. This question has been carefully considered by Brian (1957), and his conclusion that ". . . some microorganisms can produce antibiotics in soil in quantities sufficient to account for some observed biological antagonisms . . ." seems reasonable today. To the argument that most antibiotics have not been shown to act in nature, especially in nonsterile soil, Brian makes the point that microorganisms tend to live in microenvironments, such as on plant rootlets or pieces of organic debris, and it is in these highly competitive environments that antibiotic production as a defense mechanism is likely to be significant. Unfortunately, methods are not available for detecting antibiotic activity against organisms present on an isolated portion of a single rootlet, so that we are prevented from putting the question to satisfactory experimental test. What are needed here also are more sophisticated methods of experimentation in this area of microbial ecology. D. ARE THEREANY MORENEW ANTIBIOTICS? Literally thousands of antibiotics are already known, each sufficiently well characterized for us to be certain that it differs chemically or biologically from all the others. Most of these antibiotics are not medically useful, but they still must be contended with in any search for a new antibiotic, since we are not interested in discovering old antibiotics over again. Are there any more new antibiotics? This is a question that unfortunately has no answer. If we had a team of 1000 people working for
MICROBIAL ECOLOGY AND APPLIED MICROBIOLOGY
67
the next 1000 years and they found no new antibiotics, we still could not say that there are no more left to be found. We could only conclude that either there are no more new antibiotics, or there are new antibiotics and we had not found them. But if we were to find a new antibiotic tomorrow, this still would not satisfy us because this might be the last new antibiotic left to be discovered. The situation is somewhat analogous to that which faces the astronomer looking for new stars. If he does not find any it may only be that his telescope is not powerful enough-or there may be no more new stars. The question is thus logically impossible to answer. If we decide, after these considerations, to look for a new antibiotic, then we are doing this on faith. We believe that there are new antibiotics yet to be found, and we are willing to gamble time and money looking for them. I do not know whether drug company executives look at their research efforts in terms of a gamble, but if they do they must spend many disquieting days and nights. Note, however, that it is just as dangerous to say that there are no more antibiotics as it is to say that there are some waiting to be discovered. If we take the negative approach, we will, of course, never do any research at all on anything. The scientist, like the theologian, builds his life on the faith that something exists. Without this faith neither the scientist nor the theologian could exist.
E. WHATKINDSOF ORGANISMS PRODUCE ANTIBIOTICS? If we use the definition we began with, then antibiotics are produced by all groups of living organisms-higher and lower animals, higher and lower plants, fungi, and bacteria-since all of these groups produce chemical substances which act on other organisms. However, our interest here is in antibiotics as medically useful substances, which means that we are interested in chemicals which attack the bacteria, fungi, animals, and viruses which affect human beings. As is well known, even the human body itself produces agents which act on these creatures, such as the hydrochloric acid of the stomach, the lysozyme of the tears and saliva, and the antibodies, if we use our definition in its broadest sense. But let us restrict our definition to low-molecular-weight chemical substances (M.W. less than 1000). This definition includes most medically useful antibiotics and eliminates substances which are normally antigenic or are difficult to compound into useful dosage forms. Antibiotics against human pathogens which meet this restricted definition have been found to be produced by all sorts of creatures: higher plants, algae, invertebrate animals, fungi, and bacteria. Where then should we look for a new antibiotic?
68
THOMAS D. BROCK
For the past 15 years, the search in drug companies for new antibiotics has concentrated almost exclusively on a small group of ubiquitous soil bacteria called actinomycetes. Why has the search been confined to such a narrow group of organisms if antibiotic production is distributed so widely over the phylogenetic tree? There are two reasons for this. The first deals with the psychology of scientists, and especially the psychology of industrial scientists. Nothing succeeds like success, and the actinomycetes have been eminently successful. Since streptomycin, all of the new medically useful antibiotics have come from actinomycetes, with the single exception of griseofulvin. Of course, if the search has concentrated on the actinomycetes, any new antibiotics are bound to come from this group. It might also be said: if the search has been concentrated on the actinomycetes for so long, might not this group be exhausted? Again there is no answer. Antibiotic researchers operate with the faith that there are more new antibiotics to be found in this group; otherwise they would not continue to look for them. The second consideration in concentrating on actinomycetes revolves around familiarity and practicality. Antibiotic researchers have been working with actinomycetes for so long that they know most of their tricks. Reduction to practice, that is, the transfer of a new antibiotic production process from the laboratory to the pilot plant to the largescale fermenter, is relatively quick and easy with actinomycetes. Furthermore, the physical plant for producing antibiotics from actinomycetes, an expensive investment, is available. A new antibiotic produced for instance by an obligately photosynthetic organism would require a large financial investment in new glass fermenters. Such an antibiotic would really have to be tremendously valuable to justify this new investment, whereas even an ordinary actinomycete antibiotic might be profitably produced in existing equipment. The intellectual daring of the scientist must be tempered by the practicality of the business man and engineer. Because he is concerned solely with actinomycetes, the only intellectual daring available to the antibiotic researcher is to look for antinomycetes in new places. This explains the passion for obtaining soils from exotic locales, with the idea that they might produce exotic antibiotics. Yet the culture that produced chloramphenicol was isolated almost simultaneously out of soil from Venezuela and Urbana, Illinois. It should be obvious that in itself the geographical location of the soil is of minor importance and that its pedology is of major importance. What really do we know about the ecology of actinomycetes? What physiological, structural, genetic, and biochemical factors control the development of these organisms in nature? Where precisely in the soil
MICROBIAL ECOLOGY AND APPLIED MICROBIOLOGY
69
do actinomycetes grow? Is their nutrition and physiology the same in soil as in culture? What factors influence growth, sporulation, and metabolic activity in soil? Why are actinomycetes more common in some soils than in others? Why are they rarely found in large numbers in other environments, such as water or the animal intestinal tract? These are questions of a fundamental ecological nature. The answers to these questions may tell us nothing about how to find antibiotic-producing actinomycetes; yet we cannot be sure. Surely it might be of some practical value to know at least partial answers to some of these questions. The mere fact that we raise such questions reveals the depth of our ignorance.
F. FACTORS INFLUENCING THE DETECTION OF ANTIBIOTICS 1 . The Test Organism In the culture medium in which a presumptive antibiotic-producing organism is growing, the antibiotic, if present, is invisible. It can be recognized only through its action on the growth or viability of a sensitive test organism. It would be reasonable that if we wanted an antibiotic to cure brucellosis, then we should use a Brucella species as test organism. Yet this is almost never done, partially because of the laboratory hazards of working with such organisms and partially because experience has shown that antibiotics are active against a variety of bacteria and that more tractable organisms such as Escherichia coli and Bacillus subtilis can be used instead. Yet, is such an approach necessarily desirable? Bacteria do differ from each other in important ways, and these differences may involve antibiotic sensitivity. If we always use E . coli as a model for Brucella, could we ever detect an antibiotic active only against the latter? Again we must operate on faith and assume that what is good (or bad) for E . coli is good ( o r bad) for the rest. As yet we have little understanding of why bacteria differ in their sensitivity to antibiotics. If we understood these things, perhaps we could select the test organisms we would use more wisely. 2. The Assay Medium Whether an antibiotic will be detected under a given situation depends on two factors: ( I ) the intrinsic activity of the antibiotic against the test organism in the medium used for the test, and ( 2 ) the amount of antibiotic made by the antibiotic producer. These two factors frequently get mixed up or confused. The antibiotic azaserine, for instance, is highly active against E. coli, yet it was missed for years in screening programs because the medium used for the test contained aromatic amino acids
70
THOMAS D. BROCK
which reversed the antibiotic activity. Although it may seem that a rich culture medium would mimic the environment in which the pathogen grows in the human body, this probably is not so. For instance, the animal body is so deficient in purines that a purine-requiring mutant of Salmonella typhimurium is not pathogenic, although pathogenicity can be restored if purine is injected into the animal along with the pathogen (Stocker, 1959). The reason sulfanilamide is an effective drug is that folic acid levels in the animal are low enough so that drug reversal cannot take place. It seems obvious that the test conditions should attempt to mimic the nutritional and physical aspects of the environment in which pathogens grow, yet this is never done. It is illusory to assume that a peptone medium mimics the in vivo environment.
3. The Concentration of Antibiotic Produced
A large inhibition zone may be due to large amounts of a weakly active antibiotic or to small amounts of highly active antibiotic. Conversely, a small inhibition zone may not indicate a weakly active antibiotic, since the antibiotic may be very active but present only in tiny amounts. Only after the antibiotic has been purified completely can its intrinsic activity against a given test organism in a given medium be determined. From a practical point of view, it has been found that, if all antibiotic activity is lost after a 1 : l O dilution of an active culture filtrate, this antibiotic should not be worked on. In such a case it will be almost impossible to develop adequate purification methods because frequently all activity will be lost when the antibiotic is partitioned between two solvents.
4. Production of Multiple Antibiotics Many organisms produce more than one antibiotic, sometimes as many as five or six. The number of antibiotics and the amount of each are often influenced by the culture medium and by other environmental conditions. All of the antibiotics may be active against a single test organism, or different organisms may be sensitive to different antibiotics in the mixture. If the former is true, then it is obvious that the inhibition zone obtained is due to the concerted action of the several antibiotics, and this presents many complications. If the antibiotics can be separated by paper chromatography then it will be possible to assay for one in the presence of another. If the antibiotics cannot be separated, then the existence of multiple antibiotics will not even be realized. Tetracycline, for instance, was present in the chlortetracycline ( Aureomycin) fermentation from the very first, and the lack of knowledge of this fact may have caused serious economic consequences.
MICROBIAL ECOLOGY AND APPLIED MICROBIOLOGY
71
G. THE SCREENINGMETHODOF LOOKING FOR NEW ANTIBIOTICS Our approach will depend greatly on the theoretical basis on which we wish to work. If we believe that antibiotics are randomly occurring products with no role in nature, we will reason that our only way of finding them will be to look randomly in as many places as possible under as many conditions as possible. Such an approach is usually called “screening,” and it involves a large team of workers operating in semiassembly line fashion. An immediately obvious disadvantage of an assembly line is that it is difficult to change it, since so many parts of the line must be changed. Another disadvantage is that the methods used will be those which are adaptable to an assembly line, and this may determine at the very beginning that some new antibiotics will never be discovered because they will slip through the screen, no matter how fine it may be. The screening approach is unesthetic and anti-intellectual, but it has been successful in the past. Its success, however, is determined greatly by the choice of organisms and conditions under which the antibiotics will be detected, and the organisms which will be tested as presumptive antibiotic producers. Up to now the presumptive antibiotic-producing organisms have been selected on historical grounds, with the idea that lightning can strike twice in the same place and that the kinds of organisms which have proved successful in the past will continue to be successful in the future. The screening approach is not an ecological approach, and the first thing that must be done is to discard the idea that it will rationally relate to natural conditions in any way. The important thing is to design a smoothly running, highly efficient, factory-type operation. Let us consider first the organisms used to detect the new antibiotics, since their choice will determine at the beginning what will be found. As I have pointed out, the test organisms used have been mainly common bacteria, which have been used with the idea (conscious or unconscious) that they mimic or substitute for the pathogens which we really want to control. Yet in reality in the screening approach we are using our test organisms to detect new biological activities, and only after these have been found and purified do we test them on pathogens. Even then, it is not pathogens in the test tube which we are after, but pathogens in the inf ected human. why not, then, set up a screen in which a variety of test organisms is used, the idea being not that these organisms will mimic human pathogens, but that they wiIl reveal new biological activities? Why not select from such organisms as Acetobacter, Myxococcus, Brevibacterium, Arthrobacter, Azotobacter, Caulobacter, Actinoplanes, Sphaerotilus, Desulfo-
72
THOMAS D. BROCK
vibrio, Mycoplana, and a long list of others, which are indubitably not pathogens but which differ widely in physiology from the organisms usually used? And why not also use a wide variety of fungi, yeast, protozoa, and even algae? The only requirements might be that the organism is available in pure culture and that it can be handled in the laboratory in a reproducible manner. Why not discard the current idea of using dual culture media on which the test organism must grow and, in addition, the antibiotic producer must grow and produce its antibiotic? The test organisms should bc grown for the antibiotic sensitivity test on diverse culture media and incubated under a variety of environmental conditions, since it does not necessarily follow that antibiotic activity will be equal on all media. In the same way, why not discard current ideas about actinomycetes and look for antibiotic production in all sorts of organisms? Since the theoretical basis of this approach is that antibiotics are randomly distributed and exist for no good reason, why not look for them randomly with no good reason?
H. THE ECOLOGICAL APPROACH The ecological or rational approach considers that an antibiotic plays some role in the life of the organism related to competition and defense. One then goes to an environment where competition is fierce and looks for antibiotic-producing cultures. We must always remember, however, that we are concerned with treating infected humans and that our ultimate aim is to find antibiotics active against human pathogens. If we believe in the generality of the mode of action of antibiotics, then it is reasonable to look for organisms producing medically useful antibiotics in the soil, assuming that what acts on B . subtilis and Pseudomonas aeruginosa will also act on Staphylococcus aureus and Brucella abortus. However, it is also reasonable to look in other environments than the soil where competition is fierce, such as sewage treatment plants, lake and ocean sediments, and animal intestinal tracts. Believing in the generality of the mode of action of antibiotics, we can hope that antibiotics produced against the indigenous organisms will also act against human pathogens. But suppose there are antibiotics which act only on human pathogens. How could we find them? Let us consider the way in which pathogens invade the body. Except for the rare instances of wound infections, most pathogens invade by establishing themselves in epithelial and mucosal areas such as the skin, intestinal tract, and respiratory tract, which are already well supplied with harmless bacteria. The pathogen then has to compete with the normal flora before it can establish itself and invade
MICROBIAL ECOLOGY A N D APPLIED MICROBIOLOGY
73
the body. Is it not possible that in many cases the organisms of the normal flora prevent the invader from becoming established by producing antibiotics? Evidence that organisms in the respiratory tract do produce antibiotics is already known. Yet I know of no attempt at a systematic search of a wide variety of human beings for antibiotic-producing organisms. To me, such a search would be interesting even if the results were completely negative, but it seems that the chance of antibiotic production by some of these organisms is high.
I. ANOTHERECOLOGICAL APPROACH Bacteriocines are special kinds of antibiotics. They are substances produced by bacteria which are active against closely related bacteria, usually even in the same species. Bacteriocines have been found in every group of bacteria in which they have been sought. Similar types of substances are produced by protozoa (the killer factor of Paramecium) and probably by other microorganisms. From a medical point of view, bacteriocines have several disadvantages over more traditional antibiotics. They are usually highly specific, attacking certain strains and not others of the same species, and they are usually high-molecular-weight substances, probably antigenic, and could probably be administered only by injection although so few bacteriocines have been characterized that it is unwise to generalize. As to advantages, their high specificity implies that they should be completely nontoxic (although this also has not been proved). Furthermore, they usually kill sensitive bacteria rapidly upon contact, possibly even nongrowing cells. One may postulate an ecological role for bacteriocines in the invasion of niches in which closely related strains are already well established. Thus we might expect to find bacteriocine producers in areas in which the organism we wish to control already exists. But aside from these ecological speculations, we can see that bacteriocines, if amenable to development, might make the ideal wonder drugs. If we are seeking an antibiotic against Brucella why not test other Brucellae for bacteriocine production? It is almost certain that Brucella bacteriocines would be discovered, and they might then be developed medically and industrially. J. AFTERD I S c o \ 7 ~ ~ YTHEN , WHAT?
I have been concerned in this paper only with the initial discovery of antibiotic activities. Obviously any antibiotic activity produced by any organism may be due not to a new antibiotic but to an old one which our sophisticated methods have allowed us to rediscover. Once antibiotic activity has been detected, the first and foremost job is to determine
74
THOMAS D. BROCK
whether it is new or old. Briefly, antibiotics may be characterized biologically and chemically; crude materials can be characterized chemically only by methods such as determination of stability and extractability and by paper chromatography. To discard an antibiotic as old, one must have comparative biological and chemical data on all known antibiotics, and since there are thousands of these, the acquisition of such data is time-consuming and expensive. Only certain of the larger drug companies have the facilities for this, and the success of a traditional screening program can perhaps best be judged by how soon a known activity is recognized and discarded. After one has found a new antibiotic other problems arise, such as large-scale production and purification, toxicity testing, evaluation in experimental animals, and, hopefully, clinical testing in humans. Many are called and few are chosen. The tragedy frequently is that a new antibiotic is discarded as a failure after a few crude animal tests, even though months and years of effort have gone into its discovery and isolation. It should be a byword that any new antibiotic is interesting biologically and may prove useful in some unsuspected area. After years of neglect, actinomycin suddenly came under study again during the search for antitumor drugs. The antiphage antibiotic fumagillin was a failure as a medically useful antiviral agent but proved useful for amebic dysentery. A highly toxic antibiotic may have some interesting and useful pharmacological activity when given in small doses. Perhaps the greatest use today of any antibiotic screening program is that it provides simple, rapid, reproducible, and sensitive ways of detecting a natural product with biological activities. Given the existence of such a product, it should be tested and retested and tested again.
111. Summary Microbial ecology deals with the interactions of microbes with their environments, including other microorganisms. The interaction of two organisms on an agar plate is an ecological interaction and should be viewed as such. Any efforts to eliminate the folklore and sentimentality involved in looking for new antibiotics should be welcome. I have tried to divide this problem into its component parts. I have also tried to show that any decision regarding method of approach requires an analysis of the theoretical basis of the approach and a dispassionate consideration of its possible outcomes. I have also tried to show that, once a decision is made, it involves a strong element of faith. If the scientist does not believe that his approach will work, he will not be able or willing to put
MICROBIAL ECOLOGY AND APPLIED MICROBIOLOGY
75
his heart and soul into it. He may succeed, but most likely he will fail. Any decisions about new antibiotic screening programs involve lots of time and money. Drug company executives should recognize the high element of risk in any undertaking. Their money may be safer at a race track than in the hands of the scientist. Yet any gamble that is worth taking is worth taking well, and, once the money has been committed, complete faith should be expressed in the scientists, since lack of confidence may shake the scientist’s own philosophical foundations. I personally believe that the ecological approach to the search for new antibiotics has not been used enough. This approach has the following merits: ( 1) it is different from the current approach, ( 2 ) it is intellectually satisfying and would thus be more satisfying than the screening approach to the scientists who must implement it, and ( 3 ) it may lead to some unsuspected new knowledge that is of use in itself even if no new antibiotics are detected, whereas at the end of an unsuccessful screening operation little new knowledge will be available upon which to base new decisions.
REFERENCES Brian, P. W. (1957). Proc. 7th Symp. SOC. Gen. Microbid., p. 168. Cambridge Univ. Press, London and New York. Brock, T. D. ( 1966). “Principles of Microbial Ecology.” Prentice-Hall, Englewood Cliffs, New Jersey. Casas Campillo, C. 1947. Ann. Escuh Nac. Ciene. Biol. 4, 334-352. Dubos, R. (1960). “Pasteur and Modem Science.” Anchor Books, New York. Stocker, B. A. D. 1959. J. Med. Educ. 34, 354-365. Waksman, S. A., and Woodruff, H. B. 1942. Soil Sci. 53, 233-241.
This Page Intentionally Left Blank
The Ecological Approach to the Study of Activated Sludge WESLEY
0. PIPES
Department of Ciuil Engineering, Northwestern University, Evanston, Illinois
I. Introduction ..................................... A. Description of the Process. . . . . ......
A.
Bacteria
..
............................... .................. ..................
82 86 88
......
92
......
99
C. Synergisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Summary ................................ References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1.
77 78
101
Introduction
The activated sludge process is by far the most widespread and, in terms of total volume, the largest industrial application of continuous microbial culture. It is, however, different from other industrial microbial processes in three important respects. In the first place, most industrial applications of microbial culture have the objective of converting some food material into a useful product which may be either the organisms themselves or a metabolic by-product, but the objective of the activated sludge process is to degrade the organic material in the waste, and thus metabolic by-products or excess organisms produced are a nuisance. Second, most industrial microbial processes depend upon a single species of microorganism, but the activated sludge process is the most grossly contaminated culture imaginable. The third, and perhaps the most important, difference is that while most industrial microbial processes are revenue-producing operations, no one ever made any money out of activated sludge. 77
78
WESLEY 0. PIPES
For some reason, possibly having to do with one of the differences cited in the previous paragraph, the activated sludge process has been largely ignored by the applied microbiologist and most of the research studies have been conducted by either chemists or engineers. As stated by a previous contributor to this series ( Wuhrmann, 1964): “There has been, and is still, too much engineering and too little microbiology in this field of environmental sanitation.” Actually, the activated sludge process presents an opportunity for microbiologists to work on some problems of microbial growth, nutrition, metabolism, and ecology which have both intellectual interest and industrial significance. A. DESCRIPTION OF THE PROCESS Physically, the activated sludge process consists of an aeration tank followed by a sedimentation basin. A mixed microbial population grows in the aeration tank and is settled out in the sedimentation basin, and then a portion is returned to the aeration tank while the rest is wasted. All of the suspended solids in the aeration tank, including organisms and suspended material from the waste, are collectivdy known as “activated sludge.” The mixture of activated sludge and waste in the aeration tank is known as “mixed liquor.” The effectiveness of the process in purifying the waste is usually estimated by comparing the 5-day, 20°C, biochemical oxygen demand (BOD) of the influent and effluent. It is essential that the sludge be separated from the mixed liquor before discharge because the organisms in the sludge would greatly increase the BOD of the effluent by their respiratory oxygen consumption and most wastes do not contain enough organic matter to develop the needed microbial population in a few hours on a single pass basis. There are three essential biological requirements for the activated sludge process: first, a mixed population of aerobic microorganisms must be able to degrade the noxious components of the waste; second, the required population must be able to grow in the environment of the aeration tank; and, third, the organisms must grow in such a form that they will settle out in the secondary clarifier. A great many modifications of the activated sludge process have been devised (Sawyer, 1965), and many of the modified processes have been given completely different names. All of the modifications consist of either lengthening the aeration time or shortening the aeration time and reaerating the return sludge with or without the addition of other nutrients before it is returned to the aeration tank. Since the biological requirements of all of the modified processes are the same, they are all still the activated sludge process. Research on the activated sludge process should be directed toward providing solutions to practical problems. There is quite a large number
ECOLOGICAL STUDY OF ACTIVATED SLUDGE
79
of research problems whose solution would provide improved understanding of and possibly improvements in the activated sludge process. The problems which will be considered here are those of determining the conditions under which the biological requirements for the process will be met. Previous research on the activated sludge process can be divided into two categories according to the point of view of the investigator; namely, the kinetic approach and the ecological approach. Although this article is concerned primarily with the ecological approach, it is necessary to refer to the kinetic approach briefly for purposes of comparison and to show that the final solution to the questions about activated sludge will not be obtained without a synthesis of the two points of view.
B. THEKINETIC APPROACH The kinetic approach to research on the activated sludge process emphasizes the changes in chemical components of the waste during treatment. The biological nature of activated sludge is largely ignored and the sludge is considered as if it were some type of heterogeneous catalyst which increases in mass when aerated in the presence of organic wastes and decreases in mass when aerated after the organic matter has been removed from the waste. An attempt is made to describe the rate of removal of organic matter from the waste and the rate of change in the sludge mass by equations analogous to some of those used in chemical kinetics, and the stoichiometry of hypothetical chemical reactions which describe conversion of waste organic matter into sludge and oxidized products is formulated as materials balance equations. Eckenfelder and McCabe (1960) presented an exceptionally lucid account of activated sludge kinetics and demonstrated clearly how the theory can be of considerable practical value. Much of the theory of activated sludge kinetics is based on relationships developed by Hinshelwood (1946) and Monod (1949) to describe bacterial growth in pure culture. Attempts to explain why the same relationships should describe growth in a pure bacterial culture and in a mixed microbial culture have been generally unsuccessful; the best of these is in a dissertation by Gram (1956) which has had little impact because of limited distribution. One of the major problems with mathematical models of the activated sludge process derived by the kinetic approach is that one of the most important factors in the models is the concentration of oxidizable substrate in the influent waste and there is no good way to measure this quantity. In most models, BOD is used as a measure of the strength of the raw waste, the amount of oxidizable substrate available to the microorganisms in the sludge, and the pollutional potential of the effluent. BOD is treated
80
WESLEY 0. PIPES
as if it were a substance, and the formulation of some of the equations of the kinetic models of the activated sludge process implies conservation
of BOD. BOD is a parameter, not a substance; it can be reduced but not removed and it is not conserved in the sense that matter is conserved. The frustrations of trying to treat BOD as if it were a substance have led to the development of concepts such as “nonremovable BOD” and even “nonoxidizable BOD.” If something contributes to the BOD, it has to be oxidizable, and if it is oxidized, it is removed. Another of the problems with the kinetic models of the activated sludge process is that the process depends as much upon obtaining a sludge with good settling characteristics as upon removing the noxious components from the waste. So far, no one has been able to relate settling characteristics of the sludge to the other parameters of the mathematical models in a satisfactory manner, although there have been several attempts (Kraus, 1949; Orford et at., 1960). The problem here is probably that there are at least six different phenomena which can cause settling problems and these phenomena have not yet been adequately described. The kinetic approach to the study of activated sludge has resulted in improvements in the understanding of the activated sludge process, and concepts developed by these research studies are of great practical value. However, the mathematical models of the process based on the kinetic approach are based on assumptions and approximations which are not entirely accurate. There is a real need for a reexamination of these basic assumptions in order to relate the kinetic models to biological phenomena.
C. THE ECOLOGICAL APPROACH The objectives of the ecological approach are to determine what organisms play a beneficial role in activated sludge and how to design and operate an activated sludge process so as to encourage the growth of the desired organisms and discourage the growth of nuisance organisms. These studies have followed two lines of investigation; namely, (1) isolation and pure culture studies on specific organisms, and ( 2 ) variation of environmental conditions in the process with observations of the changes in the population. The best attempt at summarizing the results of the previous ecological studies of activated sludge was presented by Hawkes (1960) as a part of a symposium on waste treatment and later expanded into a book (Hawkes, 1963). He shows clearly how information derived from ecologicaI studies of activated sIudge can be of vaIue to the operator of an activated sludge process. Engineers have been reluctant over the years to make use of information derived from ecological studies of activated sludge. Undoubtedly, one of the reasons for this is that some of the studies have been so poorly con-
ECOLOGICAL STUDY OF ACTIVATED SLUDGE
81
ceived and carried out that the results are completely meaningless. A fine example of the poor quality of some of this research was presented by Barritt (1940). He mixed a small amount of milk with tap water and aerated it in the laboratory. He examined the sludge produced after 7 days’ aeration and again after 2 months’ aeration by microscopic observation and from these observations drew some rather emphatic conclusions about the activated sludge process. It is no wonder that engineers choose to ignore research of this type. There is another, more profound, reason that engineers have not made better use of the information available about the ecology of activated sludge. This is the problem of communication between the biologist and the engineer. Engineers need and expect to have quantitative data upon which to base their calculations, and most will not even read the results of qualitative studies. Many of the ecological studies of activated sludge have resulted only in descriptive material. The great challenge in this field of research at present is to produce useful quantitative data.
11. Organisms Presenf The objective of attempts to identify the organisms present in activated sludge should be not to compile a list of organisms but to determine the role of organisms in the process. Since the process is inoculated with organisms from water, soil, and sewage, the majority of species present in activated sludge probably are adventitious organisms. The organisms which do have significant roles in the process can be divided according to their roles as floc-forming organisms, saprophytes, predators, and nuisance organisms. Any particular organism may fit into more than one of these roles at a time or may change roles with a change in the condition in the plant. In a normally operating activated sludge plant, dissolved and suspended material in the waste is agglomerated with the sludge. This agglomeration is called flocculation by analogy with the water treatment process, although it is probably an entirely different phenomenon. Some flocculation may occur in a waste aerated in the absence of microorganisms, but it is generally accepted that the sludge organisms are responsible for most of the flocculation. Since the formation of a sludge with good settling qualities is a primary requisite for the process to be effective, the determination of which organisms form the floc and how to encourage them to do this is of great importance. The primary saprophytes in activated sludge are the organisms which actually break down the organic matter in the waste being treated. Some of the primary saprophytes compete with each other for the same type of
82
WESLEY 0. PIPES
organic matter, but, since there is usually a variety of types of organic matter in any given waste, there are probably a variety of primary saprophytes which do not compete with each other. It would seem logical to assume that the greater the variety of saprophytes present the greater the variety of organic compounds which can be assimilated by the sludge. In addition to the primary saprophytes, there will be secondary saprophytes which assimilate the metabolic by-products of the primary saprophytes, tertiary saprophytes, and so forth. The primary predators feed upon the saprophytes and possibly upon some of the particulate matter in the influent waste. There may be secondary predators feeding upon the primary predators, textiary predators, etc. There is some indkation that the predators may be of some importance in getting rid of saprophytes which are not floc-forming organisms or that they possibly even act as floc-forming organisms themselves. The nuisance organisms are those which interfere with the proper operation of the process when they are present in large numbers. The nuisance created in practically every case is an interference with the settling characteristics of the sludge. Organisms which grow as single cells suspended in the waste are not separated by sedimentation and are carried out in the effluent. Extensive growths of filamentous organisms often permeate the sludge, or it may form as a loose floc containing large amounts of bound water. In either case, the sludge has poor settling characteristics and is said to be “bulking.” Occasionally, a compact floc which settles rapidly will not stay settled but will rise again to the surface and be carried out in the effluent. This type of sludge is properly called rising sludge but is often confused with bulking sludge.
A. BACTERIA There have been a number of attempts to determine which bacteria have a significant role in activated sludge. According to Hawkes (1963), several of the earlier workers found intestinal bacteria to be present in the greatest numbers and considered them to be the most important organisms. However, Allen (1944), by pretreating his sludge samples to liberate individual cells from the floc particles, found that gram-negative bacilli belonging to the genera Achromobacterium, Flauobacterium, and Pseudmonas were the dominant forms present. He concluded that the intestinal bacteria were present only as adventitious forms and were of little significance in the process. The lists of bacteria from activated sludge presented in four of the more recently published reports are compared in Table I. In addition to those listed in the table, McKinney and Weichlein (1953) reported a number of intestinal bacteria such as Escherichia, Klebsiella, and Paracolon-
83
ECOLOGICAL STUDY OF ACTIVATED SLUDGE
bacterium. The other investigators apparently followed the lead of Allen (1944) in ignoring the intestinal forms. A cursory examination of Table I would lead one to believe that the significant bacteria in activated sludge belong to the genera Achromobacter, Alcaligenes, Bacillus, Flavobacterium, Micrococcus, and Pseudomonas. Micrococcus might be eliminated as probably an adventitious organism because, although all investigators found it, none found it in large numbers and it is probably always present in sewage anyway. TABLE I BACTEHIAISOLATED FROM ACTIVATEDSLUDGEa Jasewicz and McKinney and Weichlein (1953)
Achromobacter Aerobacter Alcaligenes Bacillus Bacterium Corynebacterium Comamonas Flauobacterium Microbacterium Nocardia Pseudomonus Sarcina Spirillum Zoogloea a
P P P P P
Assimilating sludge
P
D P
( lgS) Endogenous sludge -
-
-
D D
-
-
Rogovskaya and Lazareva (1959)
Dias and Bhat (1964)
-
P P P P
P P
-
P P P D -
P
P = present; D = present in large numbers.
Alcaligenes might be eliminated as a fecal organism; it was found in large concentrations only by Jasewicz and Porges (1956), and the activated sludge which they sampled was developed by aerating milk in the laboratory. This narrows the list down to the three motile, gram-negative bacilli found by Allen ( 1944) plus Bacillus. There is no assurance that this list of genera is representative of the bacterial population of activated sludge. None of the investigators examined a sufficiently large number of samples from a wide enough variety of sources to be sure that the population obtained was representative of anything. McKinney and Weichlein ( 1953) examined sludges from two plants treating domestic sewage, one plant treating an industrial waste, and one laboratory culture. Jasewicz and Porges (1956) obtained their samples from laboratory cultures which
84
WESLEY 0. PIPES
they developed by aerating milk; the “assimilating sludge” came from a culture which was being fed and the “endogenous sludge” from a culture which had been aerated several days without being fed. Rogovskaya and Lazareva (1959) examined seven different sludges developed on seven different types of waste. They actually did not say where they got the sludges, but from the facts that one of the wastes was synthetic sewage and the concentrations of suspended solids were practically the same in all seven samples, it may be inferred that the sludges were developed in laboratory culture. Dias and Bhat (1964) also examined seven different sludge samples, and they actually obtained two of the seven samples from an activated sludge plant. However, their other five samples were from laboratory cultures which were not operated at all in the same way that an activated sludge plant is operated. These four groups of investigators who reported on the bacteria which they isolated from activated sludge also used a fairly limited range of media for their isolations. Jasewicz and Porges (1956) used a milk agar, which was fairly reasonable since they were feeding milk to their activated sludge cultures. McKinney and Weichlein (1953) used nutrient agar, and Rogovskaya and Lazareva (1959) used a meat-extract agar. Dias and Bhat (1964) used sewage agar for their primary isolations, but then they identified only those organisms which survived transfer from the primary isolation plates to a proteose-peptone yeast extract broth. Thus, all of these groups isolated only bacteria which grow well in media containing high concentrations of protein or protein hydrolysis products. It is quite likely that, if a variety of media were used for the isolations, a very different picture of the bacterial population of activated sludge would be obtained. It is obvious that the significant bacteria in activated sludge must be able to form floc by themselves, be formed into floc by other organisms, or attach to particles large enough to settle well. Any organism not being included in the return sludge in greater numbers than it is present in the mixed liquor would not remain in the system long enough to build up a significant population. Thus, several investigators have looked for bacteria which are able to flocculate by themselves. Butterfield ( 1935) isolated Zoogloea ramigera from activated sludge and considered it to be the floc-forming bacterium. Later (Butterfield et al., 1937) it was shown that this organism is capable of producing a considerable degree of purification of a waste in pure culture. Heukelekian and Schulhoff ( 1938), Heukelekian and Littmann ( 1939), and Wattie ( 1943) were able to repeat and extend these observations, strengthening the belief that Zoogloea ramigera is the most significant bacterium in activated sludge.
ECOLOGICAL STUDY OF ACTIVATED SLUDGE
85
McKinney and Honvood (1952) and McKinney and Weichlein (1953) tested a number of bacteria which had been isolated from activated sludge to determine if they could form floc in pure culture. Of seventy-two different species which they isolated, they found that fourteen species belonging to the genera Escherichia, Alcaligenes, Bacillus, Pseudomonas, and Zoogloea readily flocculated in pure culture. Several of their other isolates would flocculate after prolonged aeration. They supported the theory that, since many different bacteria are capable of forming floc, it is not necessary to have Zoogloea ramigera present in order to form activated sludge. Allen ( 1944), Jasewicz and Porges ( 1956), and Rogovskaya and Lazareva ( 1959) did not even report Zoogloea ramigera among the bacteria which they found in activated sludge. However, it is not clear whether Zoogloea ramigera was not present in the sludges they examined or whether they considered it to be a growth form of some of the organisms they did report. Dias and Bhat (1964) reported that Zoogloea and Comamonas were the only bacteria present in significant numbers in the sludges which they examined. They believe that these two bacteria form the physical basis of the floc particles, and the other bacteria are present as associated organisms. They reported that all of the organisms which they isolated from activated sludge were capable of forming floc in pure culture, but Zoogloea and Comamonas formed floc much more readily. Many, perhaps all, bacteria are capable of forming floc in pure culture, but the typical growth form of Zoogloea is distinctly different in microscopic appearance from other flocculated bacteria. A series of microscopic examinations of sludge from a variety of different activated sludge plants will reveal that sometimes the typical Zoogloea growths make up most of the sludge, sometimes they are present along with other bacterial floc particles, and sometimes they are very hard to find. It would be useful to know the conditions which determine whether Zoogloea is the dominant organism in activated sludge and if it produces a more efficient or less efficient sludge than other bacteria. The nitrifying bacteria Nitrosomonas and Nitrobacter are present in activated sludge, but only a few investigators have taken the trouble to isolate them ( Hawkes, 1963). The presence of these organisms is detected by the conversion of ammonia to nitrite and nitrite to nitrate, and since they are the only agents which carry out these oxidations, the extent of their activity in the process can be determined by simple chemical analyses. It would be nice if the activity of the other organisms present could be determined as easily. One organism which seldom appears on lists of bacteria from activated sludge in reports of investigations of general surveys but which has been
86
WESLEY 0. PIPES
isolated from activated sludge a number of times is Sphaerotilus natans. Ruchhoft and Watkins ( 1928) isolated a filamentous bacterium which was present in large numbers in a treatment plant and studied it thoroughly enough to be sure of its identification as Sphaerotilus. Lackey and Wattie (1940) isolated Sphaerotilus from several different sludges and took the trouble to establish its identity, but they used an enrichment culture technique and did not collect any evidence that it was the dominant bacterium in any of the sludges that they examined. The best isolation medium for Sphaerotilus (Dondero et al., 1961) is quite different from the media that have been used for surveys of bacteria from activated sludge, and it is likely that Sphaerotilus could be isolated from almost any sludge sample if the proper technique were used. Practically all the studies of Sphaerotilus in activated sludge have sought information useful in solving the bulking problem. It has been shown that Sphaerotilus in pure culture can produce a growth very much like bulking sludge (Ruchhoft and Kachmar, 1941), and it undoubtedly is present in bulking sludge on many occasions. However, it has been shown that activated sludge can bulk without an overgrowth of filamentous organisms ( Heukelekian and Weisberg, 1956), and Lackey and Wattie (1940) stated that a number of other filamentous organisms may be responsible for bulking in certain instances. According to Lackey and Wattie ( 1940), Bacillus mycoides sometimes grows as filaments in activated sludge and it could be mistaken for Sphaerotilus in pure culture. McKinney and Horwood (1952) isolated Nocardia actinomorpha from activated sludge but listed it as a flocforming organism rather than as a filamentous organism. Probably a number of other filamentous bacteria, including many actinomycetes, could be found in activated sludge if the proper isolation techniques were employed. It would be helpful to engineers trying to solve bulking problems if someone would determine what filamentous bacteria actually are present in activated sludge and under which conditions they are likely to become the dominant organisms. B. FUNGI
The information available about the fungi in activated sludge is even sparser than information about the bacteria. Hawkes (1963) states that fungi are relatively rare in activated sludge, but he does admit that this rarity may be due to lack of reports in the literature rather than lack of fungi in activated sludge. There has been at least one general survey of fungi in activated sludge, because Cooke (1956) included samples from several activated sludge
ECOLOGICAL STUDY OF ACTIVATED SLUDGE
87
plants in his survey of fungi in polluted water. The species of molds he reported were distributed among the genera Mucor, Rhizopus, Aspergillus, Penicillium, Fusarium, Myrothecium, Gliocladium, Phialophora, Cladosporium, Margarinomyces, Aureobasidium, Geotrichum, and Trichoderma. The members of the first eight genera were present in only small numbers, and they are common enough to be considered as probable adventitious organisms in activated sludge. Members of the last five genera were reported to be present in large numbers in activated sludge, and they may play some significant role in the process. Cooke et al. (1960) reported on the yeasts isolated from the same samples. The genera Candida, Rhodotorula, Torulopsis, and Trichosporon were said to be common in activated sludge. Since yeasts are known to flocculate under certain conditions and can carry out metabolic activities which would be beneficial to the decomposition of organic waste, it would not be at all surprising to discover that they play an important role in the activated sludge process. Lackey (1949) suggested that filamentous fungi could play a role in the formation of floc by activated sludge. It is relatively easy to observe mold filaments inside floc particles which appear to be composed mostly of bacteria. Possibly the bacterial cells initially stuck to a mycelium and grew there using extramycelial material secreted by the fungus to form a floc particle, or the fungus could have invaded a floc particle initial'ly formed by some other mechanism. Unfortunately, there has been no investigation of this possible mechanism of floc formation. It is likely that fungi are the cause of bulking upon occasion but that they are often identified as Sphaerotilus (Lackey and Wattie, 1940). Genetelli and Heukelekian (1964) found a fungus causing bulking in a laboratory activated sludge culture and differentiated it from Sphaerotilus, although they did not identify which fungus it was. Hawkes (1963) reported that Geotrichum causes bulking problems at Yardley, Birmingham. Pipes and Jones (1963), in an attempt to isolate a bulking organism from activated sludge, came up with Geotrichum but worked with it for a year under the impression that it was Sphaerotilus before they discovered its real identity. One of the more interesting groups of bulking organisms are the predatory fungi. Cooke and Ludzack (1958) reported a rotifer-trapping fungus, which they identged as Zoophagus insidians, causing bulking in a laboratory activated sludge culture. Pipes and Jenkins (1965) reported the same organism from an activated sludge plant. Pipes (1966) later described the mechanism by which the rotifer-trapping fungus caused bulking and suggested that nematode-trapping fungi such as Arthrobotrys
88
WESLEY 0. PIPES
might also cause bulking. There is no evidence that predatory fungi occur frequently in activated sludge, but there is a definite possibility that they can cause a considerable nuisance when they are present. C. PROTOZOA The protozoa of activated sludge have been studied more extensively than any of the other groups of organisms present in the process. It is possible to compile long, detailed lists of the protozoa present, and some progress has been made toward finding out what they are doing there. The succession of groups of protozoa in a developing sludge has been followed (Barker, 1949), and it occurs in the following order: rhizopods, flagellates, free-swimming ciliates, crawling ciliates, and stalked ciliates. There have also been several attempts to develop a technique to use protozoa as indicators of the condition of the sludge. The extent to which protozoa participate directly in the breakdown of organic matter in the activated sludge process has been debated somewhat. Pillai and Subrahmanyan (1942, 1944) tried for several years to prove that the stalked ciliates were mainly responsible for the purification achieved by the activated sludge process and that the bacteria and fungi were of little importance. However, this contention was thoroughly disproved by the work of Barker (1946) and Heukelekian and Gurbaxani (1949). The consensus at this time is that only a few of the rhizopods and the small, free-swimming flagellates compete with bacteria directly for the organic matter in the waste. The fact that protozoa are the primary predators feeding upon the saprophytic organisms in activated sludge has been demonstrated by a number of investigators. McKinney and Gram (1956) repeated, summarized, and interpreted some of the previous studies of the predatory activity of protozoa in activated sludge. The protozoa consume unicellular organisms and particles of debris which are not included in the floc and thus prevent them from being carried out in the eHuent. There is also a suggestion that the predatory activity keeps the bacterial population “healthy.” The protozoa which are the most effective predators are the ones which associate with the floc particles and are concentrated with the return sludge. Curds (1963) reported the most convincing evidence that protozoa are effective in flocculating small particles in suspension and presented an excellent discussion of the possible mechanism of this flocculation. There is no doubt that ciliates are capable of flocculating bacterial cells and that they may be important in activated sludge because of this ability. However, since it is known that bacteria are able to form floc in pure culture, the question is the relative importance of the various possible mechanisms.
ECOLOGICAL STUDY OF ACTIVATED SLUDGE
89
There have been many attempts to develop a scheme whereby the condition of activated sludge could be determined by taking some measure of the protozoan population. Reynoldson (1942) found an inverse linear relationship between the count of Vorticellu in the sludge from one plant and the BOD of the effluent. Jenkins (1942) pointed out that this relationship may hold at one plant for a limited period of time but that it is not generally applicable for determination of activated sludge performance. Baines et ul. (1953) made a detailed study of the utility of protozoa as indicator organisms in activated sludge and concluded that the types of protozoa present could be used as a qualitative measure of sludge condition but no quantitative relationship could be developed. Hawkes (1963) maintained that by following the changes in the protozoa present in activated sludge a plant operator could predict when trouble would develop.
D.
INVERTEBRATES
Rotifers and nematodes are quite frequently present in activated sludge, and occasionally aquatic insects, insect larvae, and crustaceans also appear. Calaway (1963) states that nematodes are much more common in other types of biological waste treatment processes than in activated sludge and believes that the environment of an aeration tank is not well suited to their development. McKinney (1957) contends that the presence of rotifers in activated sludge is indicative of a highly oxidized sludge, but no evidence one way or the other has been presented on this point. It would be better if conclusions about the roles of these invertebrates were deferred until some definitive evidence is available.
111.
Ecological Factors
Since the activated sludge process is continuously inoculated with a large variety of microorganisms, the population which is able to maintain itself in the process is a function of the environment and the ecological relationships between the organisms present. The environmental conditions in the system will also influence the form of growth of the organisms and their metabolic activities. Many bacteria can grow either as single cells or clumped together, and some may be filamentous or unicellular under different conditions. Most of the imperfect yeasts and some other fungi grow as single cells, mycelia, or pseudomycelia depending upon the environment. Some protozoa may have either holozoic or holophytic types of nutrition. The extent to which a change in ecological conditions causes a population shift or a change in the growth form and/or activities of the organisms present needs to be determined. In a laboratory activated
90
WESLEY 0. PIPES
sludge culture, there is much less chance for fresh inoculation of the sludge than in a treatment plant. It is likely that the effects of changes in environmental conditions measured in laboratory experiments are primarily due to changes in the form and activities of the population already present, while effects in a treatment plant are more likely to be due to changes in the population. In kinetic studies of the activated sludge process, the response to a change in environmental conditions is measured by the BOD of the effluent. It is usually assumed that the effluent BOD measures the biologically degradable substances which were originally present in the influent but passed through the process unaffected. However, it is more likely that the effluent BOD is a measure of the assimilation of some substances not measured by the BOD determination on the influent, the assimilation of metabolic by-products of the sludge organisms, and the respiration of the organisms which were not removed by sedimentation. An increase in effluent BOD can indicate a decrease in activity by the primary saprophytes, by the secondary and tertiary saprophytes, or by the predators; a decrease in flocculation by the sludge organisms; or a population shift to organisms having poor settling characteristics. As yet, the kinetic approach offers no way to determine what an increase in effluent BOD really indicates.
A. NUTFXTION Gibson (1957) presented a very clear outline of the procedure which should be followed in an investigation of nutritional factors in microbial ecology. She also presented van Niel's postulates which should be the basis of an investigation to demonstrate that a given organism is responsible for a particular process. These postulates, which are modeled after Koch's postulates, are: (1) the organism must always be present when the relevant chemical process is occurring; ( 2 ) it must be possible to grow the organism in pure culture in the laboratory; and ( 3 ) the chemical reaction should follow when a suitable medium is inoculated with a pure culture of the organism, and it should be possible to reisolate the organism from the medium at the end of growth. These are important points to be considered in any investigation of the organisms and chemical processes of activated sludge. Unfortunately, much of the experimental work on activated sludge has not measured up to these high standards. The first step in the investigation of the effect of nutritional factors upon the activated sludge process should be a determination of the types of organic compounds present in the waste and the organisms which are able to utilize each type as a substrate. There have been a number of attempts to characterize the organic matter in sewage. In one of the more
ECOLOGICAL STUDY OF ACZIVATED SLUDGE
91
recent of these, Painter et al. ( 1961) found that less than one third of the organic carbon and only slightly more than one third of the organic nitrogen was in solution. A considerable degree of purification of sewage can be obtained merely by converting the suspended solids into a settleable form, and it would be worth while to determine which organisms feed upon the particulate matter and which metabolize soluble organic compounds. Industrial wastes which are amenable to biological treatment usually can be defined much more precisely in terms of their chemical composition than sewage can. The problems with industrial waste are very high concentrations of organic matter in the waste, the question of the degradability of the major components of the organic matter, and the ratio of nitrogen and phosphorus to the organic matter. A waste containing high concentrations (greater than 1 gm./liter) of soluble organic matter is difficult to treat by the activated sludge process. The problem is not that microorganisms will not grow in the waste but that the formation of a readily settleable sludge is hindered in some way (Heukelekian, 1949). It has not yet been determined if the high concentrations of soluble organic matter lead to the development of an entirely diflerent population than is present in normal activated sludge or if the same organisms develop but just do not form floc. The degradability of the noxious components of a waste can easily be determined by enrichment culture in the laboratory, and this technique has been used on numerous occasions (e.g., Ludzack and Schaffer, 1962). The laboratory enrichment culture studies do yield good information on the degradability of wastes and on the environmental conditions suitable for the organisms which can grow in the waste. However, they do not yield any information about the settling characteristics of the sludge which will be developed in a continuous-flow treatment plant. Domestic sewage contains larger amounts of nitrogen and phosphorus than are required for stabilization of the organic matter present, but many industrial wastes are deficient in these nutrients in proportion to the concentrations of organic matter. A series of investigations by Sawyer culminating in a classic paper (Helmers et al., 1952) clearly delineated the lowest ratios of nitrogen and phosphorus to organic matter which could be tolerated in the activated sludge process and still result in good BOD reduction. However, the effect of low nitrogen and phosphorus concentrations upon the microbial population has not been adequately described. According to Hawkes (1963), the nutrients present in the waste are the most important factor in determining the dominant population of the sludge, If this concept is true, and it is widely accepted, why is it that so many investigators develop a mixed microbial culture in the
92
WESLEY 0. PIPES
laboratory on a solution of glucose and ammonium phosphate and call it activated sludge?
B. OXYGEN SUPPLY The activated sludge process is essentially an aerobic system and depends upon aerobic microorganisms to decompose the noxious components of the waste. However, the sludge usually undergoes anaerobic periods during sedimentation and return. Also, the length of time the sludge is aerated after it is mixed with the waste seems to have a pronounced effect upon the characteristics of the sludge. Thus, there are three points to be discussed in connection with the effect of oxygen supply upon the sludge organisms: ( 1 ) the dissolved oxygen concentration ( D O ) maintained in the mixed liquor; ( 2 ) the length of the anaerobic period during sludge return; and ( 3 ) the length of time the sludge is aerated during each pass through the aeration tank. Apparently a high DO in the mixed liquor is not necessary to produce good BOD reduction by the process. Gellman (1964) reported that 1 mg./liter DO was satisfactory, but other investigators (Gaudy and Turner, 1964) found that much lower DO values did not seriously hamper BOD reduction. Wuhrmann (1964) demonstrated that the maximum size of the floc particles is a function of DO and suggested a mechanism by which low DO could cause a breakup of the floc so that it would not settle well. A number of investigators have suggested that a low DO in the mixed liquor may cause bulking by encouraging the growth of some filamentous organism (usually called Sphaerotilus), but Orford et al. (1960) could find no relationship between DO in the aeration tank and the settling characteristics of the sludge by statistical analysis of data from an extensive series of laboratory experiments. The upper limit to the DO which might be desired in the mixed liquor is determined by whether nitrification is to be encouraged. Wuhrmann (1964) has adequately reviewed the research on this problem is a previous volume of this series, and it will not be considered further herein except to indicate that such research is the best example of a combination of the kinetic and ecological approaches and has been quite successful. The dissolved oxygen concentration in the mixed liquor is the oxygen resource which the sludge microorganisms have to draw upon during the period of sedimentation and sludge return. Thus, the effect of the DO in the mixed liquor might be related to the length of the anaerobic period during sedimentation and sludge return rather than a direct effect upon the sludge organisms in the aeration tank. Apparently brief anaerobic periods do not have an adverse effect upon the sludge, but long anaerobic periods do destroy its effectiveness (Gaudy and Turner, 1964). It has
ECOLOGICAL STUDY OF ACTIVATED SLUDGE
93
been suggested that filamentous organisms can tolerate anaerobic conditions better than floc-forming organisms, and a longer anaerobic period during sludge return will favor their growth. When activated sludge is mixed with a waste and aerated, there is a fairly rapid transfer of organic matter from the waste to the sludge. There has been a great deal of speculation about the mechanism of this rapid transfer, but probably the most important mechanisms are flocculation of suspended material and assimilation and storage as a food reserve of the soluble organic compounds by the sludge organisms. The sludge seems to have some maximum capacity to take up organic matter, and, if this capacity is exceeded, the rest of the organic matter passes through the system unaffected, Aeration of the sludge restores this capacity, and the proper operation of an activated sludge plant can be interpreted in terms of maintaining a balance between the amount of organic matter taken up by the sludge and the amount of time provided for the sludge to oxidize the organic matter (Haseltine, 1956). There may be some advantage to separating the sludge from the mixed liquor and reaerating it separately before return (Grich, 1961). The time of aeration provided in relation to the amount of waste treated does have an effect upon the population of organisms present in the sludge, but the exact shift in population with increase in aeration time has not been described. Mathematical models of the activated sludge process do not describe the change in sludge characteristics with this change in aeration time either, but it is likely that, if the population shift could be described, it could be incorporated into the kinetic equations and better models of the process could be derived.
C. TEMPERATURE The wastes received by activated sludge plants usually have a temperature in the range between 0" and 30°C. There is a general impression that the process operates better at the upper end of the range than at the lower, but few data have been published to support this impression. Keefer (1962) analyzed data from plant operation and found that the effluent had a lower BOD and contained smaller amounts of suspended solids at 25°C. than at 12°C. Hurwitz et al. (1961) found that cellulose could be degraded by activated sludge at 25°C. but not at 12°C. There have been a few studies of the effect of temperature upon activated sludge cultures in the laboratory. Dougherty and McNary (1958) developed a culture on diluted orange juice and studied its adaptation to stepwise increases in temperature in the range 21"-46°C. They found that, when the temperature of the culture was raised 5 or 10 degrees, the effluent became turbid and the effluent BOD increased, but after a
94
WESLEY 0. PIPES
few days the sludge acclimated and the effluent BOD dropped back to a normal value. It was found that the higher the temperature, the longer the period of acclimation. The sludge never did regain good characteristics above 44°C. The activity of the protozoa was severely inhibited above 36"C., and no protozoa at all were present above 43°C. Ludzack et al. (1961) developed activated sludge cultures in the laboratory on slurried dog food and on a glucose-gelatin solution and studied each at 5" and 30°C. They found that temperature had little effect upon the transfer of organic matter from the feed to the waste but that the organic material accumulated in the sludge was oxidized much more rapidly at the higher temperature. They observed that the sIudge was much more likely to develop filamentous bulking at the lower temperature. On the other hand, Ruchhoft and Kachmar (1941) had observed that filamentous bulking was much more likely to occur at higher temperatures. Could it be that these two groups were observing different filamentous organisms even though they each called the organism Sphaarotilus?
D. PH Keefer and Meisel (1951) found that activated sludge could acclimate to any pH value in the range 6.0 to 9.0 but that, if the pH was above 10 or below 5, the microbial population did not develop in such a form that it could be called activated sludge. They also observed that the sludge had much better settling characteristics at a pH value of 6.5 or 7.5 than it did at a pH of 7.0. It is unlikely that an excessively high p H would cause a problem in an activated sludge plant because C 0 2 produced by the sludge organisms would rapidly lower the pH in the mixed liquor to less than 9 if it ever should rise higher. There are quite a number of low pH wastes, and some of these are introduced into activated sludge plants. The major effect of low pH on the activated sludge process appears to be the development of copious growths of fungi which replace the flocculant sludge. A few investigators still call the filamentous organism in activated sludge with a low pH Sphaerotilus, but it has been clearly shown that Sphaerotilus is even less resistant to low pH than most of the bacteria in activated sludge (Hohnl, 1955). Geotrichum appears to be a good candidate for the filamentous bulking organism that appears at low pH (Hawkes, 1963). In most cases, the filamentous organism which develops is capable of oxidizing the organic matter in the waste. The only problem is that it cannot be separated from the mixed liquor very effectively by sedimentation.
ECOLOGICAL STUDY OF ACTIVATED SLUDGE
95
E. TOXICITY Since activated sludge is a mixed microbial culture, it has amazing resistance to many poisons. Sludges which are capable of oxidizing such things as formaldehyde, phenol, and cyanide have been developed, and some of them are used in treatment plants. The problem of the toxicity of organic compounds to activated sludge is essentially the problem of determining the biologica'l degradability of the compound in question or occasionally the question of whether it is possible to degrade one organic compound in the presence of another. The problem of the toxicity of metallic ions to activated sludge is a significant one in some treatment plants, and it has been investigated extensively in the laboratory (Barth et al., 1965). Concentrations of several metals (Cr, Cu, Ni, and Zn) in the range 1-10 mg./liter will reduce the efficiency of the slludge in treating organic wastes when present continuously. Much higher concentrations of metals can be tolerated on a short-term basis. The response to slug doses of metals is an increase in the turbidity and BOD of the effluent. This observed response could be explained either by an interference with flocculation or by an inhibition of the protozoa which prey on bacteria growing as single cells but not on the bacteria in the floc particles. It has been observed that, with concentrations of metal ions around 1 mg./liter in the waste, the sludge never does become filamentous.
IV. Ecological Relationships The physical and chemical environment in the activated sludge process determines the limits upon the population which can develop and what changes it can cause in the waste being treated. However, within the limits set by the environment, the population development is controlled by the interaction of the organisms with the environment and with each other. Study of the ecological relationships of activated sludge organisms holds some of the most promising possibilities for increasing the understanding of the process and providing solutions to practical problems. Unfortunately, so little work has been done on these relationships that much of what will be covered in this section is highly speculative. The scientific discipline of quantitative ecology has developed over a period of years and has produced some notable successes and several disappointments ( Morowitz, 1965). The developments in this field have been generallly overlooked by investigators studying activated sludge, but it would appear that the time is now ripe for the concepts of quantitative ecology to be applied to the solution of practical problems of waste treatment. Slobodkin (1965) has pointed out several reasons for the
96
WESLEY 0. PIPES
failure of quantitative ecology to provide elegant mathematical models describing situations of practical significance. Those problems which ecological theory has failed to surmount do not exist in microbial ecology because of the relative simplicity of the organisms. Activated sludge might be the ideal environment for studies of quantitative ecology. A great deal of effort has been expended upon laboratory studies of activated sludge cultures trying to delineate the important parameters determining the results of treatment plant operation. Much of this research is suspect because there has been no attempt to assure that the organisms present in the laboratory cultures are the same as those which predominate in full-scale activated sludge plants and because it is obvious that the nutritional factors are quite different in the laboratory cultures, If this effort were expended on determining which organisms really have significance in the activated sludge process and what the ecological relationships between them are, there might be developed a firm theoretical basis for the kinetic models of the activated sludge process. A. COMPETITION It is a basic axiom of ecology that the direct competition between organisms for the same material and energy resources should result in the elimination of all competitors except one ( Slobodkin, 1961). Apparently, this competition is not carried to its ultimate conclusion in activated sludge, because a variety of organisms on each trophic level are present. It is likely that the continuous inoculation of the process with a large variety of microorganisms and the fact that the environment of the process varies continuously within a certain range enables some microorganisms which would be eliminated if a true steady state were attained to maintain a sizeable population in the sludge. If the climax population of the activated sludge process were known, it would then be possible to evaluate the contribution of the subclimax species and determine if the process should be operated to approach more nearly the steady-state population or to encourage the subclimax population. If it were possible to specify which organisms are desired on each trophic level and if the factors which control the outcome of competition on each level were known, then the activated sludge process could be designed and operated to encourage the dominance of the desired population. In the competition for the nutrients in the waste several environmental factors such as pH or temperature could determine the outcome, or, given a variety of organisms adapted to the same environment, the characteristics of the individual species such as specific growth rate, protoplasm synthesized per unit quantity of nutrient utilized, the abiIity to store reserve material, or resistance to predation could dr-
ECOLOGICAL STUDY OF ACTIVATED SLUDGE
97
termine which species becomes dominant. A quantitative study of the ecological relationships should determine which of these factors is critical in any given situation, 1. Competition for Substrates There is a competition on the lowest trophic level in activated sludge between bacteria, fungi, and saprophytic protozoa, If excess food is available, the saprophytic protozoa, mostly small flagellates, show up in large numbers, but they seem to be eliminated by the bacteria when food becomes scarce (Barker, 1949). However, since bacteria and fungi in the sludge are not identifiable microscopically, little is known even about the identity of these organisms and less about the outcome of the competition between them or the factors which might control it. It is possible that floc-forming organisms have a natural advantage in competition for particulate organic matter, but this has never been demonstrated conclusively. Actually, it has never been proved that the organisms which are able to flocculate organic matter are the ones that utilize it as substrate, a\lthough this seems to be a reasonable assumption. The outcome of the competition for soluble organic matter may be determined by the surface area-to-volume ratio of the various competing organisms, or, on the other hand, the ability to store large quantities of a food reserve may provide some competitive advantage. The competition between Zoogloea ramigera and Sphaerotilus natans might provide an interesting and significant problem for Iaboratory study. They both grow better on simple soluble substrates than on complex substrates (Hohnl, 1955; Dugan and Lungren, 1960), and they both are able to store large quantities of poly-P-hydroxybutyric acid ( Rouf and Stokes, 1962; Crabtree et al., 1965). Sphaerotilus seems to prefer carbohydrates and Zoogloea proteinaceous matter, so the study might actually show that they do not compete for the same nutrients and that it would be beneficial to have both of them present in activated sludge. The competition for soluble organic compounds may be a competition between primary saprophytes for organic matter in the waste or between secondary saprophytes for the metabolic by-products of other organisms present. It may prove to be difficult to classify some organisms as primary or secondary saprophytes, because the by-products of some microorganisms are the same as some of the organic compounds originally present in the waste. As yet, nothing is known about the metabolic byproducts of activated sludge organisms, so it is difficult to even speculate about factors controlling the competition between secondary saprophytes . In a waste which spends several hours in a sewer before it reaches
98
WESLEY 0. PIPES
the treatment plant, much of the organic matter of the waste may already be in the form of microbial cells before it enters the aeration tank. The organisms which grow in the waste in sewers are not likely to be the same ones which are best suited to the activated sludge process because of the great differences in the two environments. The primary competition in activated sludge may be between predators rather than between ssprophytes. In any case, competition between predators is likely to be an important aspect of activated sludge ecology, The succession of protozoa observed in a developing sludge population indicates that the factors which control the outcome of the competition between predators shift with time. However, whether the change occurs because of changes in the saprophytic population or because of interactions between the predators themselves is not known. 2. Other Competitions The other material resources of the environment for which there may be a competition among activated sludge microorganisms include nitrogen, phosphorus, and oxygen, Nutrients such as sulfur, potassium, magnesium, and the trace elements can be assimilated in inorganic form by most free living microorganisms, and the water used to carry the waste into the aeration tank probably contains adequate amounts of these substances. At least, there has never been a clearly demonstrated case of limitation of microbial activities in activated sludge by low concentrations of these other nutrients, If the nitrogen content of a waste is low in comparison with the amount of oxidizable substrate available, either the organisms present will not assimiliate all of the organic matter in the waste or the competition between the primary saprophytes will cause a population shift to those organisms having lower nitrogen requirements per unit mass of protoplasm synthesized. The observed effects of a nitrogen deficiency upon activated sludge process are a decreased rate of sludge growth, deterioration of the settling characteristics of the sludge, and a decrease in the BOD reduction by the process (Helmers et al., 1952). All of these effects could be explained either by a population shift or by a change in the activities of the organisms already present. The effect of low phosphate concentration upon activated sludge in laboratory culture was studied by Greenberg et al. (1955). They observed that, when the phosphorus concentration in the synthetic sewage being fed to their cultures was less than 2 mg./liter, there was a population shift from floc-forming organisms to filamentous organisms. They believed that the filamentous organism which became dominant was
ECOLOGICAL STUDY OF ACTIVATED SLUDGE
99
Sphaerotilus, but did not indicate that they made any serious attempt to determine its identity. Several investigators have suggested that organisms having the greatest surface area-to-volume ratio would have an advantage in competition for small concentrations of dissolved oxygen ( Ruchhoft and Kachmar, 1941 ) . Thus, bacteria growing as single cells would be favored over filamentous organisms, which in turn would have an advantage over organisms inside floc particles. On the other hand, it has been suggested that the primary effect of low DO is to inactivate the protozoa which feed on single-celled or filamentous organisms but not upon organisms in floc particles (Pillai and Subrahmanyan, 1943). On the basis of information available at present, either one of these hypotheses is feasible.
B. PREDATION When the noxious components of a waste are organic compounds which can serve as substrates for the growth of a variety of microorganisms, biological waste treatment can be viewed as an attempt to dissipate the potential energy of the organic structures. The first conversion of the organic matter of the waste to the protoplasm of the saprophytes dissipates a certain fraction of this energy, but some of it remains as potential energy in the protoplasm synthesized. The conversion of the saprophytes to the protoplasm of the primary predators dissipates a fraction of this remaining energy, and so forth. From this point of view, it would appear that the more predation in activated sludge, the better. However, excessive predation may destroy the population of saprophytes needed for the first conversion. Slobodkin ( 1961) has described the theory of prey-predator relationships and experimental techniques which have been used to study thesc relationships in the laboratory. The relationship between a prey-predator pair is unstable; i.e., unless the environment is reinoculated at frequent intervals or there is some place for the prey to hide, one or both of the organisms is eliminated. Activated sludge is reinoculated continuously and the floc partides provide a hiding place for some of the saprophytes, so it should be adaptable to studies of these relationships. Unfortunately, not much work has been done on the effect of selective predation upon the competition between saprophytic bacteria and fungi. Excessive numbers of predators in activated sludge can lead not only to serious depletion of the population of saprophytes but also to development of poor settling characteristics of the sludge. Lackey (1949) described a case of bulking due to excessive growth of stalked ciliates, which are normally considered beneficial because of their predatory
100
WESLEY 0. PIPES
activity. Excessive numbers of rotifers in the sludge can lead to the invasion of the sludge by a predatory fungus which can cause bulking (Pipes and Jenkins, 1965).
C. SYNERGISMS When two or more organisms are introduced into the same environment, they may have little effect upon each other, they may compete for the material resources of the environment, or they may produce a synergistic effect which is different from the sum of the effects they produce when separated. Synergisms occur with different levels of association between organisms, The organisms involved in the synergism may be capable of growing independently in the environment, or they may depend upon each other to such a degree that neither would be present without the other. The changes in a waste being treated by the activated sludge process are due to the sum of the effects of the individual organisms present plus the synergistic effects. There have been a few attempts to separate individual species from the activated sludge population and determine if they can reduce BOD or flocculate in pure culture (e.g., Dias and Bhat, 1964). Most activated sludge bacteria studied in this manner can reduce BOD and can form fioc by themselves, but it is usually observed that a mixed microbial culture is more effective than pure cultures of any of the organisms studied. There have been no attempts to combine pure cultures of activated sludge organisms for any purpose other than studying the effect of protozoa predation upon a bacterial population. There are a number of possible synergisms which may be important in activated sludge. Some organisms may flocculate particulate organic matter which is then digested by other organisms and thereby converted into a form which can be assimilated by the floc-forming organisms. One organism may produce a metabolic by-product which is a growth factor for another organism. Actually, the whole activated sludge process may be considered as a synergism of the entire population.
V. Summary The activated sludge process is the most efficient but also the most expensive method of purifying a waste which is amenable to biological treatment. Despite over 50 years of experience with treatment plants and laboratory and plant research, it is still considered to be unpredictable and difficult to control in many situations. There is a great need for research on activated sludge which will provide answers to practical operating problems,
ECOLOGICAL STUDY OF ACTIVATED SLUDGE
101
Investigators studying activated sludge have tended to follow one of two approaches : the kinetic approach, which emphasizes the chemical changes which occur in the waste and ignores the biological nature of the sludge, and the ecological approach, which concentrates upon the microbial populations in the sludge. The kinetic approach has yielded many valuable concepts and much data upon which the rational design of an activated sludge system can be based, but it has also fostered the acceptance of some erroneous concepts and has failed to solve several operating problems. The ecological approach has failed to provide anything but a few vague generalizations and much worthless speculation, but this is probably due to the quality of the research which has been carried out rather than to any basic defect in the approach. Investigators using the kinetic approach usually evaluate the performance of the process by comparing the biochemical oxygen demand (BOD) of the effluent with that of the influent. It is often assumed that the effluent BOD measures the amount of biologically degradable substances which were originally present in the influent but passed through the process unaffected, but it actually measures oxygen consumption due to other phenomena. Mathematical models of the activated sludge process do not describe the settling characteristics of the sludge nor do they take into consideration the effect of the variation in ecological factors. The results of research on the activated sludge process using the kinetic approach need to be related to the biological phenomena responsibIe for the effects measured. Ecological theory has developed in recent years to the point where it should be of value in describing quantitatively the relationships between the activated sludge organisms. Since the activated sludge population is composed primarily of microorganisms, many of the problems which have hampered the development of quantitative ecology will not be encountered in investigations of the ecology of activated sludge. The elucidation of the ecological relationships which control the microbial population of activated sludge should provide the basis for further improvements in the kinetic approach to activated sludge, but a great deal of work needs to be done before these improvements will be forthcoming. REFERENCES Allen, L. A. (1944). J. Hyg. 43, 424-431. Baines, S., Hawkes, H. A,, Hewitt, C . H., and Jenkins, S. H. (1953). Sewage Ind. Wastes 25, 1023-1033. Barker, A. N. (1946). Ann. Appl. Biol. 33, 314-325. Barker, A. N. (1949). J. Znst. Sewage Purif. pp. 7-22. Barritt, N. W. (1940). Ann. Appl. B i d . 27, 151-158.
102
WESLEY 0. PIPES
Barth, E. F., Ettinger, M. B., Salotto, B. V., and McDermott, G . M. (1965). 1. Water Pollution Control Federation 37, 86-96. Butterfield, C. T. (1935). Public Health Rept. (US.)50, 671-684. Butterfield, C. T., Ruchhoft, C. C., and McNamee, P. D. (1937). Sewage Works 1. 9, 173-196. Calaway, W. T. (1963). J. Water Pollution Control Federation 35, 1006-1016. Cooke, W. B. (1956). Syndowia 1, 146-175. Cooke, W. B., and Ludzack, F. J. (1958). Sewage Ind. Wastes 30, 1490-1495. Cooke, W. B., Phaff, H. J., Miller, M. W., Shifrine, M., and Knapp, E. P. (1960). Mycologia 52, 210-230. Crabtree, K., McCoy, E., Boyle, W. C., and Rohlich, G. A. (1965). Appl. Microbiol. 13, 218-226. Curds, C. R. (1963). 1. Gen. Microbiol. 33, 359-383. Dias, F. F., and Bhat, J. V. (1964). Appl. Microbiol. 12, 412-417. Dondero, N. C., Phillips, R. A., and Heukelekian, H. (1961). Appl. Microbiol. 9, 219-227. Dougherty, M. H., and McNary, R. R. (1958). Sewage Ind. Wastes 30, 1263-1265. Dugan, P. R., and Lungren, D. G. (1960). Appl. Microbiol. 8, 357-362. Eckenfelder, W. W., and McCabe, J. (1960). In “Waste Treatment” (P.C.G. Isaac, ed.), pp. 156-187. Pergamon Press, Oxford. Gaudy, A. F., and Turner, B. G. (1964). 1. Water Pollution Control Federation 36, 767-781. Gellman, I. (1964). Pulp Paper Mag. Can. 65, 6-14. Genetelli, E. J., and Heukelekian, H. (1964). 1. Water Pollution Control Federation 36, 643-649. Gibson, J. (1957). In “Microbial Ecology” (R. E. 0. Williams and C. C. Spicer, eds.), pp. 22-41. Cambridge Univ. Press, London and New York. Gram, A. L. (1956). Reaction kinetics of aerobic biological processes, Rept. No. 2, Institute of Engineering Research Ser. 90. Univ. of California, Berkeley, California. Greenberg, A. E., Klein, G., and Kaufman, W. J. (1955). Sewage Ind. Wastes 27, 277-282. Grich, E. R. ( 1961). 1. Water Pollution Control Federation 33, 856-863. Haseltine, T. R. ( 1956). In “Biological Treatment of Sewage and Industrial Wastes” (W. W. Eckenfelder and J. McCabe, eds.), Vol. 1, pp. 257-270. Reinhold, New York. Hawkes, H. A. (1960). In “Waste Treatment” (P. C. G. Isaac, ed.), pp. 52-98. Pergamon Press, Oxford. Hawkes, H. A. (1963). “The Ecology of Waste Water Treatment.” Macmillan, New York. Helmers, E. N., Frame, J. D., Greenberg, A. E., and Sawyer, C. N. (1952). Sewage Ind. Wastes 24, 496-507. Heukelekian, H. (1949). Ind. Eng. Chem. 41, 743-769. Heukelekian, H., and Gurbaxani, M. (1949). Sewage Works J. 21, 811-818. Heukelekian, H., and Littmann, M. L. (1939). Sewage Works J. 11, 752-763. Heukelekian, H., and Schulhoff, H. B. (1938). Sewage Works J. 10, 43-48. Heukelekian, H., and Weisberg, E. (1956). Sewage Id.Wastes 28, 558-574. Hinshelwood, C. N. (1946). “The Chemical Kinetics of the Bacterial Cell.” Oxford Univ. Press (Clarendon), London and New York. Hohnl, G. (1955). Arch. Mikrobio2. 23, 207-250.
ECOLOGICAL STUDY OF ACTIVATED SLUDGE
103
Hurwitz, E., Beck, A. J., Sakellariou, E., and Krup, M. (1961). J. Water Pollution Control Federation 33, 1070-1075. Jasewicz, L., and Porges, N. (1956). Sewnge Ind. Wastes 28, 1130-1136. Jenkins, S. H. (1942). Nature 150, 607. Keefer, C . E. (1962). J. Water Pollution Control Federation 34, 1186-1196. Keefer, C. E., and Meisel, J. (1951). Sewage Ind. Wastes 23, 982-991. Kraus, L. S. ( 1949). Sewage Works J. 21, 613-622. Lackey, J. B. (1949). Sewage Works J. 21, 659-665. Lackey, J. B., and Wattie, E. (1940). Sewage Works J. 12, 669-684. Ludzack, F. J., and Schaffer, R. B. (1962). J. Water Pollution Control Federation 34, 320-341. Ludzack, F. J., Schaffer, R. B., and Ettinger, M. B. (1961). J. Water Pollution Control Federation 33, 141-156. McKinney, R. E. (1957). Appl. Microhiol. 1, 167-174. McKinney, R. E., and Gram, A. (1956). Sewage Ind. Wastes 28, 1219-1231. McKinney, R. E., and Horwood, M. P. (1952). Sewage Ind. Wastes 24, 117-123. McKinney, R. E., and Weichlein, R. G . (1953). Appl. Microbiol. 1, 259-261. Monod, J. ( 1949). Ann. Reu. hlicrobiol. 3, 371-394. Morowitz, H. J. ( 1965). In “Theoretical and Mathematical Biology” (T. H. Waterman and H. J. Morowitz, eds.), pp. 24-35. Ginn (Blaisdell), Boston, Massachusetts. Orford, H. E., Heukelekian, H., and Isenberg, E. (1960). 3rd Conf. Biol. Waste Treat., Manhattan Coll., New York, N.Y., 1960. Preprints pp. 68-84. Painter, H. A,, Viney, M., and Bywaters, A. (1961). J. Inst. Sewage Purif. pp. 302-310. Pillai, S. C., and Subrahmanyan, V. (1942). Nature 150, 525. PiUai, S. C., and Subrahmanyan, V. (1943). Sci. Cult. (Calcutta) 8, 376-378. Pillai, S. C., and Subrahmanyan, V. (1944). Nature 154, 179-180. Pipes, W. 0. (1966). Purdue Uniu., Eng. Bull., Ext. Ser. (in press). Pipes, W. O., and Jenkins, D. (1965). Intern. J. Air Water Pollution 9, 495-500. Pipes, W. O., and Jones, P. H. (1963). Biotechnol. Bioeng. 5, 287-307. Reynoldson, T. B. (1942). Nature 149, 608-609. Rogovskaya, T. I., and Lazareva, M. F. (1959). Microbiologiya 28, 530-538. Rouf, M. A., and Stokes, J. L. (1962). J. Bacterial. 83, 343-347. Ruchhoft, C. C., and Kachmar, J. F. (1941). Sewage Works J. 13, 3-32. Ruchhoft, C. C., and Watkins, J. H. (1928). Sewage Works J. 1, 52-57. Sawyer, C. N. (1965). J. Water Pollution Control Federation 37, 151-162. Slobodkin, L. B. (1961). “Growth and Regulation of Animal Populations.” Holt, New York. Slobodkin, L. B. (1965). Am. Scientist 53, 347-357. Wattie, E. (1943). Sewage Works J . 15, 476-490. Wuhrmann, K. (1964). Aduan. Appl. Microbiol. 6, 119-150.
This Page Intentionally Left Blank
Control of Bacteria in Nondomestic Water Supplies CECILW . CHAMBERS AND NORMAN A . CLARKE U S. Department of Health. Education. and Welfare. Federal Water Pollution Control Administration. Robert A . Tuft Sanitary Engineering Center. Public Health Service. Cincinnati. Ohio
. .
Introduction ........ .......................... Water as a Source of terial Contamination . . . . . . . . A . General Considerations ........................ B. Some Waterborne Bacteria and Their Significance I11. Water as a Bacteriological Medium . . . . . . . . . . . . . . . . . A . Types of Bacteria and General Problems . . . . . . . . . B. Examples of Growth in Water . . . . . . . . . . . . . IV . Areas Where Contaminants Multiply . . . . . . . . . . . . . . . . A. Filters ...................................... B. Minimal Flow Areas and Other Sources . . . . . . . . . V . Biological Factors Affecting Control . . . . . . . . . . . . . . . . A. Bacteriophage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Antibiotic Effects ............................ C . Predation ................................... VI . Physical Methods of Control ..... .............. A . Temperature . . . . . . . . . . . . . . B. Filtration and Flocculation .................... C . Ultraviolet Light . . . . . . . . . . ............. D . Miscellaneous Physical Metho VII . Chemical Methods of Control ...................... A. Factors to Consider in Se ........... B. Chlorination . . . . . . . . . . . . . . C . Other Oxidizing Agents ....................... D . Silver . . . . . . . . . . . . . . . . . E . Quaternary Ammonium C F. Excess Lime ................................. G . Miscellaneous Chemical Methods . . . . . . . . . . . . . . . H . Problems of Analytical Control . . . . . VIII . Economics of Control . . . . . . . . . . . . . . . . . . . . . . . . . . IX . Evaluation of Effectiveness of Control Methods . . . . Bacteriological Criteria ........................ X . General Comments and Conclusions . . . . . . . . . . . . . . . . References .......................
I I1
I
.
105 106 106 107 108 108 109 111 111 113 115 115 115 115 116 116 117 118 120 121 121 123 129 130 131 131 132 133 135 136 136 138 139
Introduction
This discussion is based on information presented in relation to the control of pseudomonads in water supplies (Chambers. 1965) and has been broadened to include other waterborne bacteria. What does control consist of? To the pharmaceutical manufacturer 105
106
CECIL W. CHAMBERS AND NORMAN A. CLARKE
preparing water for subsequent intravenous use, control means complete elimination of all organisms and any microbiological metabolic products such as pyrogens. By way of contrast, an engineer dealing with an industrial-process water supply may be interested only in keeping the numbers of organisms within tolerable limits for the intended use. Thus, because of the wide variety of waters to be considered, some definition of the term “control” seems desirable. For purposes of this discussion, control is defined as tailoring the bacteriological quality of water to specifications dictated by the intended use. Some examples of specific problems and consequences of the presence of undesirable bacteria in water will be discussed. It is obviously impractical, however, to attempt to enumerate all the different types of situations where difficulties could develop and suggest specific solutions for each problem. The designation “nondomestic water supplies” includes recreational and irrigation waters and various types of industrial waters used for cooling purposes, processing of dairy and other food products, and the manufacture of paper, plastics, cosmetics, and a variety of other items. Halophilic organisms and saline waters are not discussed, and the control of bacteria in drinking water and swimming pools is not considered per se. Many problems encountered with potable and swimming pool waters and the methods available for their solution are equally applicable, however, to industrial and other waters. For example, the fundamental factors governing the successful use of chlorine for the prevention of aftergrowths in distribution systems are the same for potable water and water used for any other purpose. The primary purposes of this review are to ( 1 ) alert those responsible for the control of bacteria in water to the numerous factors to be considered, and ( 2 ) provide information useful to those faced with the problem of developing their own system for control of bacteria in water.
II. Water as a Source of Bacterial Contamination
A. GENERALCONSIDERATIONS The bacteriological requirements for many nondomestic waters, such as those used in the manufacture of cosmetics, pharmaceutical prepa-
rations, and other precisely formulated products, may be more stringent than those for drinking water. Specifications regarding the coliform content of drinking water are clearly stated in the Public Health Service Drinking Water Standards (1962), but there are no clearcut criteria for total numbers of noncoliform bacteria. Some tap waters consistently meet the coliform requirements but contain considerable numbers of other
CONTROL OF BACTERIA IN NONDOMESTIC WATER
107
bacteria. Accordingly, it cannot be assumed that acceptable drinking water is free of other bacteria that may be undesirable in many manufactured products. The opposite is often true. Psychrophilic bacteria such as pseudomonads are frequently found in potable water that is free of coliform bacteria. These organisms can cause severe problems in many manufacturing uses of water. For example, pseudomonads present in a municipal water supply were responsible for spoilage of cottage cheese (Seiberling and Harper, 1955). Because of such problems, it is frequently more difficult to produce water suitable for food processing than it is to produce potable water. In some food processing, the problem is further complicated by the fact that a convenient method, such as chlorination, may produce “off-flavors” in the finished product (Welch and Folinazzo, 1959). B. SOME WATERBORNE BACTERIA AND THEIRSIGNIFICANCE The desirability of excluding pathogenic organisms from water is selfevident. Of the other bacteria considered, Pseudomonos along with related genera such as Xanthomonas, Flavobacterium, and Achrmobacter are frequently the most serious offenders in many manufacturing and industrial uses of water. Regardless of past views concerning the pathogenic status of Pseudomonas aeruginosa, this species can no longer be relegated to a position of minor importance. Bejuki (1965) considers the pathogenicity of P . aeruginosa to be well documented, and its role in producing human enteritis is clear. In one outbreak, for example, 9 infants, of a total of 24 at risk, died as a result of ingesting milk contaminated by water containing P. aeruginosa (Hunter and Ensign, 1947). The genus Pseudomonos includes almost 50% of all bacterial plant pathogens, and Xanthmonas about 25% (Burkholder, 1948). In recent years, the problem of possible transmission of these and other plant pathogens by irrigation water has begun to receive more attention (Kelman et al., 1957; Hoadley and McCoy, 1965). The role of irrigation water in the transmission of human enteric diseases has been the subject of considerable investigation (Dunlop, 1952; Norman and Kabler, 1953). Recently improved procedures ( Spino, 1966) have resulted in recovery of salmonellae from polluted waters where the fecal and total coliform densities were as low as 2.2 and 22 per ml., respectively. Even the metabolic products of bacteria can be a serious problem in waters used to prepare solutions for injection (Honeywell et al., 1962), and viable pseudomonads have been particularly troublesome in cosmetics and ophthalmic solutions ( Tennenbaum, 1965). Problems in dairy
108
CECIL W. CHAMBERS AND NORMAN
A.
CLARKE
products are frequently caused by Pseudomonas fragi (Parker et al., 1953). Morrison and Hammer (1941) found this species in 9.7% of Iowa dairy-plant water supplies sampled. Burgwald et u1. ( 1952) reported problems with psychrophiles in a municipal water supply used in the production of reconstituted milk. Mossel and Ingram (1955) found that, at low temperatures, bacterial spoilage of foods is “characterized by psychrophilic strains of Pseudomonas, Achromobacter, and Flavobacterium. The relationship of pseudomonads to the degradation of plasticizers and other materials seems to be well established (Bejuki, 1961, 1965). Sphaerotilus natans is a serious nuisance in flowing water and can destroy the recreational value of natural waters. Fundamental factors relating to growth and other characteristics of Sphaerotilus have been discussed by Dondero (1961). Some of the iron bacteria, such as the Sphaerotilus-Leptothrix group and Crenothrix, cause industrial water problems ( Mulder, 1964) . Sulfate-reducing bacteria and Crenothrix are troublesome, frequently causing “ r e d water difficulties ( Palmer, 1961; Lewis, 1965). Jarrett (1965) estimates that “losses as a result of abandonment of wells infested with iron bacteria amount to approximately one-quarter million dollars each year in North Carolina.” Harrison and Heukelekian (1958) reviewed in detail a wide variety of chemical, nutritional, and physical factors that are very important in understanding the problems of controlling slime-forming organisms.
111. Water as a Bacteriological Medium A. TYPESOF BACTERIA AND GENERAL PROBLEMS
Tne extent to which water can serve as a bacterial growth medium is one of the least recognized facets of bacteriology. Water, even nominally high-quality distilled water, is far from free of viable bacteria, and the growth potential of such water is generally underestimated ( Leifson, 1962). Many bacteria reproduce in water; among the genera that will grow in water of unquestioned potable quality are: ( 1) Pseudomonas, ( 2 ) Xanthomonas, ( 3 ) Achromobacter, ( 4 ) Escherichiu, ( 5 ) Aerobacter, ( 6 ) Streptococcus, ( 7 ) Desulfovibrio, and ( 8 ) Crenothrir. Some of these grow surprisingly well in synthetic waters designed to simulate natural waters, prepared with distilled water and ACS-CP grade chemicals. In this respect pseudomonads are the most ubiquitous. Pseudomonads are frequently found in surprisingly large numbers in potable waters. Witter (1961) refers to Pseudomonas as the predominant psychrophilic genus and considers that water and soil are well estab-
CONTROL OF BACTERIA I N NONDOMESTIC WATER
109
lished as the natural sources of psychrophilic bacteria. He states that they have been “repeatedly and consistently found in every conceivable water source.” Why are pseudomonads so prevalent in water when sporulating bacteria, which are more resistant, are not usually found in troublesome numbers? This apparently results from the innate ability of many species of Pseudomonas to produce enzymes that enable them to utilize traces of a wide variety of compounds as their sole source of carbon, including compounds ordinarily considered germicidal and those as diverse as thymol, cresols, chlorophenols, and nitrophenols (Tabak et al., 1964). These and many other more readily assimilable sources of carbon, when present in water, yield surprisingly high populations when inoculated with pseudomonads. In addition to being adaptable to diverse sources of carbon, these and some other species grow very well at temperatures slightly above freezing and proliferate to a remarkable degree at 0°C. Ingraham and Stokes (1959) concluded that the ability to grow well at 0°C. is restricted mostly to strains of Pseudomonas, others being mainly Flavobacterium, Achromobacter, and Micrococcus. It is most unfortunate that such levels of growth do occur at temperatures from near 0” to 20°C. because these are the temperatures at which the largest volumes of water are used. For this reason, storage at low temperature cannot be considered an effective means of maintaining a low bacterial count in many waters. OF GROWTH IN WATER B. EXAMPLES
The problems of bacterial growth in water have long been recognized. Baylis ( 1930) stored filtered chlorinated water containing approximately 1 p.p.m. of oxidizable material, which he considered “fairly low when compared with many water supplies,” in Pyrex1 containers at 20°C. Initial total bacterial counts were 10 per ml.; they remained essentially constant for 48 hours, and then increased rapidly. Within 2 to 3 weeks the total counts in most samples exceeded 1,000,000 per ml.; there was no increase in Escherichia coli. Gilcreas and Kelly (1955) reported greater than 100-fold increases in E. coli after periods of storage comparable to those used by Baylis. Nankivell (1911) found that when well water from chalk areas, inoculated with 460 E. coli per ml., was stored, the counts were 165,000 and 280,000 per ml. at the end of the second and fourth days, respectively. Alexander (1944) concluded that many kinds of 1 Mention of commercial products here and elsewhere does not imply endorsement by the Public Health Service.
110
CECIL W. CHAMBERS AND NORMAN A. CLARKE
microorganisms thrive in pipe systems in what might be considered a “perfect” water. When E. coli was inoculated into buffered dilution water and incubated at 35°C. for 48 hours, initial counts of 9 to 20 per ml. increased to as much as 21,000 per ml. (Chambers et al., 1957). Eisman et al. (1949) reported that bacterial counts in excess of 1,000,000 per ml. may occur when deionized water, intended as a substitute for distilled water and initially containing 6,000 to 12,000 bacteria per ml., is stored for 24 hours. Geldreich and Clark (1965) reported pseudomonad counts ranging from 21 to 51 per ml. in distilled water from lines subject to constant use. TABLE I TOTALBACTERIALCOUNTS PER MILLILITERIN DISTILLED WATER STORED AT ROOM TEMPERATURE IN GLASS-STOPPERED PYREXBOTTLE&’
“Spcxial’’ waterc Storage time, days
2 4 6 9
21
27 45 52 120 a
*
0
Freshly drawn laboratory-line distilled water no inoculum
inoculated with 0.1% laboratory-line distilled water
2,400 15,000 39,000 52,000 49,000 44,000 27,000 30,000 13,000
130,000 250,000 280,000 180,000 230,000 210,000 270,000 83,000
2,000
“Special” waterc no inoculum Not counted 700 Not counted 40,000 35,000 37,000 35,000
3,000 Not counted
Reproduced by courtesy of Rhines ( 1965). Membrane filter method, M-Plate Count Broth; incubated 24 hours at 35°C. See text.
Leifson (1962) and Rhines (1965) found a surprising degree of growth when distilled water from an all-glass system is stored. Rhines heated water from a distilled-water line in a building, after adding HBSOl and KMn04, and distilled it into a final retort with venting of 50% of the output. Water from the retort was distilled through a packed column, the bottom and top layers being, respectively, Berl saddles (porcelain) and quartz pebbles. Fifty per cent of the output from the column condensed and was returned to the retort; the remaining 50% was recovered by the Vycor condenser that followed the packed column. Results obtained in growth studies with this water (designated “special”) and distilled water from a laboratory pipe line are presented in Table I. The predominant organisms in all samples were pseudomonads. In general, nearly total
CONTROL OF BACTERIA IN NONDOMESTIC WATER
111
absence of bacteria in distilled water may be the basis for suspecting that the water is toxic (Geldreich and Clark, 1965).
IV. Areas Where Contaminants Multiply A. FILTERS Bacteria often multiply to a remarkable degree in filter beds, ion exchange units, and related percolation-type devices. A number of reasons exist for this, prominent among which is the tendency for traces of particulate and soluble organic matter to be retained on the filter materials by either physical entrapment or precipitation. Continued use, even with waters having low organic content, leads to extensive buildup of such organic matter. Bacteria, especially Pseudomonas and other genera that seem to be present in water almost invariably and that thrive at medium to very low ambient temperatures, may develop to such an extent that the surfaces of sand grains or other materials in the filter bed become coated with bacterial growth. The authors have experienced difficulty with a gravel, sand, and activated-carbon filter used to dechlorinate tap water prior to distillation. After several months’ use it became increasingly difficult to produce a satisfactory water for critical halogen germicide studies. The trouble apparently was due to volatile substances derived from accumulations of organic matter and growths in the filter bed. Periodic drainage, followed by filling and leaching with 50% sodium hydroxide and thorough flushing, relieved the trouble. Distilled water when passed through a glass column filled with activated carbon alone has frequently shown marked increases in bacterial colony count. Pseudomom invariably was dominant and usually was present almost to the exclusion of other genera (Chambers, 1965). Geldreich and Clark (1965) also have referred to growth-promoting materials released from an exhausted carbon filter. Willis (1957) found that anaerobic organisms persist in sand filters and serve as inocula to produce aftergrowths in drinking water. The numbers of these organisms present in chlorinated, filtered effluent leaving the plant were low, but they increased markedly in the distribution system. He suggested that organisms escaping destruction in the plant may be “stimulated by low concentrations of disinfectant, and emphasized the fact that their subsequent survival and multiplication depend on the amount of organic matter present in the water. Alexander (1944) also discussed the problem of aftergrowths.
112
CECIL W. CHAMBERS AND NORMAN A. CLARKE
Ion exchange media of various types constitute one of the greatest potential sources from which bacterial contaminants gain access to a variety of water supplies. These are extremely useful materials, and subsequent comments are not intended in any way as either an indictment or a deterrent to their use. The problems are of particular interest to pharmaceutical manufacturers. Eisman et al. (1949) investigated synthetic cation and anion exchange resins. Their experience indicated that the presence or absence of sizeable numbers of bacteria depended on how frequently the units were used. When freshly regenerated, the units removed most or all of the small numbers of bacteria present in the incoming water; however, when not regenerated frequently, the units became a “culture” medium for bacteria that were flushed out when operation of the unit was resumed. Incoming water containing a maximum of 90 organisms per ml. yielded an effluent that contained from 6,000 to 12,000 organisms per ml. They indicated that when this water was stored for 24 hours it could contain more than 1,000,000 organisms per ml. Satisfactory control of the bacterial content of such waters was maintained by using the units daily and regenerating every 3 or 4 days. They found that predominant organisms were “Pseudornonus-types normally present in fresh water.” Jeffrey and Fish (1964) referred to similar problems with deionizers in which P . aeruginosu and other members of this genus were frequently the cause, and Tennenbaum (1965) reported high counts of gram-negative organisms in deionized water. Fowler et al. (1960) reported problems with reactor cooling water. The water from the ion exchange filter contained 108 bacteria per ml., apparently a species of P s e u d m n a s in “pure” culture. They also considered the possibility that radiation resistance or mutation was a factor, which suggests interesting potential problems in such usage. Proper backwashing and changing the resin produced satisfactory water. In a report on ion exchange softening (Panel Discussion, 1949), the bacterial buildup in many types of resins was considered. Bacterial counts were made at different depths within the exchange layers; as many as 6,000,000 to 10,000,000 viable cells per ml. of exchange material were reported (the panelist considered it unnecessary to reduce the figure to a gravimetric base). Influent counts varied from 15 to 900 per ml.; those in the softened effluents ranged from 13 to 18,000 per ml. The conclusion reached was that, “unless controlled, heavy bacterial growths will develop in all cation-exchange softeners.” Chlorination or treatment with formaldehyde are among the methods suggested for con-
CONTROL OF BACTERIA IN NONDOMESTIC WATER
113
trol. Care must be exercised, however, to select a germicide that is compatible with the resin, otherwise it may be damaged. B. MINIMALFLOWAREAS AND OTHERSOURCES Dead ends in distribution systems are prime locations for development of bacteria. The gradual settling and precipitation of organic matter frequently results in an accumulation of nutrients suitable for the growth of many types of microorganisms. The oxygen depletion together with CO, produced by growth of these organisms leads to conditions suitable for the growth of sulfate-reducing bacteria and other nuisance organisms, thus setting a vicious and somewhat self-perpetuating cycle in motion. Furthermore, chlorine is depleted by “demand-producing substances in sediments in lines subject to sluggish flow. In outlying dead ends of such systems, there frequently is no chlorine, even though the water was originally chlorinated. Bacteria multiply rapidly under such conditions. Baylis (1930) investigated many facets of the aftergrowth problem. The ability of bacteria to multiply in pipe sediment was determined, and very significant levels of growth were reported. He pointed out that in dead ends, sediment accumulation can be so thick that even though the water contains a nominal amount of chlorine there may be a zone of chlorine-free water within the sediment “where bacteria may grow abundantly.” Because of sediment resulting from settling out of dead microorganisms, these problems are greater in waters treated by chlorination without prior flocculation or filtration. Shannon and Wallace (1944) determined that chlorinated or ammoniachlorine-treated water leaving the plant at temperatures above 55°F. had total 20°C. bacterial counts ranging from 42 to 110 per ml., but when sampled in dead-end areas the counts varied from 700 to 20,000 per ml. The problem of bacterial growth in jute packing has long been recognized. Spaulding (1931) found that the coli-aerogenes count of jute packing, based on incubating 0.1 gm. of jute in 100 ml. of water for 24 hours, was 285,000,000 per pound. Additional growth-promoting potential of the jute was indicated by a further five- to ten-fold increase if incubation was continued for 48 hours or more. Jute joints in mains were not sterilized when the line was filled with chlorine sufficiently strong to leave a residual of 180 p.p.m. after standing for 48 hours. Although jute can be sterilized, it is difficult to prevent reinoculation by sediment carried in the water. Calvert (1948) reported that an initial chlorine concentration of 700 p.p.m. was used to treat a repaired main, followed by many more treatments. Satisfactory disinfection was not attained until more than 90 days had elapsed, and then only after a more penetrating
114
CECIL W. CHAMBERS AND NORMAN A. CLARKE
germicide than chlorine was used. Recommendations for disinfection of mains are available (Am. Water Works Assoc., 1948), including procedures for disinfection of vegetable-derivative packing material. The use of joints of impervious materials, where compatible with the installation, apparently would help resolve this problem. Some of the sulfur joint compounds may be suitable although potentially they would be subject to problems with sulfur bacteria. Even rubber has been subject to attack by bacteria (Zobell and Beckwith, 1944; Leeflang, 1963), although Zobell and Beckwith apparently concluded that this was not a serious problem in pipe joints because of the small surfaces exposed. On the other hand, in certain manufacturing or other operations where large rubber surfaces are exposed to water for long periods of time a greater possibility of significant growth exists (Zobell and Beckwith, 1944). Growth of nuisance and other organisms in wells is well documented. Honeywell et al. (1962) investigated problems with Pseudomoms and other genera in wells and dead ends. Redwood storage tanks have been reported to be a source of problems owing to leaching of bacterial nutrients from the wood (Jones and Greenberg, 1964). Jones referred to data provided by the California Redwood Association that show the following percentages of various compounds in the soluble leaching from redwood: Cyclitols, 30; tannin, 25; nontannin phenolics, 18; polysaccharides, 1.5; and simple sugars, 0.5. These data further indicate that “extractives” may constitute 8 to 40% of the dry weight of redwood (heartwood), and that this may be as high as 60% in the surface zone. The growth potential of such compounds is evident from the work of Tabak et uZ. (1964), and many others, relating to bacterial utilization of phenolic compounds. Bacterial degradation of asphalt has been reported by Traxler (1962), and Phillips and Traxler (1963); this may possibly be a factor where asphaltic paints are used. Preservatives and sealers are used in many wooden structures, such as cooling towers, storage vats, and standpipes in industrial operations, and some of these formulations may contribute nutrients that result in bacterial multiplication. Jones and Greenberg (1964) reported that leaching of redwood incident to continued use seemed to resolve the problem after about a year. Much by way of preventive control can be accomplished by judicious selection of materials when new construction is in the planning stage. Many of the examples of bacterial growth presented have occurred in drinking water or water of even higher quality. When such levels of growth occur in potable water, little imagination is needed to visualize the growth potential of many waters of lesser purity that are used in numerous industrial processes.
CONTROL OF BACTERIA IN NONDOMESTIC WATER
115
V. Biological Factors Affecting Control A. BACXERIOPHAGE Bacteriophage probably has been responsible for the reduction of bacterial populations in certain specialized situations, such as the rapid dieoff of Vibrio cholerae in the Ganges River. Beard (1933) concluded that it does not seem possible that bacteriophage is likely to participate significantly in the reduction of bacterial numbers in water or sewage, and Flu (1941) expressed a similar opinion. Thomas (1935) referred to a bacteriophage in water that is virulent for plant pathogens, while Silvey and Roach (1964) concluded “it is likely that the termination of a rather dense population of any one of the microorganisms in fresh water might be caused by a viral invasion.” While there appears to be little question that bacteriophage participates in natural purification, the magnitude or degree to which this activity is significant under practical conditions has not been clearly established. B. ANTIBIOTICEFFECTS Reitler and Seligmann (1957) stated that P. aeruginosa is not rare in waters with relatively low coliform counts. They suggest that low coliaerogenes counts may be due to an “inhibitory” effect of P . aeruginosa on coliforms in water heavily loaded with organic matter. Hutchinson et al. ( 1943) determined the “anti-coliform”activity of strains of Pseudomonas, Actinomyces, Sarcina, Micrococcus, Flavobacterium, yeast, and unidentified non-spore-forming gram-negative rods, in water containing E . coli, and reported a reduction in the numbers of E . coli. Weindling (1956) provides a realistic appraisal of the status of antibiotic effects as follows: “A survey of the literature offers no clear-cut evidence that antibiotics act generally, in nature, like magic bullets that destroy human, animal, and plant pathogens wherever antagonistic activities of microbes occur.”
C. PREDATION From a practical standpoint the part played by predators in controlling or reducing the numbers of bacteria in water is primarily pertinent only where relatively polluted waters are considered. With certain types of treatment, mostly storage under specified conditions, protozoa may feed heavily on bacteria. While many protozoa consume limited numbers of bacteria, significant activity is Iargely confined to certain genera. Members of the genus Oicomonas, for example, are particularly active in this respect; according to Calaway and Lackey (1962) “these organisms are voracious feeders on bacteria.” Butterfield (1929, 1933) reported on the
116
CECIL W. CHAMBERS AND NORMAN A. CLARKE
predatory activities of Colpidium, and Bartsch and Ingram (1959) refer to ciliates as important bacteria “eaters” whose activity can be used to advantage if polluted water can be stored for awhile. Kryiasides (1931) reported that protozoa destroyed V. cholerae and Salmonella typhosa in Berlin tap water in 4 to 6 days. The review of Kidder (1951) provides valuable information on the nutritional requirements of protozoa. Knowledge regarding the degree of pollution associated with different protozoa, especially when considered in relation to the other types of organisms present, can be a useful indicator of the progress of natural purification. In general, however, control of bacteria in these kinds of waters is intimately associated with the engineering aspects of treatment. Accordingly, the role of protozoa and other predators should be studied and understood in relation to the treatment process as a whole.
VI.
Physical Methods of Control
A. TEMPERATURE Little consideration can be given to low-temperature storage as a means of maintaining a small bacterial population in water over any considerable time because of the low-temperature growth characteristics of organisms such as pseudomonads. Freezing markedly reduces bacterial populations, but it is impractical for most purposes; Rahn (1945) indicated that it is impossible to sterilize water by freezing. Heat is an effective and often convenient means of killing bacteria in water. Heating water to 100°C. will quickly kill all vegetative bacteria, and if this temperature is maintained sufficiently long it will kill very resistant spore-formers even though the time required may be excessive; Esty and Meyer (1922) encountered spores of Clostridium botulinum that were killed only after 330-minute exposure at 100°C. In numerous manufacturing operations, such as the production of cosmetic preparations, it is common practice to heat water to destroy bacteria (Tennenbaum, 1965). Pasteurization at 143°F. for 30 minutes suffices for many manufacturing uses. Where time and space are a problem, high temperature pasteurization at 161°F. for 15 seconds should be considered (Goldstein et al., 1960). Construction plans for the unit used by Goldstein have been published (US.Public Health Service, 1959). Seiberling and Harper (1955) reported on a successful pasteurizer design which utilized high-temperature short-time exposure where psychrophilic organisms were present in the city water supply. This design was implemented for use in two dairies having problems with psychrophiles in cottage-cheese wash water; P . frugi was particularly troublesome. Both plants installed heat exchangers for pasteurization of the water at 175°F.
CONTROL OF B A m R L 4 IN NONDOMESTIC WATER
117
with a 20-second holding time. After approximately 10 years of operation, Harper (1965) states that “this system of control has been extremely satisfactory in these plants and continues in use today.” Deindoerfer and Humphrey ( 1959a,b) reported on design principles of continuous sterilizers, and suggested formulas for calculating heat sterilization times in liquids when the temperature varies in a continuous process. In any method of controlling bacteria in water by use of time-temperature combinations, short of those that assure complete sterilization, thennoduric and thermophilic organisms must be considered. Malaney et al. (1962) pointed out the possibility that in dairy operations thermoduric and thermophilic bacteria in the raw water may complicate the use of a continuous-flow water pasteurizer. While control by chemical methods is discussed in Section VII, it is appropriate to consider one aspect of chemical treatment here. In some situations, a gradual buildup of thermoduric or thermophilic bacteria occurs. Under these conditions, periodic substitution of a chemical germicide such as chlorine, in lieu of heat, frequently resolves the problem. Use of chlorine is particularly advantageous for this purpose if a chemical germicide in the water is undesirable from the standpoint of its effect on the product, because it can either be removed with activated carbon or be neutralized with sulfur dioxide or other reducing agents (Am. Water Works Assoc., 1950); the end products of such neutralization yield no detectable tastes or odors.
B. FILTRATION AND FLOCCULATION Filtration is a relatively simple and remarkably effective means of removing bacteria from water. For some purposes, where a nominal reduction in bacterial population is all that is desired, slow or rapid sand filtration provides all the treatment needed. Diatomaceous earth filters are also effective and are commercially available in a variety of sizes and designs. Goldstein et al. (1960) evaluated the efficiency of a slow sand filter in removal of coliform bacteria. The coliform content (M.P.N.) of the influent vaned from 4.6 to 4,600 per ml. whereas that of the effluent ranged from 0.045 to 33 per ml. Gilcreas and Kelly (1955) determined the efficiency of slow and rapid sand filters and reported that rapid sand filtration removed approximately 75% of the bacteria, whereas the corresponding removal by a slow sand filter was about 98%. When flocculated supernatant was passed through the rapid sand filter, removal increased to about 98%. Black and Spaulding (1944) investigated diatomaceous earth filters under a wide variety of conditions. Results varied according to influent water quality and experimental conditions, but, in general,
118
CECIL W. CHAMBERS AND NORMAN A. CLARKE
relatively high levels of bacteria were removed. In one of Black‘s tests an influent raw water having a total 20°C. colony count of 10,OOO per ml. yielded an effluent with a count of 400 per ml. Black‘s work provides information on what can be accomplished with a variety of filter aids with both raw and pretreated (flocculated) water. Almost complete removal of bacteria occurred in some tests with combined filtration and flocculation. Design criteria for rapid sand filters were reviewed recently (Panel Discussion, 1959a), and Robeck et al. (1964) reported results of some new approaches to combined flocculation and filtration. Flocculation without filtration can also be very effective when carefully controlled. Chang et d. (1958) investigated the efficiency of twostage flocculation in reducing both the coliform and total bacterial content of Ohio River water. They varied the temperature, turbidity, and pH of the water, and the dosage and nature of the flocculant. Data obtained in these studies are presented in Table 11. Mallmann and Kahler (1948) presented data indicating that the sludge blanket process, in conjunction with lime-alum and alum-clay flocculation, as used in lime softening, was very efficient in removing bacteria. Langlier and Ludwig (1949) reported detailed data on the chemical and physical mechanisms of flocculation. For specialized uses, water can be made completely free of bacteria by filtering through membrane filters. A limiting requirement, however, is that the suspended solids content of the water be very low; otherwise, the method is impractical because of premature clogging of the filters. Such solids can readily be removed, however, by multistep filtration.
LIGHT C. ULTRAVIOLET Chambers ( 1961) summarized the germicidal properties of ultraviolet light as follows: “The use of this disinfectant is limited to special situations where other methods are not considered feasible. Because nothing is added to the water there is a minimum possibility of taste and odor problems, but no protective residual germicide is provided. “The germicidal effect of U. V. is mostly limited to the output of wavelengths in the vicinity of 2570 Angstrom units. It is a good germicidal agent, but its successful use depends on adequate engineering to assure that sufficient U. V. energy actually reaches the desired point of application. The germicidal efficiency of the tubes decreases with use because the glass gradually loses its transmission efficiency. The light output should be determined at regular intervals with U. V. meters designed for the proper wavelength. The cold cathode tubes have better life expectancy and are less prone to blacken at low temperatures. Tubes should be cleaned frequently.
8
TABLE I1 EFFICIENCY OF TWO-STAGE FLOCCULATION IN REMOVING NATIVEBACTERIA FROM RAWOHIORIVERWATERAT VARYING TE~~PERATURE~.~ Temperature, "C.
Initial turbidity, p.p.m.
25
16-240 1-5
15
140-255 1-5
-
-
5
40-135 1-5 I
Per cent Removal Stage of flocculation
Coliform bacteria
Total bacteria
Final turbidity, p.p.m.
1st 2nd Combined 1st 2nd Combined 1st 2nd Combined
99.8 93.8 99.99 94.4 82.4 99.9 98.8 61.6 99.95
99.8 94.8 99.99 99.3 78.0 99.8 98.7 88.4 99.8
1-5 0.1 0.1 1-5 0.1 0.1 15 0.1-1 0.1-1
3P
Final pH
Floc formation
6.7-7.3 7.3-7.8
Very good Good -
F 2m *z
6.7-7.4 7.3-7.7
Very good
z
6.7-7.4 7.3-7.7
Very good Good
-
-
-
Good
-
-
From Chang et al. (1958). Reproduced from Am. J . Public Health, 48, p. 161, with permission of the copyright holder The American Public Health Association, Inc. b 1st stage: 25 p.p.m. Al,(SO,),; 2nd stage: 25 p.p.m. FeC13. 5
M
3 3 #
2
5
F13
120
CECIL W. CHAMBERS AND NORMAN A. CLARKE
“U. V. light will not penetrate most materials and has only limited ability to penetrate clear water. The water to be treated must be free of turbidity and color. Ordinary glass should not be interposed between the water and light source because it filters out most of the rays of germicidal wavelengths. If immersion type tubes are used, particular care must be taken to remove any deposits that form on the glass.” Huff et aZ. (1965) investigated the use of an ultraviolet light unit for disinfecting water on a ship during a 3-month world cruise under practical operating conditions. The results obtained were later supplemented with data from laboratory studies to provide a more rigorous determination of the effect on waters containing controlled turbidity, color, and iron. Test organisms were E . coli and Aerobacter aerogenes for evaluation of bactericidal efficiency with waterborne vegetative bacteria, while Bacillus cereus spores were used to determine the dosage necessary with resistant nonvegetative forms. Results indicated that the unit produced satisfactory water if the intensity of ultraviolet exposure was maintained above a specified critical level and the designated flow rate was not exceeded.
D. MISCELLANEOUS PHYSICAL METHODS Impoundment and natural purification can improve surface waters considerably, especially with regard to reducing the numbers of pathogens present. Although impoundment improves the bacteriological quality of waters under some conditions, it can result in deterioration of subsurface supplies in certain types of underlying strata. Control, therefore, must sometimes start at a point far from the location of the troublesome growths. When alluvial and other organic deposits accumulate in a reservoir, subsequent decomposition results in high COZ ( H2C0,) concentrations in water filtering through these deposits. Under these conditions Crenothrix and Beggiatoa have sometimes caused problems of such magnitude in waters pumped from wells subject to this type of infiltration that it was necessary to abandon a new well and several tunnels ( Ackerman and Lynde, 1944). Similar problems have resulted from careless surface disposal of organic wastes ( Whipple et al., 1927). Careful selection of sites for sanitary landfills and other waste disposal areas should do much to control this problem. The difficulty with Crenothrix and Gallionella has its origin in the fact that these organisms are generally considered to utilize carbon only from carbon dioxide and to derive their energy from the oxidation of the lower carbonates of iron and manganese (Wilson, 1945). Beggiatoa causes problems in waters containing significant amounts of hydrogen sulfide, and some investigators feel that it may obtain its energy from the oxidation of hydrogen sulfide and grow as an autotroph. In supply lines, the
CONTROL OF BACTERIA I N NONDOMESTIC WATER
121
problem with Beggiatoa is usually confined to “dead ends”; one solution is regular flushing of such areas to clear out the organic matter from which other organisms produce hydrogen sulfide and carbon dioxide. Members of the genus Thiobacillus oxidize sulfur and incompletely oxidized inorganic sulfur compounds. Thiobacillus ferroxiduns oxidizes ferrous iron in streams receiving acid mine wastes and causes red-water trouble when the ferric hydrate precipitates ( Starkey, 1956). Abatement usually consists of mine sealing or other measures to control drainage. When surface waters are a source of supply, placement of multiple intakes at different depths takes advantage of the fact that the organic content of water varies significantly with depth. This condition often varies a t different times within a 24-hour interval (Wilson, 1932). Proper consideration of these factors provides a worthwhile measure of control by reducing available organic nutrients that would support bacterial growth. Baylis (1930) has appropriately said: “It is easy to remove microorganisms and avoid other particles by filtration, but it is not easy to reduce the organic content of many waters to the point where it will not support bacterial growths.” Sphaerotilus is a severely troublesome slime-producer that frequently grows in flowing water; it is an attached form and extracts food from large volumes of water containing extremely low concentrations of nutrients (Amberg et al., 1962). These growths are particularly troublesome from an esthetic standpoint in recreational waters and have caused severe problems in fouling commercial fishing nets. In some instances such problems have been resolved by changing from continuous discharge of sulfite liquor wastes to intermittent release (Amberg et d., 1962). McKeown (1963a,b) reported that scouring by increased flow rates (2.5 f.p.s.) prevents adherence, and he confirmed the value of intermittent discharge; control was also accomplished by withholding essential nutrients. Shannon and Wallace (1944) suggest that breaks in mains, new construction, and cross-connections are a source of “reinfection” with a variety of bacteria. Finally, if adequate volumes of suitable water are available, the most obvious physical control method of all is to combine waters of varying bacterial quality in ratios that provide the maximum yield of water of a quality adequate for the purpose considered.
VII. Chemical Methods of Control A. FACTORS TO CONSIDER IN SELECTING A GERMICIDE Many things influence the efficiency of chemical germicides, Chemical and physical conditions of the water have profound effects. If storage of the water is considered, whenever possible the use of marginal doses of
122
CECIL
w.
CHAMBEHS AND NORMAN A. CLARKE
germicides should be avoided, because with time any chemical germicide applied is subject to some loss of efficiency. Where water is stored before use, such losses may destroy the bactericide or reduce the concentration to a level at which microbial growth occurs. Growth stimulation by sublethal dosage is also a potential possibility. Germicides may be grouped in the following four general classes with respect to their suitability for most water treatment applications : ( 1 ) Those which, at some concentration, are subject to biological degradation. This group includes organic compounds such as phenols, cresols, and chlorophenols. It is important to use these in concentrations sufficiently high to avoid losses resulting from bacterial activity. Pseudomonads adapt quickly to some germicides as a source of carbon. Problems of this nature usually develop after the germicide has been used for a while and the organism’s metabolic “appetite” for the compound gradually increases. ( 2 ) Bactericidal agents for which the organisms develop an adaptive tolerance, without actual biodegradation, and thus withstand exposure to increasing concentrations of a compound. This phenomenon has been observed with some quaternary ammonium compounds ( Q.A.C.) . MacGregor and Elliker (1958) adapted a Q.A.C.-sensitive species of Pseudomonas to tolerate increased concentrations of the same Q.A.C. The increased resistance of the adapted strain over the parent Q.A.C.-sensitive strain was verified in parallel germicide tests in water. Chaplin (1952) adapted a strain of Serratia marcesens, in which growth was originally suppressed by less than 100 p.p.m. of alkyl dimethyl benzyl ammonium chloride, to grow in the same medium containing 100,000 p.p.m. of the test compound. ( 3 ) Compounds which are efficient bacteriostatic agents at relatively low concentrations; many of these are also germicidal with increased exposure time or concentration factors. The heavy-metal germicides such as silver ( McCulloch, 1945) are frequently bacteriostatic, although they are by no means the only ones. For some industrial uses, bacteriostatic agents are very useful because they inhibit growth. Bacteriostatic formulations are particularly good if inhibition of further increases above the levels present at the time of treatment is all that is desired. For example, cooling waters may contain bacteria in numbers not considered troublesome. If not controlled, however, these organisms serve as inocula to produce undesirable slimes in the system. For such purposes a bacteriostatic agent is an excellent choice, especially one that retains its effectiveness in the presence of an increasing buildup of organic matter in water that is recirculated. ( 4 ) Germicides without readily demonstrable bacteriostatic effects.
CONTROL OF BACTERIA IN NONDOMESTIC WATER
123
The halogens, chlorine, iodine, and bromine and other active oxidizing agents are the main members of this group. These bactericides are usually selected when a very rapid kill is desired. Some, such as chlorine dioxide and ozone, are excellent choices in some food processing operations where undesirable chlorophenol tastes and odors resulting from chlorination are a problem. General factors to be considered in selecting a germicide are toxicity, convenience, and suitability for the overall operation. Combining several bactericidal agents in a single formulation sometimes increases efficiency. In some usages, the inhibitory effect of a water treatment germicide, especially a good bacteriostatic agent, interferes with the growth of desired organisms. Finally, it is highly desirable, though not always attainable, to select a germicide for which a simple chemical test is available for quantitatively determining the amount of residual germicide present in the water. The test should measure the amount of active agent present in terms of germicidal effectiveness in the particular water used. For example, the starch iodide test for chlorine does not differentiate between free available chlorine and combined available chlorine, yet the difference in germicidal effectiveness of these two forms of chlorine is 25- to 100-fold ( Butterfield and Wattie, 1946).
B. CHLORINATION 1 . Background Information Because much more water is treated with chlorine than with any other germicide, it is particularly important to understand the fundamental behavior of chlorine. The germicidal factor in free available chlorine is hypochlorous acid ( HOCl) which is formed according to the following reaction when chlorine is added to water: Clz HzO $ HOCl H+ C1- (Fair et al., 1948). They point out that under practical operating conditions this hydrolysis of chlorine is essentially complete; measurable molecular chlorine (Cl,) is not produced unless the pH is below 3.0 or the chlorine concentration is 1000 p.p.m. or more. Free available chlorine can be derived from either liquid chlorine or hypochlorites. The HOCl ionizes in water as follows: HOCl s H + OC1-; the OC1- ion is essentially nongermicidal. The bactericidal efficiency of both free available and combined available chlorine (chloramines) is directly related to the pH of the water. Increasing the pH reduces germicidal activity; the effect of pH on free available chlorine is shown in Figure 1. The amount of free available chlorine as HOCl decreases as the pH increases (Table 111). A comparison of results presented in Figure 1and Table I11 reveals the relation-
+
+
+ +
124
CECIL W. CHAMBERS AND NORMAN A. CLARKE
ship between germicidal efficiency and the amount of free available chlorine present as HOC1. Breakpoint chlorination is the most effective germicidal technique. According to Butterfield ( 1948a) increasing increments of chlorine (Cl,)
CI, EXPOSURE TIME 5 min. CHLORAMINE 2 0 min.
CHLORAMINE pH 7.0
CHLORINE, ppm
FIG. 1. The bactericidal effect of free and combined available chlorine on
Pseudomonas aeruginosa. From Buttefield et al. ( 1943, 1946). Reproduced courtesy Public Health Rept. (US.)
added to water containing 0.9 p.p.m. of ammonia nitrogen, under the conditions indicated in Figure 2, result in corresponding increases in combined available chlorine recoverable by test until the point designated as the “hump” is reached and there is no change in the ammonia nitrogen concentration. After the “hump” is attained, adding chlorine reduces the concentration of both combined available chlorine and ammonia nitrogen. When approximately 9.0 p.p.m. of chlorine has been added, the
125
CONTROL OF BACTERIA IN NONDOMESTIC WATER
water contains no residual chlorine or ammonia nitrogen. This is the “breakpoint.” The chlorine residual present after “breakpoint” is initially free available chlorine. In most waters, free available chlorine is gradually lost by slow continued reaction with traces of residual oxidizable TABLE I11 PERCENTAGE OF FREECHLORINE AS HOCP
PH
HOCI, %
4 5 6 7 8 9 10 11
100 99.7 96.8 75.2 23.3 2.9 0.30 0.030
Free chlorine/ p.p.m. HOCI, p.p.m. 1.000 1.003 1.033 1.33 4.3 34 331 3,300
a From Fair et al. (1948, p. 1052). Reproduced from J. Am. Water Works Assoc., 40, p. 1052, with permission of the copyright holder American Water Works Association.
0.9
0.7
z
4 z
0 2 1.5
z
6 -I
6 3
e
W v) ).3
n
‘.I
CHLORINE ADDED, ppm
FIG.2. Ideal residual chlorine curve (ammonia solution). From Butterfield ( 1948a). Reproduced from J . Am. Water Works Assoc. 40, p. 1306, with permission of the copyright holder American Water Works Association.
126
CECIL W. CHAMBERS AND NORMAN A. CLARKE
material not destroyed in the breakpoint process. This characteristic of free available chlorine must, therefore, be weighed against the greater stability of chloramines in deciding which is the more suitable for use in a given situation. Hazey (1951) presented data showing the wide variations in chlorine demand that occur in water on an hourly basis over a 24-hour period. Chlorine ammonia treatment, resulting in the formation of chloramines, is frequently used instead of or in combination with free available chlorine. Because chloramines are a combined form of chlorine, a unit of chlorine as chloramines is not germicidally as efficient as a unit of free available chlorine. Why then consider chloramines? The reason is that because of the continuing chlorine demand of reducing substances in many waters, which continue to react with chlorine after the initial demand has been satisfied, it is difficult to maintain a free available chlorine residual for very long. It is frequently advisable to kill the bacteria initially with free available chlorine, and then apply chloramine to provide some residual of germicidal agent that will continue to act over a longer period of time. The following reactions depict the formation of chloramines from hypochlorous acid and ammonia:
--
NH, + HOCl NH,Cl + HOCl NHC1, + HOCl
+ + +
NH,Cl H,O NHCl, H,O NCI, H,O
These reactions are pH and temperature dependent (Fair et al., 1948). Wyss (1956) considered the question of adaptive tolerance to chlorine and concluded that “although coliform organisms have been subjected to killing by chlorine for over 40 years there is no evidence that there has been any adaptation to great tolerance.” He concluded that “the last survivors are chance escapees of the lethal process rather than more resistant organisms.” Farkas-Himsley ( 1964), however, reported the isolation of chlorineresistant mutants of E . coli which, in tests at 30°C., survived 10 minutes’ exposure to 20 p.p.m. of chlorine at pH 7.2. This unusually extended survival, however, as well as other findings reported, raises serious questions regarding the methods and procedures used in this study. 2. Practical Use of Chlorine Butterfield et at. (1943; Butterfield and Wattie, 1946) evaluated the effectiveness of free available chlorine and chloramine. Figure 1 shows the effect of variations in pH on the survival of P . aeruginosa exposed to free available chlorine. Results obtained with chloramine at a very
CONTROL OF BACTERIA IN NONDOMESTIC WATER
127
favorable pH (7.0) are included for comparison to demonstrate the superior germicidal activity of free available chlorine. Figure 3 shows the effect of free available chlorine on P. aeruginosa, E. coli, and S. typhosa at an unfavorable pH (9.8). The effectiveness of chlorination in the control of coliform organisms is particularly imporI oc
i I
I
I
I
I I 2Oo-25OC
I
I
9c
8C
7C
A
." >
6C
P I3 In 50 t 2
W V
a
2 40 30
20
10
0
FIG. 3. The bactericidal effect of free available chlorine on Pseudornonas aeruginosa, Escherichia coli, and Salmonella typhosa. From Butterfield et al. (1943). Reproduced courtesy Public Health R e p . ( U . S . ) .
tant in water used for paper and pulp manufacture (Sandborn, 1944). Pseudomonas aeruginosa is one of the more resistant vegetative forms frequently found in large numbers in waters that, except for the presence of these organisms, would be considered very pure. Various personal
128
CECIL W. CHAMBERS AND NORMAN A. CLARKE
communications as well as the data in Table I indicate that it is a very common contaminant in distilled water. Olson et aL (1955) referred to probIems with psychrophiles and recommended chlorination of water used to manufacture dairy products. Hays et al, (1963) pointed out the advantages of acidification of chlorine-treated waters for control of Pseudomonas fluorescens, P. fragi and Alkaligenes metalkaligenes in food-plant water supplies. Effective chlorine concentrations were established for each full pH value from 5 to 11 inclusive, As indicated before, where high chlorine residuals are objectionable in food processing, the water can be dechlorinated with sulfur dioxide (Alexander, 1944) or other chlorine removal agents (Am. Water Works Assoc., 1950). Blair ( 1954) described successful procedures for control of Crenothrix in distribution lines. Control of slimes with chlorine is not recommended for cooling systems because of excessive chlorine losses resulting from aeration and sunlight ( Nason, 1938 ). Sandborn ( 1944) analyzed slime growths from 340 pulp and paper mills and reported that in 52% of the plants coliform bacteria were either prevalent or the principal members of the slime flora; Aerobacter was the principal slime-forming genus in 94% of 175 samples examined for coliform bacteria. He concluded that E. coli, Alkaligenes fecalis, P. fluorescens, and Pseudomonas viscosa are often controllable with chlorine residuals of from 0.4 to 0.8 p.p.m. which is comparable to the findings reported by Butterfield et al. (1943) in Figures 1 and 3, although more than 1.0 p p m . was necessary to control the more resistant mucoid variants. Dead ends, elbows, and pockets in systems sometimes present problems and may necessitate the use of germicides that can penetrate slime. Honeywell et al. (1962) reported good success in eliminating pyrogens, attributed to Pseudomonas and other genera, from well waters by chlorination of the wells. The problem did not recur, and as an added bonus productivity of the wells improved markedly. Harris (1960) described several methods for chlorinating wells, lines, and tanks to eliminate bacterial contamination. A broad general discussion of the value and limitations of chlorine was presented in a panel discussion (1959b). Griffin (1947) suggested that about 2.0 p.p.m. of chlorine will “breakpoint” most waters that are relatively free of pollution. He considered the prime capability of the process to be its ability to destroy all types of waterborne bacteria. Guidelines designed to provide an adequate margin of safety in germicidal treatment with free and combined available chlorine have been published by Butterfield ( 1948b) and Snow (1956). If the cysticidal recommendations
CONTROL OF BACTERIA IN NONDOMESTIC WATER
129
of the latter are followed, a wide margin of safety should be provided for vegetative bacteria in all but the most difficult situations. C. OTHEROXIDIZING AGENTS 1. Chlorine Dioxide
This compound is a particularly potent germicide and has been reported to be more lethal than chlorine for both vegetative bacteria and spores. Ingols and Ridenour (1948) concluded that, in contrast to chlorine, it does not react with ammonia. The effect of pH is the opposite of that with chlorine; increased pH apparently enhances the effectiveness of chlorine dioxide, whereas lowered pH lessens its effectiveness (Benarde et al., 1965). Ridenour and Armbruster (1949) determined the germicidal activity of chlorine dioxide on species of Shigella, Salmonella, Staphylococcus, Pseudomonas, and Aerobacter at pH 7.0 and 9.5, and at temperatures of 5" and 20°C. The nature of its oxidizing properties makes it particularly attractive for destroying the organic content of water. Phenolic tastes and odors resulting from chlorination of many waters are a problem in waters used in some food processing operations. These are avoided with chlorine dioxide. Welch and Folinazzo (1959) reported that it is particularly effective in controlling bacterial growths and slime formation in water used in pea and corn canning operations. The generation and use of chlorine dioxide, and equations relating thereto, have been presented by Granstrom and Lee (1957, 1958). Although the usual method of treatment with chlorine dioxide is somewhat more difficult than that with chlorine, especially on a small scale with limited personnel resources, a stabilized product containing 5% chlorine dioxide in water is now available (Lacy, 1963).
2. Bromine and Iodine Although excellent germicides, bromine and iodine probably have limited application for the purposes considered in this discussion. Like chlorine, they hydrolyze, forming hypoiodous and hypobromous acid, both of which are highly germicidal. As with hypochlorous acid, increasing pH decreases germicidal efficiency. Iodine does not produce iodamines under ordinary conditions. Bromamines can be produced, but they differ from chloramines in that they have about the same germicidal efficiency as the parent element. Bromamines and free available bromine cannot coexist more than momentarily at pH levels usually found in water (Johannesson, 1960). The factors governing the use of bromine have
130
CECIL W. CHAMBERS AND NORMAN A. CLARKE
been presented by Brooke (1951), Johannesson (1960), and Goodenough (1964). The germicidal properties of iodine have been described by Chambers et al. (1952), and Chang and Morris ( 1953) have recommended iodine dosages that should be adequate to cover the most difficult situations. The use of iodine for treatment of small water supplies has been suggested by Chang (1966) as being more practical than chlorination; he also described a simple automatic dosing device.
3. Ozone Ozone is a very powerful oxidizing agent, but it leaves no lasting protective residual in the water. In a review, Hann (1956) indicated that it has good bactericidal potency and is effective in removing tastes and odors. Turbidity interferes with the germicidal action. Iron and manganese react to consume ozone and produce insoluble precipitates that necessitate the filtration of water for most uses if the iron or manganese concentration exceeds 0.2 p.p.m.
D. SILVER Silver is available in a variety of forms for use in water treatment. In addition to being bactericidal, it is one of the most highly bacteriostatic agents available (McCulloch, 1945). It has a pronounced tendency to adsorb on surfaces, and this may have advantages for some industrial uses where slimes are a problem. It is relatively susceptible to interfering substances in water. Zimmerman (1952a,b) evaluated many of the factors affecting the germicidal activity of low concentrations of silver. He concluded that the water must be clear and free of impurities if satisfactory disinfection is to be attained. The silver apparently is retained on the bacterial cell wall and does not diffuse into the cell. The action of the silver seems to block reproduction, and a markedly bacteriostatic effect was reported. Wuhrmann and Zobrist (1958) also reported studies on silver in natural waters and determined the effects of various interference factors. Evaluation of the germicidal efficiency of low concentrations of silver has suffered from lack of analytical methods. In a recent laboratory study this problem was resolved by using Ag"Om in conjunction with a scintillation counter to secure basic information on germicidal activity and adsorption phenomena (Chambers et al., 1962). Synthetic or distilled water was used to avoid or minimize interferences. Under these conditions a modified rhodanine test worked reasonably well, but with natural waters more interference with this chemical test was encountered. The
CONTROL OF BACTERIA IN NONDOMESTIC WATER
131
general problems encountered in the use of silver for disinfection of water are reviewed by Woodward (1963).
E. QUATERNARY AMMONIUM COMPOUNDS Quaternary ammonium compounds (Q.A.C.) are surface active agents and are good germicides for some purposes. Gram-positive bacteria tend to be more susceptible to Q.A.C. than do gram-negative organisms. They have marked bacteriostatic properties which are especially apparent with thermoduric micrococci and spore formers ( Scarlett, 1962). Q.A.C. are susceptible to interference in hard water (Chambers et al., 1955; TABLE IV EFFECTOF HARDNESS ON 50 P.P.M. OF ALKYL DIMETHYL BENZYLAMMONIUM CHLORIDEa
"/o E. coli surviving Compounds added8 MgSO4 MgC4 Mg(HCO, 12 CaSO, CaC12 c a ( HCO,) NaHC0,C a
-
PH
at 22°C. after
Initial
Final
2 min.
5 min.
10 min.
8.1 8.9 8.3 8.2 8.3 7.4
7.3 8.2 8.3
85 90 11 81 92 77 0
22 24 0.85 49 68 48
0.076 0.10 0.0003 2.3 6.6 8.9
7.5
7.5 8.0 7.4 7.9
From Chambers et ul. (1955, p. 548). Reproduced courtesy Public Health Rept.
(US.).
1, All solutions are equivalent to 375 p.p.m. CaCO, in distilled water except soft water control for NaHCO, which contains bicarbonate ion concentration equivalent to that in the tests with Ca( HCO,), or Mg( HCO,),. Data from soft water control tests not tabulated in Chambers et al. ( 1955).
Cousins and Clegg, 1956; Scarlett, 1962). Different compounds vary considerably in this respect; some of those susceptible to hardness interference are thoroughly effective in soft water, as shown by the trends presented in TabIe IV. With a few exceptions, most bactericidal studies of Q.A.C. have been conducted with relatively high concentrations of the active agent. Parker et al. ( 1953), however, compared alkyl dimethyl benzyl ammonium chloride and chlorine using both germicides at a concentration of 15 p.p.m., and Shay et al. (1965) recently evaluated four different Q.A.C. at a concentration of 7 p.p.m.
F. EXCESS LIME The germicidal effect of hydroxyl (OH-) ions is sufficient to kill most vegetative bacteria. If plant processes are such that soft water is de-
132
CECIL W. CHAMBERS AND NORMAN A. CLARKE
sirable, lime softening may be utilized to take advantage of bacterial reductions resulting from the effect of lime floc and high pH. Wattie and Chambers (1943) and Riehl et at. (1952) determined the germicidal efficiency of excess lime. The exposure times required to kill P . aeruginosa
0
I
2
3 4 TIME IN HOURS
5
6
FIG.4. The bactericidal effect of hydroxyl ions on Pseudomonas aeruginosa. From Wattie and Chambers (1943). Reproduced from J. Am. Water Works Assoc. 35, p. 714, with permission of the copyright holder American Water Works Association.
with excess lime at different pH’s are shown in Figure 4. Mallmann and Kahler (1948) reported remarkably good removal of bacteria by the floc produced in lime softening.
G. MISCELLANEOUS CHEMICAL METHODS The high microbiocidal properties of sodium pentachlorophenate have long been recognized for effectiveness in controlling slime fornation
CONTROL OF BACTERIA IN NONDOMESTIC WATER
133
(Nason, 1938). He indicated that it is stable on aeration and is not volatilized in spray systems, and that corrosion problems are minimized because it does not break down in alkaline waters. Sandborn (1944) evaluated 125 compounds for control of slime-forming coliform bacteria. He concluded that trichlorophenate fractions, metallic salts of chlorophenols, chlorinated isopropyl phenols, and alkyl derivatives of halogenated phenols are effective. Zabel and O’Neil (1957) determined the inhibitory concentrations of 44 arsenical compounds for A. aerogenes and Bacillus mycoides. Lederer and Delaney (1960) conducted field studies in paper mill “white” water and confirmed the effectiveness reported in laboratory evaluations of several hundred organomercurials. They found di ( phenylmercuric ) ammonium salts to be highly toxic and di( phenylmercuric ) -ammonium propionate to be one of the most powerful antimicrobial agents yet tested against the common slime-forming organisms. Many of these miscellaneous compounds have wide utility in industrial applications. Stability in the presence of organic matter is a desirable attribute of many of them. Numerous compounds in the group are toxic to man, however, and this must be considered in judging their suitability for a given use.
H. PROBLEMS OF ANALYTICAL CONTROL 1. Chlorine Four tests are commonly considered for the determination of residual chlorine. The starch iodide test is generally used only for relatively high concentrations of chlorine. The sample is acidified, iodine being released by the addition of potassium iodide. The iodine is then titrated with a standardized solution of sodium thiosulfate by use of a starch indicator. This method measures total chlorine present and does not differentiate between free available and combined available chlorine. Its utility is further limited by its Iack of specificity for chlorine; other oxidants may also release iodine under the conditions of the test. The ortho-tolidine test, if read quickly as a “flash test, can be used to determine free available chlorine at concentrations as low as 0.1 p.p.m. Color that develops slowly is due to combined chlorine. The ortho-tolidine-arsenite test, when carefully applied, can be used to determine the respective amounts of free available chlorine, combined available chlorine, and false color resulting from interfering substances in the water. The most accurate method for determining the concentrations of the various forms of chlorine, at levels used in water treatment, is by means of the amperometric titrator. Interfering substances are less of a factor
134
CECIL
W.
CHAMBERS AND NORXfAN A. CLARKE
with this test than with the other three. This method can also be used to differentiate monochloramine and dichloramine. “Standard Methods for the Examination of Water and Wastewater” (Am. Public Health ASSOC., 1965) is an excellent source for information on the routine use of these tests and includes a bibliography of original references. 2. Chlorine Dioxide Many of the characteristics of chlorine dioxide in water are not well understood. The reason for this is that there is at present no completely satisfactory test for determining low residuals of chlorine dioxide in water. This has hampered the use of this excellent water-treatment germicide. The problems in determining chlorine dioxide residuals have been thoroughly reviewed by Feuss ( 1964 ) . 3. Silver
The concentration of silver in water, at practical use levels, is difficult to determine. Interferences or failure to recover the silver quantitatively are serious problems. Chambers and Proctor (1960) investigated analytical methods for determining silver and encountered severe problems in all of the tests used. Where the quality of the water and the form of silver permit its use, the spot test reported by Renn and Chesney (19531956) has certain practical advantages.
4. Quuternay Ammonium Compounds In the past, maximum economy in the use of Q.A.C. has been hampered by lack of an adequate chemical test that shows the amount of germicidally effective Q.A.C. in the presence of interference resulting from hardness or organic material in the water. Law et al. (1964), however, report a chemical test that shows reductions in Q.A.C. residual resulting from the addition of organic matter that, to a reasonable degree, parallel losses in germicidal efficiency as determined by bactericidal tests. This test apparently does not reflect interference due to hardness because 18 p.p.m. of a Q.A.C. resulted in zero survival in 30 seconds in distilled water, while more than 200 p.p.m. was required to secure a comparable result in hard water; the chemical test reflected the amount of Q.A.C. originally added to both waters.
5. Iodine and Bromine Conditions governing tests for residual iodine in water have been presented by Kramer et al. (1952); they concluded that the amperometric test was the best. Johannesson (1960) reviewed tests for residual bromine and considered amperometric titration superior to other tests available.
CONTROL OF BACTERIA IN NONDOMESTIC WATER
135
Palin (1961) described colorimetric tests that are adapted for use with bromine.
VIII. Economics of Control Where large volumes of water having characteristics compatible with chlorination are to be treated, liquid chlorine unquestionably provides the least expensive germicidal protection. Purely on the basis of cost of material, it is followed closely by liquid bromine and calcium hypochlorite. Cost comparisons based solely on the value of materials used, however, can be very misleading. Such factors as convenience for a specified use, compatibility with interfering substances in the water, effect on marketability of the product being processed, amortization of initial outlay for dosing mechanisms, and expenses in terms of time allocated for operation and maintenance of equipment should be investigated. All these factors must be evaluated in relation to each other before a knowledgeable estimate of the true cost of a unit of applied germicidal activity can be provided. Once these factors have been established the cost of some of the more widely used germicidal materials becomes relevant. The basic costs of ultraviolet light and ozone are governed by electric rates in a given area. The ultraviolet-light unit used by Huff et al. (1965) has an output of 13.8 watts and a rated capacity of 500 gallons per hour. Hann (1956) considered ozone to be relatively inexpensive, but not as economical as chlorine. He calculated the power requirement at 10 to 15 kilowatt hours per pound of ozone produced, depending on the size of the installation. Chambers (1961) calculated the cost of a p.p.m. of each of the following germicides in terms of the price of 1.0 pep.”. of applied molecular chlorine as liquid chlorine at $0.0625 per pound, multiplied by the factor following the respective germicide?: Liquid bromine, 3.5; chlorine dioxide (from NaC102 and CIz), 13; iodine as metallic iodine, 18; and silver, 233. Because of the various modes of application, the cost of using silver probably varies more than that of any of the other germicides considered. Many of the commercial formulations are prepared by the user as a concentrated stock solution that is introduced into the water periodically as a “slug” or allowed to trickle in intermittently or continuously. Some are available as briquettes or slow-dissolving pellets, which are suspended in the water to become “self-dosing.” The cost of these dosing mechanisms is relatively small. The cost ratios of a p.p.m. of some representative formulated materiaIs 2
Based on prices to industrial users at bulk rates,
136
CECIL W. CHAMBERS AND NORMAN A. CLARKE
compared with 1.0 p.p.m. of liquid chlorine are: Ca( OCI),, 7.03; sodium pentachlorophenate, 5; sodium orthophenylphenate ( tetrahydrate), 8; alkyl dimethyl benzyl ammonium chloride, 72; and 2-bromo4-hydroxyacetophenone, 80. It is reemphasized that there are great differences in the relative efficiency (use concentrations) of different germicides. In many instances, what appears to be an expensive formulation may be the most economical when all factors are considered. Goldstein et al. (1960) reported an average power consumption of 37.7 kilowatt hours per 1000 gallons of water treated with an electric rapid pasteurizer operated at a rate of 250 to 270 gallons per day. The cost would have been lower if operation had been continuous because there was considerable heat loss in starting.
IX. Evaluation of Effectiveness of Control Methods BACTERIOLOGICAL CRITERIA 1. Sample Collection Bottles for sample collection should be free of toxic agents. Silver (Chambers et al., 1960, 1982) is adsorbed by glass and is not effectively removed by ordinary rigorous cleansing methods. Some of the silver is subsequently released and may be bacteriostatic. This can result in errors in later samples collected in the same bottles; it can be particularly important in industrial waters where disinfectant concentrations may be relatively high. If such germicides are investigated in the laboratory, glassware should be segregated to avoid interference with other work. When water being sampled is treated with a chemical germicide, all sampIe bottles should contain a nontoxic neutralizer that will instantly arrest both bactericidal and bacteriostatic action. Unfortunately, satisfactory neutralizers are not available for some disinfectants. The time between sampling and bacteriological analysis should be minimal; if samples cannot be examined quickly, they should be stored in ice.
2. Bacterial Count The total bacterial colony count is probably the best method for determining the bacterial content of many industrial waters. Exceptions would be in situations where differential counts with special media yield information regarding species of unusual significance to a particular As equivalent HOCI. When liquid C1, is added to water half the chlorine is lost HC1: C1, + H,O $ HOCl H + + C1-. When Ca( OCl), is added to water there is no loss: Ca(OCI), + Ca++ 2OC1-; when the OC1- ions combine with H + ions from water the result is H + OCI- $ HOCl (Fair et al., 1948)3
as
+
+ +
CONTROL O F BACTERIA IN NONDOMESTIC WATER
137
process. In certain situations, microscopic examination may provide qualitative clues to troublesome problems caused by certain slime-forming organisms. Colony counts may reflect only a small percentage of the organisms surviving treatment. Black and Spaulding ( 1944) showed count differences as great as 100-fold in parallel inoculations of filtered raw water when agar plates were incubated at 20” and 37°C. Jewel1 (1942) considers that the standard plate count may represent only a fraction of the surviving organisms, and Favero et al. (1964) pointed out the importance of extending incubation time beyond the usual 24 to 48 hours under some conditions. Many organisms causing serious problems grow only on special media incubated under specified conditions; even then, the colony count may bear little relation to the practical problem. Crenothrix and Sphaerotilus are good examples of this, as are anaerobes and actinomycetes. Colony counts of such genera as Aerobacter, Escherichia, and Pseudmonas may provide a reasonably accurate indication of the bacterial content of the water. With these and other organisms the membrane filter technique, where applicable, should be considered for its ability to give an earlier warning of impending problems. 3. Coliform Zndex
The coliform index is valuable for determining the extent to which members of the coli-aerogenes group are controlled. The test is certainly significant whenever the index is high. A low or negative index could be misleading if interpreted as indicating a water of low total bacterial content. Such a finding may merely reflect conditions unfavorable to coliforms and represent a shift in microbial populations. Silvey and Roach (1964) indicated that if there is not much pollution in the water, the gram-negative genera will be mostly Alkaligenes, Aerobacter, and Pseudomonas; if sewage is present, Escherichia and associated types increase. McCulloch (1945) pointed out that when P . aeruginosa, which he refers to as an “active antagonist,” is present in water, it is sometimes unaccompanied by other bacteria. 4. Miscellaneous Procedures
In some manufacturing processes where water is recirculated, total bacterial colony counts are useful on samples taken at regular intervals of one to several hours, when the counts are correlated with simultaneous chemical tests for residual germicide. These analyses can reveal, under actual operating conditions, the minimum effective concentration of germicide. This information can provide the basis for determining the most economical use of the bactericidal agent; added savings are reflected in some operations in reduced frequency of “clean-ups.”
138
CECIL W. CHAMBERS AND NORMAN A. CLARKE
In some processes, evaluation of a variety of germicides in the same water as that used in the plant leads to the selection of the best germicide. Cousins and Clegg (1956) recognized the problems of hardness with Q.A.C. and pointed out the desirability of bacteriological tests to evaluate water treatment germicides under use conditions. The test reported by Chambers (1956) is well suited to such purposes. In addition, where suppression of growth provides the level of control desired, determination of satisfactory bacteriostatic concentrations may be all that is necessary. For such tests, the lack of an adequate neutralizer is not critical. It is advantageous to use test organisms recently isolated because there are some indications that these are more resistant than stock strains (Heathman et al., 1936; Riehl et al., 1952) and are therefore more representative of actual operating conditions. The test can be modified for use with organisms naturally present in the water. If this is done, vigorous agitation of the test water should be maintained to avoid “pooling of the concentrated stock germicide at the point of addition.
X. General Comments and Conclusions It is readily apparent that remarkedly pure water has considerable potential as a bacterial medium. Some bacteria, particularly Pseudomonas and related genera, are highly adaptable, require little in the way of nutrients, and grow very well at temperatures near 0°C. Preventative control consists of keeping organic content to minimum levels. This includes measures to prevent decomposition of organic matter in impoundments or in surface or subsurface disposal areas, which can deplete the oxygen content and increase the COS (H2C03) content of water percolating into subsurface strata. This leads to conditions that result in the growth of Crenothrix, Gallionella, sulfate-reducing bacteria, and other nuisance organisms. Some physical methods of control are remarkably effective, particularly filtration and coagulation or a combination of both. Impoundment, controlled flow, and dilution or diversion of flow of waters containing H2S or forms of iron that support the growth of nuisance bacteria are also important control procedures. Heat, properly applied, probably is the one infallible method of completely killing all organisms in water. In chemical control, one of the most important factors is the correct selection of a germicide to match the water use anticipated. A mixed formulation of germicidal agents is advantageous for some purposes. Where thermophilic or thermoduric organisms are a problem, a chemical germicide may be a better choice than heat. Adaptive resistance to some chemical germicides does occur. Residual bacteriostatic properties may
CONTROL OF BACTERIA IN NONDOMESTIC WATER
139
be a disadvantage for some usages, but advantageous for others. Indiscriminate overdosing with a chemical germicide is economically unwise and, in the case of bactericidal agents that are relatively immune to organic demands, may interfere with oxidation of wastes when subsequently discharged to waste disposal units, especially if they are small. In the use of the halogen elements and chlorine dioxide, a thorough understanding of their reactions with chemicals in the water is particularly important. Selection of the correct test for determining residual germicide is likewise critical. With chlorine, in particular, the form of the residual is of the utmost importance. Finally, in research with free available chlorine, the water must be devoid of chlorine demand. The amount of free available chlorine must be verified in the test water initially, before the bacteria are added and, again, after the bacteria have been added, at the time the last sample is withdrawn for determination of survival. A parallel test, without bacteria, must be included to verify the absence of chlorine demand in the test water or glassware and to provide a basis for comparison with the bactericidal test to determine how much, if any, chlorine demand was attributabIe to the test organisms added.
REFERENCES Ackerman, T. V., and Lynde, E. J. (1944). 1. Am. W d e r Works Assoc. 36, 315-322. Alexander, L. J. (1944). J . Am. Water Works Assoc. 36, 1349-1355. Amberg, H. R., Cormack, J. F., and Rivers, M. R. (1962). Tappi 45, 770-779. Am. Public Health Assoc. (1965). “Standard Methods for the Examination of Water and Waste Water,” 12th ed., pp. 91-112. Am. Public Health Assoc., New York. Am. Water Works Assoc. (1948). 1. Am. Water Works Assoc. 40, 131-138. Am. Water Works Assoc. (1950). “Water Quality and Treatment,” 2nd ed., pp. 221222. Am. Water Works Assoc., New York. Bartsch, A. F., and Ingram, W. M. (1959). Public Works 90, 7, 104-110. Baylis, J. R. (1930). Water Works &. Sewerage 77, 335-338. Beard, P. J. (1933). J. Infect. Diseases 52, 420-426. Bejuki, W. M. (1961). In “Developments in Industrial Microbiology” ( S . Rich, ed.), 2, pp. 263-270. PIenum Press, New York. Bejuki, W. M. (1965). “Pseudomonas: Its Ubiquity and Role in Industrial Microbiological Test Procedures.” Presented in Analytical Microbiology Group Round Table, Am. SOC.Microbiol., Atlantic City, New Jersey, 1965. (Mimeo. ) Benarde, M. A,, Israel, B. M., Olivieri, V. P., and Granstrom, M. L. (1965). Appl. Microbiol. 13, 776-780. Black, H. H., and Spaulding, C. H. (1944). J. Am. Water Works Assoc. 36, 12081221. Blair, G. Y. (1954). 1. Am. Water Works Assoc. 46, 681-683. Brooke, M. ( 1951 ). 1. Am. Water Works Assoc. 43, 847-848. Burgwald, L. H., Paxton, J. A., and Gould, I. A. (1952). Milk Dealer 42, 2, 50. Burkholder, W. H. (1948). Ann. Reu. Microbiol. 2, 392.
140
CECIL W. CHAMBERS AND NORMAN A. CLARKE
Butterfield, C. T. (1929). Public Health Rept. ( U S . ) 44, 2865-2872. Butterfield, C. T. (1933). Public Health Rept. ( U . S . ) 48, 814-818. Butterfield, C. T. ( 1948a). J. Am, Water Works Assoc. 40, 1305-1312. Butterfield, C. T. (194813). Public Health Rept. ( U . S . ) 63, 934-940. Butterfield, C. T., and Wattie, E. (1946). Public Health Rept. ( U . S . ) 61, 157-192. Butterfield, C. T., Wattie, E., Megregian, S., and Chambers, C. W. (1943). Public Health Rept. (US.)58, 1837-1866. Calaway, W. T., and Lackey, J. B. (1962). “Florida Engineering Series,” No. 3, p. 25. Univ. of Florida, Gainesville, Florida. Calvert, C. K. (1948). J. Am. Water Works Assoc. 40, 125-130. Chambers, C. W. (1956). J. Milk Food Technol. 19, 183-187. Chambers, C. W. (1961). Zllinois Univ., Agr. Erpt. Sta., Cir. 71, 27-42. Chambcrs, C. W. (1965). “Control of Pseudomonads in Water Supplies.” Presented in Analytical Microbiology Group Round Table, Am. SOC.Microbiol., Atlantic City, New Jersey, 1965. (Mimeo.) Chambers, C. W., and Proctor, C. M. (1960). Tech. Rept. W60-4, pp. 1-18. U.S. Public Health Service, Taft Sanit. Eng. Center, Cincinnati, Ohio. Chambers, C. W., Kabler, P. W., Malaney, G., and Bryant, A. (1952). Soap Sanit. Chem. 28, 10, 149. Chambers, C. W., Kabler, P. W., Bryant, A. R., Chambers, L. A., and Ettinger, M. (1955). Public Health Rept. ( U S . ) 70, 545-553. Chambers, C. W., Tabak, H. H., and Kabler, P. W. (1957). J. Bacteriol. 73, 77-84. Chambers, C. W., Proctor, C. M., and Kabler, P. W. (1962). J. Am. Water Works ASSOC. 54, 208-216. Chang, S. L. (1966). Organo Oficial de la Asoc. Inter-Anier. de Ingenieria Sanit. (in press). Chang, S. L., and Morris, J. C. (1953). Zncl. Eng. Chem. 45, 1109-1112. Chang, S. L., Stevenson, R. E., Bryant, A. R., Woodward, R. L., and Kabler, P. W. (1958). Am. J. Public Health 48, 159-169. Chaplin, C. E. (1952). J. Bactmiol. 63, 453-458. Cousins, C. M., and Clegg, L. F. L. (1956). J. Appl. Bacteriol. 19, 250-255. Deindoerfer, F. H., and Humphrey, A. E. ( 1959a). Appl. A4icrobiol. 7, 256-264. Deindoerfer, F. H., and Humphrey, A. E. (1959b). Appl. Microhiol. 7, 264-270. Dondero, N. C. (1961). Advan. Appl. Microbiol. 3, 77-107. Dunlop, S. G. (1952). Sanitarian (Denoer) 15, 107-110. Eisman, P. C., Kull, F. C., and Meyer, R. L. (1949). J. Am. Pharm. Assoc., Sci. E d . 38, 88-91. Esty, J. R., and Meyer, K. F. (1922). J. Infect. Diseases 31, 650-663. Fair, G. M., Morris, J . C., Chang, S. L., Weil, I., and Burden, R. P. (1948). J. Am. Water Works Assoc. 40, 1051-1061. Farkas-Himsley, H. (1964). Appl. Microbiol. 12, 1-6. Favero, M. S., Drake, C. S., and Randall, G. B. (1964). Public Health Rept. ( U . S . ) 79, 61-70. Feuss, J. V. ( 1964). 3. Am. Water Works Assoc. 56, 607-615. Flu, P. C. (1941). Antonie van Leeuwenhoek, J . Microhiol. Serol. 7, 39-60. Fowler, E. B., Christenson, C. W., Jurney, E. T., and Schafer, W. D. (1960). Nucleonics 18, 4, 102-105. Geldreich, E. E., and Clark, H. F. (1965). J. Milk Food Technol. 28, 351-355. Gilcreas, F. W., and Kelly, S. M. (1955). I. Am. Wnter Works Assoc. 47, 683-694. Goldstein, M., McCabe, L. J., and Woodward, R. L. (1960). J. Am, Water Works ASSOC.52, 247-254.
CONTROL OF BACTERIA
IN
NONDOMESTIC WATER
141
Goodenough, R. D. ( 1964). Swimming Pool Age 38,4,25. Granstrom, M. L., and Lee, G. F. (1957). Public Works 88, 12, 90-92. Granstrom, M. L., and Lee, G. F. (1958). J. Am. Water Works Assoc. 50, 1453-1466. Griffin, A. E. (1947). J. New Engl. Water Works Assoc. 61, 145-150. Hann, V. A. (1956). J. Am. Water Works Assoc. 48, 1316-1320. Harper, W. J. (1965). Personal communication. Ohio State Univ., Dept. of Dairy Technol., Columbus, Ohio. Harris, W. C., Jr. (1960). Water Works Eng. 113, 893. Harrison, M., and Heukelekian, H. ( 1958). Sewage Ind. Wastes 30, 1278-1302. Hays, H., Elliker, P. R., and Sandine, W. E. (1963). 1. Milk Food Technol. 26, 147-150. Hazey, G. J. (1951). J. Am. Water Works Assoc. 43, 292-298. Heathman, L. S., Pierce, G. O., and Kabler, P. W. (1936). Public Health Rept. (U.S.) 51, 1367-1387. Hoadley, A. W., and McCoy, E. (1965). Appl. Microbiol. 13, 575-578. Honeywell, G. E., Rueber, F. M., and Stapert, E. M. (1962). In “Developments in Industrial Microbiology” (C. Koda, ed.), Vol. 3, pp. 306-312. Plenum Press, New York. Huff, C. B., Smith, H. F., Boring, W. D., and Clarke, N. A. (1965). Public Health Rept. ( U . S . ) 80, 695-705. Hunter, C. A., and Ensign, P. R. (1947). Am. 1. Public Health 37, 1166-1169. Hutchinson, D., Weaver, R. H., and Scherago, M. ( 1943). J. Bacteriol. 45, 29. Ingols, R. S., and Ridenour, G. M. (1948). J. Am. Water Works Assoc. 40, 12071227. Ingraham, J. L., and Stokes, J. L. (1959). Bacteriol. Reu. 23, 97-108. Jarrett, J. M. (1965). Personal communication. Dir. Sanit. Eng. Div., North Carolina State Board of Health, Raleigh, North Carolina. Jeffrey, L. P., and Fish, K. H., Jr. (1964). Am. J. Hosp. Pharm. 21, 497-500. Jewell, A. B. (1942). Southwest Water Works I . 23, 13-14. Johannesson, J. K. (1960). Am. J. Public Health 50, 1731-1736. Jones, F. E., and Greenberg, A. E. (1964). 1. Am. Water Works Assoc. 56, 14891493. Kelman, A., Person, L. H., and Hebert, T. T. (1957). Plant Disease Reptr. 41, 798802. Kidder, G. W. ( 1951). Ann. Reu. Microbiol. 5, 140. Kramer, H. P., Moore, W. A., and Ballinger, D. G. (1952). Anal. Chem. 24, 18921894. Kryiasides, K. (1931). Z . H y g . Infektionskrankh. 112, 350-364. Lacy, I. 0. (1963). Public Works 94, 3, 91-93. Langelier, W. F., and Ludwig, H. F. (1949). J. Am. Water Works Assoc. 41, 163181.
Law, A. B., McNulty, P. J., and Rakus, L. M. (1964). Mfg. Chemist Aerosol News ( B T . ) 35, 11, 88-96. Lederer, S. J., and Delaney, W. J. (1960). Tappi 43, 160-166. Leeflang, K. W. H. ( 1963). 1. Am. Water Works Assoc. 55, 1523-1535. Leifson, E. ( 1962). Intern. Bull. Bacteriol. Nomenclat. Taxonomy 12, 133-170. Lewis, R. F. (1965). J. Am. Water Works Assoc. 57, 1011-1015. McCulloch, E. C. (1945). “Disinfection and Sterilization,” pp. 34 and 280. Lea & Febiger, Philadelphia, Pennsylvania. MacGregor, D. R., and Elliker, P. R. (1958). Can. I. Microbiol. 4, 499-503. McKeown, J. J. (1963a). Ind. Water 6.Wastes 8, 3, 19-22.
142
CECIL W. CHAMBERS AND NORMAN A. CLARKE
McKeown, J. J. (1963b). Ind. Water b Wastes 8, 4, 30-33. Malaney, G. W., Weiser, H. H., Turner, R. O., and Van Horn, M. (1962). Appl. Microbiol. 10, 44-51. Mallmann, W. L., and Kahler, D. (1948). J. Awl. Water Works Assoc. 40, 615-624. Morrison, H. B., and Hammer, B. W. (1941). J. Dairy Sci. 24, 9-18. Mossel, D. A. A., and Ingram, M. (1955). 1. Appl. Bacteriol. 18, 232-268. Mulder, E. G. (1964). J. Appl. Bacteriol. 27, 151-173. Nankivell, A. T. ( 1911). 1. Hyg. 11, 235-258. Nason, H. K. (1938). J. Am. Water Works Assoc. 30, 437-452. Norman, N. N., and Kabler, P. W. (1953). Sewage Ind. Wastes 25, 605-609. Olson, J. C., Jr., Parker, R. B., and Mueller, W. S. (1955). J. Milk Food Technol. 18, 200-203. Palin, A. T. (1961). Water Sewage Works 108, 461-462. Palmer, C. M. ( 1961). J. Am. Water Works Assoc. 53, 1297-1312. Panel Discussion. ( 1949). J. Am. Water Works Assoc. 41, 933-947. Panel Discussion. ( 1959a). 3. Am. Water Works Assoc. 51, 1433-1472. Panel Discussion. (195913). J. Am. Water Works Assoc. 51, 215-233. Parker, R. B., Caldwell, A. L., and Elliker, P. R. (1953). J. Milk Food Technol. 16, 136-139. Phillips, U. A., and Traxler, R. W. (1963). Appl. Microbiol. 11, 235-238. “Public Health Service Drinking Water Standards.” ( 1962). U.S. PubZic Health Sero., Publ. 956, 1-61. Rahn, 0. (1945). Bacteriol Rev. 9, 1-47. Reitler, R., and Seligmann, R. (1957). J. Appl. Bucteriol. 20, 145-150. Renn, C. E., and Chesney, W. E. (1953-1956). Reports to Salem-Brosius, Inc. on Research on Hyla System of Water Disinfection. Johns Hopkins Univ., Baltimore, Maryland. Rhines, C. E. (1965). Unpublished data. U.S. Public Health Service, Taft Sanit. Eng. Center, Cincinnati, Ohio. Ridenour, G. M., and Armbruster, E. H. (1949). J. Am. Water Works Assoc. 41, 537-550. Riehl, M. L., Weiser, H. H., and Rheins, B. T. ( 1952). 1. Am. Water Works Assoc. 44, 466-470. Robeck, G. G., Dostal, K. A., and Woodward, R. L. (1964). J. Am. Water Works Assoc. 56, 198-213. Sandbom, J. R. (1944). J. Bucteriol. 48, 211-217. Scarlett, C. A. (1962). 1. SOC. Dairy Technol. 15, 155-164. Seiberling, D. A., and Harper, W. J. ( 1955). J. Dairy Sn’. 38, 588, Abt.-M-47. Shannon, A. M., and Wallace, W. M. (1944). 1. Am. Water Works Assoc. 36, 13561364. Shay, E. G., Clarke, P. H., and Crawford, R. (1965). Soap Chern. Specialties 41, 5, 126. Silvey, J. K. G., and Roach, A. W. (1964). J. Am. Water Works Assoc. 56, 60-72. Snow, W. B. (1956). 3. Am. Water Works Assoc. 48, 1510-1514. Spaulding, C. H. (1931). Am. 1. Public Health 21, 1380-1383. Spino, D. F. (1966). Appl. Microbiol. (in press). Starkey, R. L. (1956). Id.Eng. Chem. 48, 1429-1437. Tabak, H. H., Chambers, C. W., and Kabler, P. W. (1964). J. Bacteriol. 87, 910-919. Tennenbaum, S. ( 1965). “Pseudomoms in Cosmetics and Pharmaceuticals.” Presented in Analytical Microbiology Group Round Table, Am. SOC.Microbiol., Atlantic City, New Jersey, 1965. (Mimeo.)
CONTROL OF BACTERIA I N NONDOMESTIC WATER
143
Thomas, R. C. (1935). PhytopathoZogy 25, 371-372. Traxler, R. W. (1962). Biotechnol. Bioeng. 4, 369-376. U.S. Public Health Service. (1959). Mimeographed Report. Taft Sanit. Eng. Center, Cincinnati, Ohio. Wattie, E., and Chambers, C. W. ( 1943). 1. Am. Water Works Assoc. 35, 709-720. Weindling, R. (1956). Id.Eng. Chem. 48, 1407-1410. Welch, J. L., and Folinazzo, J. F. (1959). Food Technol. 13, 179-182. Whipple, G. C., Fair, G . M., and Whipple, M. C. (1927). “Microscopy of Drinking Water,” 4th ed., 5th printing, pp. 442-443. Wiley, New York. Willis, A. T. (1957). 1. Appl. Bacterial. 20, 61-64. Wilson, C. (1932). 1. Am. Water Works Assoc. 24, 1792-1799. Wilson, C. (1945). 1. Am. Water Works Assoc. 37, 52-58. Witter, L. D. (1961). I. Dairy Sci. 44, 983-1015. Woodward, R. L. (1963). J. Am. Water Works Assoc. 55, 881-886. Wuhrmann, K., and Zobrist, F. (1958). Schweitz. 2. Hydrol. 20, 218-254. Wyss, 0. (1956). Ind. Erg. Chem. 48, 1404-1406. Zabel, R. A., and O’Neil, F. W. (1957). Tuppi 40, 911-914. Zimmerman, W. (1952a). Z. Hyg. Infektionskrankh. 135, 403-413. Zimmerman, W. ( 1952b). Z. Hyg. Infektionskrankh. 135, 414-420. Zobell, C. E., and Beckwith, J. D. (1944). J. Am. Water Works Assoc. 36, 439-453.
This Page Intentionally Left Blank
The Presence of Human Enteric Viruses in Sewage and Their Removal by Conventional Sewage Treatment Methods STEPHENALAN KOLLINS~ Department of Environmental Sciences, College of Agriculture and Environmental Science, Rutgers University, New Brunswick, New Jersey
Introduction ..................................... The Nature of Enteric Viruses ...................... Enteric Viruses in Feces .......................... Transmission of Viruses Through Water . . . . . . . . . . . . . . A. Poliomyelitis ................................. B. Infectious Hepatitis .......................... V. Presence of Viruses in Sewage ...................... VI. Removal of Viruses by Sewage Treatment Methods . . . . A. Storage ..................................... B. Primary Sedimentation ........................ C. Trickling Filters ............................. D. Activated Sludge ..................... E. Disinfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . I. 11. 111. IV.
.............
VII. Summary
.................
References . . . . . .
1.
............. ................. ............. .................
145 146 159 162 162 164 170 175 175 177 179 180 185 188 188 189 191 191
introduction
The control of communicable diseases represents one of man’s most challenging problems. The agents of these diseases are many in fom and number, their individual importance being dependent upon prevailing conditions in the community. It is generally accepted that the welfare of our communities is enhanced by the removal of these infectious agents from our environment or by their effective control. Although contagious diseases are spread in many ways, the universal consumption of water presents a potential hazard which cannot be ignored. The nature of the supply and distribution of our water resources makes it essential that man use his surface waters for both water supply and sewage disposal. Large numbers of pathogenic organisms, bacteria and viruses as well as protozoa and parasitic worms, are excreted in feces. It is obvious, then, that in the absence of adequate waste treatment and water treatment practices a potential for the spread of enteric disease exists, for, should the agents 1
Present Address: College of Medicine, University of Cincinnati, Cincinnati, Ohio. 145
146
STEPHEN ALAN KOLLINS
pass through the waste treatment plant unharmed and be capable of surviving for some time in water, they may find their way into the domestic water supply of a community downstream. Contamination of a domestic water supply could result in an extensive spread of the infectious agent and a subsequent epidemic. In this paper I shall consider the significance of one such group of pathogenic agents, the enteric viruses. These are excreted in the feces in large numbers and are found commonly in urban sewage, especially during late summer and early fall. The infections they cause are widespread throughout the population, with the highest morbidity rate in infants and young children (Clarke and Chang, 1959). I shall be concerned with the nature of the enteric viruses and of the diseases they cause, the possible role water may play in their spread, their incidence and survival in feces and sewage, and their removal or inactivation by conventional sewage treatment practices.
II. The Nature of Enteric Viruses Viruses are ultramicroscopic entities capable of passing through filters which will retain ordinary bacteria. They are obligate intracellular parasites, incapable of proliferating in an extracellular environment. Viruses have been known to infect man, other vertebrates, plants, insects, and bacteria. Those infecting bacteria have been termed bacterial viruses (or bacteriophages), while those infecting man and other vertebrates are known as animal viruses. The Rickettsia, basophilic viruses, and pleuropneumonia-like organisms ( PPLO) are usually considered, both physically and biologically, intermediates between the bacteria and animal viruses. Unfortunately, a binomial system of nomenclature as is commonly used to classify bacteria is not readily applicable to animal vimses. There is not, as yet, sufficient knowledge of virus properties nor of the relationships viruses hold to one another. Furthermore, probably only a small fraction of all the animal viruses have yet been discovered (Rhodes and VanRooyen, 1962). Perhaps the most reasonable classifications of animal viruses are those which assign viruses with similar properties to groups designated by distinguishing names and the suffix “virus.” Such a classification is shown in Table I. Under this system of nomenclature the viruses of current concern fall within three groups: the adenovirus group, the enterovirus group, and the hepatitis group. Collectively I shall refer to them as “enteric viruses.” The nature of the infective process of virus infections is not unlike that of pathogenic bacteria or protozoa. The route of entry into the host may
REMOVAL OF HUMAN ENTERIC VIRUSES I N SEWAGE
147
be determined by the location of susceptible cells, by the portal of exit from the previous host, by the mode of transmission, and by the ability of the virus to survive outside the body. The high degree of cell specificity of some viruses may limit both the route of entry into the host and the means of spread within the host. The nature of the virus and its particular host help to determine the pathway infection will take. The transmission of viruses to fresh hosts, on which the survival of the virus depends, wiII usually be related to the route by which the virus leaves the body of the infected patient. While viruses producing localized lesions on the skin or mucosa will obviously be disseminated from the exudates or secretions from such surfaces, the excretion of viruses from generalized infections will depend largely upon the site in the host (the target organ) in which the virus proliferates. Focal lesions in the mouth associated with diseases such as herpes simplex, measles, chickenpox, and smallpox release virus to the saliva, but the process is inherently different from that in generalized infections such as mumps or rabies which become localized in the salivary gland (Anderson et al., 1962). In a similar manner a number of viral diseases, such as those caused by inffuenza or reoviruses, result in the release of virus to the feces, but these infections are inherently different from enteric virus infections. Rhodes and VanRooyen (1962) state that “adenoviruses have been isolated from adenoids and tonsils grown in tissue cultures, and, in the acute phase of the illness, from pathologic specimens of throat secretions, conjunctiva1 exudate, mesenteric lymph nodes and stools.” Though one would expect viruses associated with respiratory ailments to be most easily isolated from throat secretions, adenoviruses are more readily isolated from the stools than from throat specimens. Many, in fact, have been isolated only from stools (Berg, 1964). Although the virus is capable of establishing an infection of the adjoining lymph glands after localized multiplication in the conjunctival sacs and upper respiratory tract, it is not known if blood invasion occurs. The possibility of successful passage of swallowed secretions through the alimentary tract is supported by the observation that some strains may even multiply in the intestinal wall and produce gastroenteritis and mesenteric adenitis (W. Smith, 1963). Formerly referred to as A.P.C. viruses ( adenoidal-pharyngeal-conjunctival), the adenoviruses have been associated with a number of clinical syndromes, most of which are typically spread by direct and indirect contact, droplets, and fomites (D. T. Smith and Conant, 1960). Pharyngoconjunctival fever, mainly a disease of the summer months, has been spread among Canadian children while swimming in pools (Omsby and Aitchison, 1955). Similar observations have been made in the United
TABLE I MAJORVmus INFECTIONS OF MANIN 1962: ARRANGED ACCOFIDING TO BIOLOGICGROUPOF CAUSAL AGEWP Biologic group of causal agent Herpesvirus
Commonly used name of species of virus Herpes simplex
1
B virus
1 1 1
Varicella-zoster Pseudorabies Poxvirus
Number of antigenic types
Major clinical features Herpes Myelitis Varicella, zoster Pseudorabies
Variola Vaccinia cowpox Molluscum
1 1 1
Smallpox, alastrim Vaccinia cowpox Molluscum contagiosum
Measles group
Measles Rubella
1 1
Measles German measles
Myxovirus
Influenza viruses A, A', Asian Influenza viruses B and C Mumps Newcastle disease Parainfluenza viruses types 1, 2, 3, 4, and Sendai
3
2 1 1 5
Influenza Influenza Mumps Conjunctivitis Respiratory illness
Adenovirus
Adenovirus types 1-24
1
24
Respiratory and eye infections
Reovirus
Reovirus types 1, 2, and 3
3
Respiratory infection
Cytomegalovirus
Salivary gland virus (cytomegalic inclusion disease virus)
2?
Cytomegalic inclusion disease
Hepatitis group
Infectious hepatitis Serum hepatitis
l?
Infectious hepatitis Serus hepatitis
l?
Arborvirus Group A (Casals)
Group B (Casals )
Equine encephalitis (Eastern, Western, Venezuelan ) Arborviruses from Africa, Asia, South America St. Louis subgroup St. Louis encephalitis Japanese B encephalitis Murray Valley encephalitis West Nile Ilheus Dengue subgroup Dengue types 1-4 Yellow fever subgroup Yellow fever Uganda S Russian encephalitis subgroup Spring summer encephalitis (Europe, U.S.S.R., Malaya) Hemorrhagic fever ( U.S.S.R.) Diphasic fever (U.S.S.R.) Louping ill ( Scotland) Powassan (Eastern Canada) Kyasanur Forest disease (India) Other group B viruses Ntaya and other viruses from South America and Africa
3
Encephalitis
6
Subclinical; fever; dengue-like illness
1 1 1 1 1
Encephalitis Encephalitis Encephalitis Dengue-like illness Fever
3
2!
99
r 0
21
3
F
z 4
Dengue
1 1
Yellow fever Subclinical
1 1 1 1 1 1
5
Encephalitis Hemorrhagic fever Meningoencephalitis Encephalitis Encephalitis Hemorrhagic fever Subclinical; fever; influenzalike illness
E! F1
z
0
8 s
4t 2
3
fi zW
G 0
TABLE I (Continued) Commonly used name of species of virus
Biologic group of causal agent
Number of antigenic tvpes
Major clinical features
Group C
Brazilian viruses
6
Fever
Unassigned to groups A. B. or C
Rift Valley fever Colorado tick fever Sandfly fever types 1 and 2 Viruses from Africa, South America, U.S.A., U.S.S.R.
1 1 2
Generalized infection Tick fever Sandfly fever Subclinical; fever, encephalitis; hemorrhagic fever
Rabiesvirus Lymphocytic choriomeningitis Enterooiriis
Rabies Lymphocytic choriomeningitis Poliovirus types 1, 2, and 3 Coxsackie A Coxsackie B ECHO virus Foot and mouth disease
Encephalomyocarditis
MM-Columbia SK Encephalomyocarditis Mengo
Papova virus
Warts
Miscellaneous
Common cold viruses ( Salisbury ) Respiratory syncytial agent Eaton’s agent of atypical pneumoniab
a b
15
1 1 3 24
6 28 Several
Rabies Virus meningitis Polio Fever; herpangina Pleurodynia; meningitis; myocarditis; pericarditis Meningitis; rashes; respiratory illness Fever, vesicles
l? 1 1
Fevers; nervous illness Encephalitis; myocarditis Encephalitis; myocarditis
1
Warts
Several
1 1
Common cold Common cold Common cold; atypical pneumonia
Taken from Rhodes and VanRooyen (1962). Total of antigenically distinct viruses infecting man, approximately 200. This may be a pleuropneumonia-like organism.
REMOVAL OF HUMAN ENTERIC VIRUSES IN SEWAGE
151
States, and an outbreak of pharyngoconjunctival fever caused by a type 3 adenovirus was attributed to swimming pool contacts in 1954 ( McLean, 1963). Water-borne spread in swimming pools depends upon ( 1 ) the local concentration of infecting agents near their source, ( 2 ) the general concentration of infecting agents in the pool, ( 3 ) the rate of kill in the water, and (4) the proximity of contacts ( McLean, 1963). Studies have shown enteric (ECHO) viruses to be present in unchlorinated wading pools; however, in swimming pools using the same municipal water supply system but chlorinating to 0.2 p.p.m. free residual chlorine, no agents were demonstrable ( Kelly and Sanderson, 1961). Because adenoviruses are much less resistant to chlorination than the enteroviruses and the virus of infectious hepatitis ( McLean, 1963), their spread in swimming pools may be (and probably is) dependent upon intimate personal contact. In addition, the diseases produced by adenoviruses in adults are often too mild to be brought to the attention of a physician and hence may go undiagnosed. These are probably the major reasons why there have been no clearly documented cases of water-borne adenovirus infection. A classification of the adenovirus group and a summary of the clinical syndromes caused by this group are seen in Tables I1 and 111. As will be discussed later, the great variety seen in the clinical manifestations of a single group of viruses is typical of all enteric virus infections. The enterovirus group, as the name implies, are frequently present in the feces, as they proliferate intensely in the enteric tract. They are also commonly seen in throat washings. The viruses which have been accepted into this group are the poliovirus types, coxsackie A and B viruses, the ECHO viruses of human and animal origin, and Theiler’s mouse virus. The Teschen virus and the virus of foot and mouth disease also possess many of the properties of the enteroviruses but have not been universally accepted as members of the group (Rhodes and Van Rooyen, 1962). Those of special concern to us here are the polioviruses, coxsackie A and B viruses, and the ECHO viruses. The Committee on the Enteroviruses ( N . I. H.) includes the following criteria in their definition of the enterovirus group: They must ( 1 ) inhabit the gastrointestinal tract of man, ( 2 ) be cytopathic for monkey and human tissue cultures, ( 3 ) be unrelated to other agents seen in the throat and intestine of man such as the influenza, herpes simplex, and hepatitis viruses, (4) cause infection in man as indicated by the presence of neutralizing antibodies in pooled human y-globulins as well as in individual serum samples from which the agent was isolated, ( 5 ) cause pathogenic effects in tissue culture, suckling mice, and monkeys, and ( 6) have a characteristic size, resistance to ether, seasonal prevalence, and epidemiological pattern.
TABLE I1 TYPES^
ADENOVIRUS
Prototype Type 1
2 3 4 5 6 7 7a 8 9 10 11
12 13 14 15 16 17 18 19
strain Human Origin Ad. 71 Ad. 6 G.B. RI-67 Ad. 75 Ton. 99 Comen S-1058 Trim. Hicks
J.J. Slobitski Huie A.A.
DeWitt Ch. 38 Ch. 79 Ch. 22 D.C. 587
Source of Prototype Strain Isolated by
Specimen
Type of Case
NIHb NIH NIH Hilleman and Werner hTIH
Adenoid Adenoid Nasal washing Throat washing Adenoid
Hypertrophied tonsils and adenoids Hypertrophied tonsils and adenoids Common cold volunteer Primary atypical pneumonia Hypertrophied tonsils and adenoids
NIH Berge NIH Jawetz Kibrick NIH Kibrick Kibrick NIH (Rosen) van der Veen Murray and Chang Murray and Chang Murray and Chang NIH Bell et al.
Tonsil Throat washing Throat swab Eye swab Stool Eye swab Stool Stool Stool Throat swab Eye swab Eye swab Eye swab Anal swab Conjunctival secretion
Hypertrophied tonsils and adenoids Pharyngitis Undifferentiated respiratory infection Epidemic keratoconjunctivitis Arthritis, rheumatoid?, myelitis? Conjunctivitis Paralytic polio (type 1 also recovered) ? Nonparalytic polio WeIl child Acute respiratory disease Conjunctivitis ( early trachoma?) Conjunctivitis (early trachoma?) Conjunctivitis (early trachoma?) Niemann-Pick disease? Trachoma
3
Lm_!
4
5* 2: R 0
E 3
20 21 22 23
931 1645 2711 2732
Bell Bell Bell Bell
24
3153
Bell et
et al. et al.
et al. et al. UI!.
conjunctival conjunctival conjunctival conjunctival
secretion secretion secretion secretion
Trachoma Trachoma Trachoma Trachoma
conjunctival secretion
Trachoma
Simian Origin
c-1c M-lc
Bertha S.V.l
Sabin and NIH Hull
M-2
cv2
NI H
M-3
Abin.
NIH
M-4
2043
Rightsel and McLean
a 6 c
Taken from Rhodes and VanRooyen ( 1962). NIH, National Institutes of Health, Bethesda, Md., U.S.A. C, chimpanzee; M, monkey.
Chimpanzee stool Cynomolgus kidney tissue culture Cynomolgus kidney tissue culture Air sample from monkey room Monkey kidney tissue culture
8 X
c
5Z
!9s M,
2
$
c,
8
TABLE I11 CLINICALSYNDROMES CAUSEDBY ADENOVIRUSES~ Syndrome
Occupational group affected
Acute respiratory disease Pharyngoconjunctival fever Nonbacterial exudative pharyngitis Minor respiratory illness and croup
Servicemen Children and civilian adults Servicemen Children and civilian adults
Virus pneumonia Sporadic conjunctivitis Epidemic keratoconjunctivitis a
From Rhodes and VanRooyen ( 1962 1.
More common
Less common
3
types 4, 7,7a
types 3, 14 1,4,7,7a, 14
9
3
3,4,7
1,2, 3, 4,5, 7,7a
14
Servicemen, children, and civilian adults
3, 4,7,7a
1, 2,18
Children and civilian adults Workers in heavy industry; may spread to family contacts and general community
2, 3, 6, 7, 9 8
5Z w
8 G
z,
REMOVAL OF HUMAN ENTERIC VIRUSES I N SEWAGE
155
The pathological and clinical aspects of infections caused by each of these viruses need not be discussed in detail, but rather the general description of enterovirus infection given by Rhodes and Van Rooyen (1962) may be quoted: These viruses enter the body by the mouth or upper respiratory tract and undergo multiplication in the tonsils and Peyer’s patches. From these primary sites the virus spreads to the regional lymph nodes where further multiplication takes place. The blood may then be invaded and, following viremia, the virus localizes in one or more of the following target organs: meninges, ganglionic nerve cells of the brain or spinal cord, striated muscle, myocardium or skin. The precise site of localization depends to some extent on the strain of the enterovirus. . . . It is probable that in the great majority of the cases, enterovirus infection involves only the stage of localized multiplication in the gastrointestinal tract and perhaps proliferation in lymph glands, the process remaining symptomless.
Tables IV through VIII show commonly accepted classifications of the enterovirus groups and Table IX the common clinical features arising as a result of virus localization. The production of several clinical diseases is clearly evident here, as with the adenoviruses. Each individual virus has been seen to produce diseases greatly varying in manifestations and severity, while each of these diseases may, in turn, be produced by several viruses. In addition to this complexity, the large proportion of illnesses falling below levels of clinical detectability make it very difficult to study their epidemiology. The large ratio of abortive and nonparalytic cases of poliomyelitis to paralytic cases, as well as the widespread distribution of poliovirus among healthy individuals, indicates that contact with an ambulatory carrier is inevitable, The most generally accepted epidemiology implicates person-to-person contact of more than a casual nature, with subclinical cases and carriers playing a major role (Rivers, 1948). This does not rule out the possibility of water-borne spread and, as will be discussed, cases of possible water-borne transmission of poliovirus are known. None of the other enteroviruses have been involved in wateror sewage-borne outbreaks, either because no such outbreaks occur or because current epidemiological techniques are not sensitive enough to detect them (Clarke and Kabler, 1964). The occurrence of these other viruses in large numbers in sewage has been repeatedly demonstrated and will be described further. It seems ironic that the only clearly documented cases of water-borne spread of viruses have involved infectious hepatitis virus, a virus not culturable under any known laboratory conditions. Although its presence in sewage cannot be demonstrated without the use of human volunteers, numerous outbreaks have been traced to consumption of polluted water. This disease has largely been one of personal contact, particularly associated with conditions of communal living and feeding. It is usually
TABLE IV THE ENTEROVIRUS GROUP:MAIN PROPERTIES~ Growth
Group Poliovirus ( Poliovirus hominis ) Theiler’s virus (Poliovirus muris ) Coxsackie virus group A
Coxsackie virus group ECHO virus (human strains) ECHO virus ( animal strains ) Foot and mouth disease virus a
Antigenic types
Infection in man
Ether resistance
Monkey CNS
Suckling mice
in
tissue culture
Types 1, 2, and 3
All forms of polio
+
+
-
+
Two chief variants, TO and GD VII
None
+
-
+
Strains vary
24 types
Herpangina; fevers; rashes; aseptic meningitis; paralysis Myalgia; myocarditis; pericarditis; fevers Meningitis; rashes; fevers None
Mostly negative
+
Strains vary
( myosi,tis)
Mostly negative Mostly negative
(fat, pancreas) Only ECHO 9 pathogenic
-
-
+ + +
-
+
+
6 types 28 types Many types from bovines, porcines, dogs, monkeys Several
From Rhodes and VanRooyen (1962).
Vesicles; fever
+ + + +
+
+
TABLE V COXSACKIE GROUPAa Type 1
2 3
4 5 6 7 8 9 10 11 12 13 14 15 16
17 18 19 20 21b 22c 240 a 0
c
Prototype strain
Geographic origin
Tonipkins Fleetwood Olson High Point Swartz Gdula Parker Donovan Bozek Kowalik Belgium-1 Texas-12 Flores G-14 G-9 G-10 G-12 C-13 NIH-8663 IH-35 Kuykendall Chulman Joseph
Coxsackie, New York Delaware New York North Carolina New York New York New York New York New York New York Belgium Texas Mexico South Africa South Africa South Africa South Africa South Africa Washington, D.C. New York California New York South Africa
Material yielding virus Polio patient, stool Polio patients, stools Aseptic meningitis, stool Sewage Polio patient, stool Aseptic meningitis, stool Aseptic meningitis, stool Polio patient, stool Aseptic meningitis, stool Aseptic meningitis, stool Epidemic myalgia Flies Stool ? ? ?
? ? ? Infectious hepatitis, stools Stool Stool Stool
Investigator Dalldorf Dalldorf Dalldorf Melnick Dalldorf Dalldorf DaUdorf Dalldorf Dalldorf Dalldorf Cumen Melnick Sickles Gear Gear Gear Gear Gear Huebner Sickles Lennette Sickles Gear
2+ 8
8
E 2 M
5 e
ji
c!
in
c1
'
m"
3 $
Taken from Rhodes and VanRooyen ( 1962). Also known as the Coe virus. The Vispo strain was designated by some workers as Coxsackie A type 23. This virus is now regarded as a strain of ECHO type 9. CR -4
TABLE VI COXSACKIE GROUPBa Type 1 2 3 4 5
6 a
Prototype strain Conn-5 Ohio-1 Nancy J.V.B. Faulkner S chmitt
Geographic origin
Material yielding virus
Investigator
Connecticut Ohio Connecticut New York Kentucky Philippine Islands
Asceptic meningitis, stool Summer grippe, stool Minor febrile illness, stool Stool Suspected polio, stool Healthy adult, stool
Melnick hielnick hfelnick Sickles Steigman Hammon
Taken from Rhodes and VanRooyen ( 1962).
REMOVAL OF HUMAN ENTERIC VIRUSES I N SEWAGE
159
spread by the fecal-oral route but may sometimes be transmitted parenterally. Most cases occur in children and young adults, with incubation periods of from 15 to 50 days. Outbreaks are common in schools, hospitals, mental institutions, summer camps, mining communities, and military establishments. The disease is world wide in distribution and in the tropics it is prevalent year round. In temperate climates it occurs primarily in the fall and winter months. Person-to-person transmission is indicated as the most important means of spread by the extremely high morbidity rates under conditions of close personal contact, poor sanitation, and lack of personal hygiene. The recognition of water-borne epidemics, however, has led to an increased awareness of the significance of this mode of transmission (W.H.O., 1964). Foods, especially milk (Murphy and Petrie, 1946) and shellfish (Henderson, 1961), have also been incriminated, the latter becoming contaminated as the result of sewage poIIution of the shellfish beds.
111. Enteric Viruses in Feces Poliovirus was found in the feces in frank cases of poliomyelitis as early as 1912 in Sweden and 1915 in this country and its presence has been verified repeatedly (Carlson et al., 1942). After an attack of acute disease the virus remains in the enteric tract longer than in the throat. It can be recovered from stools for at least 2 to 3 weeks and may persist up to 12 weeks after onset. Contacts of patients with paralytic disease, especially those in the same family, have been known to excrete the virus for up to 2 months and not show any clinical symptoms (Gear and Measrock, 1946). In an outbreak involving institutionalized patients little effort was required to recover the virus from apparently healthy contacts, both children and adults ( Kramer, 1939). Even more surprising may be the recovery of poliovirus from both tonsils and feces of noncontacts during an interepidemic period (Kessel and Moore, 1945). Because the period in question immediately preceded an epidemic, the authors attributed their recoveries to a period of quiescent dissemination of the virus just prior to the outbreak of clinical cases. In all areas in which an adequate investigation has been made poliovirus infection has been found. The prevalence varies from population to population and even within a single population as a function of living conditions. Under conditions of overcrowding and poor hygiene one could expect up to 90% of the population to be infected by the age of 6 years. Reinfection would be common and the virus would eventually prevail throughout the population (Rhodes and VanRooyen, 1962). Even in normal populations during nonepidemic times children less
c,
Q,
TABLE VII ECHO VIRUSES~
Type
1 2 3 4
5 6 7 8 9 1o c 11 12 13 14 15 16 17 18 19 20 21
Prototype strain Farouk Comelis Momsey Pesascek Noyce D’Amori Wallace Bryson Hill Lang Gregory Travis Hamphill Tow CH 96-51 Harrington CHHE-29 Metcalf Burke JV-1 Farina
Geocxaphic origin
Illness in person yielding prototype virus (all from stools)b
EgYPt Connecticut Connecticut Connecticut Maine Rhode Island Ohio Ohio Ohio Ohio Ohio Philippine Islands Philippine Islands Rhode Island West Virginia LMassachusetts Mexico City Ohio Ohio Washington, D. C. Massachusetts
Pione Aseptic meningitis Aseptic meningitis Aseptic meningitis Aseptic meningitis Aseptic meningitis None None None None None None None Aseptic meningitis None Aseptic meningitis None Diarrhea Diarrhea Fever Aseptic meningitis
0
Investigator Melnick Melnick hlelnick Melnick Melnick Melnick Ranios-Alvarez, Sabin Ramos-Alvarez, Sabin Ramos-Alvarez, Sabin Ramos-Alvarez, Sabin Ramos-Alvarez, Sabin Hammon, Ludwig Hammon, Ludwig Melnick Ormsbee, Melnick Kibrick, Enders Ramos-Alvarez, Sabin Ramos-Alvarez, Sabin Ramos-Alvarez, Sabin Rosen Enders, Kibrick
8w
8
+z$ fc
E
2
22 23 24 25
26 27 28 a
b 0
Harris Williamson De Camp JV-4 Coronel Bacon 2060
Ohio Ohio Ohio Washington, D. C. Philippine Islands Philippine Islands Great Lakes
Taken from Rhodes and VanRooyen (1962). Prototype ECHO virus type 28 was isolated from the nasopharynx. Now Reovirus.
Diarrhea Diarrhea Diarrhea Diarrhea None None Mild respiratory
Sabin Sabin Sabin Rosen Hammon Hammon Pelon
d
8 !3 9
E 2 k
g
bm
162
STEPHEN ALAN KOLLINS
TABLE VIII POLIOVIRLJSES~ Type 1 2 3 a
Prototype strain Brunhilde Lansing Leon
Geographic origin
Material yielding virus
Investigator
Maryland Michigan California
Stools (paralytic) CNS (fatal) CNS (fatal)
Howe and Bodian Armstrong Kessel
Taken from Rhodes and VanRooyen ( 1962).
than 5 years of age are commonly symptomless carriers. ECHO viruses are widespread during the summer season in the normal population. In an outbreak of aseptic meningitis, lOO%of the hospitalized patients were releasing ECHO viruses in their feces (Karzon et al., 1956). In various surveys of warmer climates it has been found that up to 15% of the children excrete ECHO viruses in the feces at any given time. In the tropics 3040% of the children excrete one or more enteroviruses at any given time during the year, while in the temperate lands infection is commoner in the warmer (summer and fall) months (Rhodes and Van Rooyen, 1962). Although group B Coxsackie viruses are rarely found in the feces, 1 to 7% of normal persons excrete group A viruses. Other investigators have found enteroviruses of one group or another in as many as 60% of stool specimens from children. IV. Transmission of Viruses Through Water
A. POLIOMYELITIS In discussing the possible water-borne dissemination of poliovirus in 1943, Maxcy proposed the following criteria for water-borne diseases: (1) The prevalence of the disease must be correlated with a poor sanitary environment including, but not necessarily depending directly upon, water supplies subject to human fecal pollution. ( 2 ) Under these conditions one would expect cases to be excessive in number and scattered in time, place, and persons (except as modified by immunity). ( 3 ) A population using the suspected water supply should show a higher incidence than other populations using other water supplies very similar in all respects. An increase in morbidity should be seen in populations in which there is a breakdown of proper water treatment, and, conversely, a decrease should occur in populations in which proper treatment is installed while there is no other change in living conditions. (4)The only medium to which all patients have been exposed must be a water supply contaminated with human fecal material. At the time these criteria were framed they were not truly documented
SPECTRUM
OF
TABLE IX CLINICAL FEATURES CAUSED BY ENTEROVIRUSES PATHOGENIC FOR M A N ~
Causal agents Clinical feature Paralysis Aseptic meningitis Summer fevers Rashes (maculopapular or vesicular ) Myocarditis
3+
Coxsackie
Polio L2,3 L2,3
A A2,7,9 A2,4,7,9
B
ECHO
B2, 3,4, 5 B1 to 5
E l , 2,4, 6, 7,9, 11, 12, 16 El, 2, 3, 4, 5, 6, 7, 9, 11, 12, 13, 14, 15, 16, 18, 19,20,21
1,2,3
Several types A2, 4, 9, 16
B1 t o 5 B1, 3, 4, 5
Several types E2,4, 6,9, 14, 16, 18
Pericarditis Herpangina Epidemic pleurodynia and myalgia Diarrhea Respiratory illness Taken from Rhodes and VanRooyen (1962).
!2 9
A2, 4, 5,6, 8; 10 B1 to 5
B1, 3
! 9
B2,3,4, 5
A2 1
5 fZ 3i
B2, 3, 4, 5
L2,3
r $
E2,7,8, 11, 12,14,18, 19 E6, 8, 11, 20, 28
*3
8
164
STEPHEN ALAN KOLLINS
for poliovirus diseases (Maxcy, 1943). In fact, in a later paper, Maxcy himself felt that the principal mode of transmission is personal contact and that only under special conditions could spread occur via accessory or indirect methods (Maxcy, 1949). Because of the nature and severity of the paralytic forms of the disease there has been considerable interest in the epidemiology of poliomyelitis. Epidemics were known in Europe before 1907, the year in which the first poliomyelitis epidemic was recorded in the United States. As early as 1929 it was felt that water-borne spread was possible (Kelly, 1957). By 1940 it was noted that the incidence of polio was high in communities using swimming facilities polluted with sewage. These high incidences were more notable and more persistent than in cities of the same size or larger not using such bathing water, implicating the bathing water as a vector of transmission (Ellsworth, 1940). There have been only two well-documented outbreaks of polio which are considered to have been water borne, In 1952 in Huskerville, Nebraska (Bancroft et al., 1957), contamination of the water distribution system by the back siphonage of faulty plumbing was held responsible for an outbreak. A high incidence in people drinking water near the source of contamination, with a decrease in incidence with distance from the source, provided epidemiological evidence that the outbreak was water borne. The decrease in incidence was attributed to dilution and disinfection with chlorine. Even though coliform organisms were present in the drinking water, the evidence was only epidemiological and merely suggestive owing to the explosive nature of the outbreak. Another outbreak occurred in Edmonton, Alberta, Canada, in 1953 (Clarke and Chang, 1959). Here again the evidence is epidemiological, but pollution of the Saskatchewan river (the water source for Edmonton) by failure of the chlorination facilities of a sewage treatment plant at a town 20 miles upstream is held responsible for the outbreak. A total of 322 cases and 16 deaths were reported.
B. INFECTIOUS HEPATITIS Although infectious hepatitis is transmitted largely by personal contact, the only clear and well-defined water-borne outbreaks of enteric viruses have involved its spread. It is probably the most common waterborne disease in the United States today even though the number of documented cases is small (Clarke et al., 1964). In addition to those cases reported by Clarke and Chang in 1959 (see Table X ) more recent outbreaks have been reported (Henderson, 1961; Poczenik et al., 1956; Poskanzer and Beadenkopf, 1961; Mosley et d,1959; Vogt, 1961; Randel
REMOVAL OF HUMAN ENTERIC VIRUSES IN SEWAGE
165
and B o w , 1962). Because the agent is not culturable under laboratory conditions we must rely upon epidemiological evidence. The following discussion of an outbreak in a summer camp, the New Delhi catastrophe, and the Raritan Bay incident serves well to point out the potential dangers in municipal systems as well as in the pollution of waters used for cultivation of shellfish. Neefe and Stokes (1945; Neefe et al., 1957) reported an explosive outbreak of infectious hepatitis in a summer camp in 1945. Their paper excellently demonstrated the considerations to be taken in evaluating epidemic conditions. Using human volunteers they found that the chief agent of the disease was probably the feces of infected patients. The evidence indicated the disease was water borne. Because of the explosive nature of the outbreak it was assumed that many persons were exposed to and infected from a common source at nearly the same time. For this reason transmission through air or by fomites, biting insects, or direct contact was considered of little importance. Possible vectors were food, milk, bathing water, and drinking water. All but the last failed to account for one or more of the main features of the epidemic. Five features of the unusually high incidence rates must be noted: (1)During the late summer of 1944, 350 of 572 summer campers developed hepatitis within 13 weeks; in 344 of the 350 the onset occurred within a 7-week period. ( 2 ) There was a higher total incidence in the females than in the males. (3) There was a predominance of early-appearing cases among the girls and later-appearing cases among the boys. ( 4 ) Infections occurred simultaneously in a number of patients who had not been in personal contact with one another. ( 5 ) The infectious agent was acquired at the camp with apparent ease, in contrast to the apparent lack of ease with which it was acquired by those in contact with the infected persons away from the camp. The well that was used as a water supply was located within 150 ft. of three cesspools. The soil was primarily limestone, with from a few inches to 4-6 ft. of top soil covering a layer of “hard pan.” There were cracks and fissures in the bed rock, permitting drainage to ground water. The cesspools never overflowed but drained into the ground. Both the well and the reservoir were inaccessible except underground. The well showed bacteriological evidence of fecal contamination on two different occasions. In volunteers water from this well caused an illness associated with hepatic dysfunction and an immunity to the agent excreted in the feces of camp patients. Presumably this illness was produced by the same agent infecting the young campers. The cesspool receiving wastes from the girls’ infirmary as well as from the cabin in which the first two
TABLE X REPORTED OUTBREAKS OF WATERBORNE INFECTIOUS HE PAT IT IS^ Total cases
Year
Place
1944
Children’s camp near Philadelphia, Pa.
350
1945-46
Small town on Tidal River, Pa.
52
1952
Mountainous farming area in Pa.
22’
1952
Summer
Water supply incriminated
Water treatment
Evidence of con tamination
Domestic
camp
in
102
1956
Two Arctic villages in Alaska
21
1956
Davies County, Ky.
18
1940
German troop installation in Paris
84
1943
Sanatorium in Sweden
178
Tenn.
Driven well contaminated by cesspool 75 ft. away Sewage pollution of river at point of intake Drilled well polluted by cesspool 50 ft. away Spring contaminated by sewage from broken sewer Various natural waters with fecal contamination Wells contaminated by sewage from septic tanks Foreign Distribution system contaminated by sewage from damaged sewers Conduit contaminated bv defective drain
RTone
Bacterial; viral agent demonstrated
Municipal
Epidemiological
None
Bacterial
Chlorination
Bacterial; dye tracing
None
Coliform organisms in most samples
None
Coliform organisms in most samples
Municipal
Epidemiological
None
Bacterial
1944-45
Hotel in mountainous region in northern Sweden
34
1945
American troop encampment in Italy
100
1946
American military camp in a German city
1947
Sewage-contaminated
Inadequate
Bacterial
Well water with fecal pollution
None
Bacterial
31
Raw water contaminated by sewage pollution near intake
Municipal
Bacterial
Two American ordnance units in Germany
26
Contaminated stream water intended for industrial use
None
Bacterial
1947-48
American military camp in Berlin, Germany
52
Distribution system contaminated by sewage
Municipal
Bacterial
1950
British troop encampment in Hong Kong
81
Treated water contaminated by polluted water
Municipal
Epidemiological
Source with heavy fecal contamination
Municipal
lake water
30,000-
1956-57
A factory in Kalmer County, Sweden
46
Well water contaminated by sewer 50 ft. away
None
High C1- ion and high C1, and alum demand of water Bacterial
1958
Student picnic in Australia
6
River water contaminated by sewage effluent
None
Bacterial
1955-56
New Delhi, India
50,000
a
Taken from Clarke and Chang (1959).
b
Nine of these cases were traced to the well water.
168
STEPHEN ALAN KOLLINS
cases developed probably was polluting the girls’ well through underground drainage. The delay in the cases among the boys was due to the holding of water from the well before transfer to their section of the camp. The noticeable ease of infection among the campers was probably due to ingestion of the virus, whereas personal contacts away from the camp did not ingest the virus. In addition to demonstrating the water-borne transmission of infectious hepatitis and pointing out obvious potential problems existing in these epidemics, the work of Neefe and Stokes (1945; Neefe et al., 1947) was significant in that it showed that 1 p.p.m. free residual chlorine with a 30-minute contact time was not sufficient to inactivate the viral agent. This matter will be discussed further. Perhaps much more alarming is the record presented by Dennis (1959) of the New Delhi epidemic of 1955-1956. This is the largest epidemic of infectious hepatitis in history. The disease is normally endemic in Delhi at a level of 300-400 cases per 100,000 population per year (in the United States the level is less than 20 per 100,000 population). This is suggestive of a large reservoir of infection and of a very high virus density in the sewage. During the 6-week epidemic more than 7000 cases were reported, Sampling indicated that more than 29,000 cases of jaundice were unreported in the population of 1,700,000. Dennis estimates the total number of infections at 1,000,000. The epidemic resulted from massive contamination by sewage of the city’s raw water supply; the raw water might have been as much as 50% sewage! None of the cases of infectious hepatitis were traced to one of the two water treatment plants. This plant had been prechlorinating to a level of 0.7 p.p.m. residual free chlorine. The second treatment plant, which normally produced bacteriologically safe water, was the cause of the epidemic. Because of the very large demand for chlorine of the raw water, the chlorination procedures produced only low levels of combined residual chlorine. There are no data available for this plant during the week of most severe pollution, but residuals of 0.15 and 0.2 p.p.m. were seen in the weeks preceding and following this period respectively. Although no record of the bacteriological quality of the water at that time exists today, it has been stated that the treated water was free of colifonn bacteria. This lends some doubt to the validity of the coliform standard, as well as to the efficacy of flocculation and chlorination procedures in treating heavily polluted waters. It should be noted, on the other hand, that control was begun late and enacted slowly and to an inadequate level. In a properly operated plant such a high level of pollution would necessitate the immediate addition of even more flocculant and more disinfec-
REMOVAL OF HUMAN ENTERIC VIRUSES IN SEWAGE
169
tant than was added near the end of the pollution period in Delhi. Once again, the severity of the disease requires us to be aware of the dangers. During the winter and spring of 1961 a large number of cases of infectious hepatitis developed in the northeastern portion of the United States (Henderson, 1961). A total of 3883 cases were reported. Many of those infected had eaten clams some time prior to the onset of illness. Of those cases studied (a total of 554) the patients were predominantly men in their 20s and 30’s and generally in above-average socioeconomic brackets. About 50% of these men had eaten raw clams 10-60 days prior to onset. In the New Jersey area less than 10% of the general population in similar economic brackets gave similar histories of having eaten clams during the previous 120-day period. It thus appeared that the consumption of raw clams was linked to the cases of hepatitis. The source of these clams was as yet undetermined. However, the incidence of raw clam consumers among the hepatitis patients was highest in those states and even counties on or near the Raritan Bay. Because the non-clam-eating patients showed a more diffuse pattern without such a notable geographic concentration, these waters were closed to clam harvesting on May 1, 1961. That same year oysters were found to be the source of an outbreak in Mississippi, where most of the primary patients were aged 20 years or more. The literature contains an account of a similar outbreak in Sweden, spread by oysters (Rhodes and Van Rooyen, 1962). It is difficult to determine whether the shellfish in question served to concentrate the virus from the polluted water or permitted true multiplication. The expected high degree of cell specificity for viruses in general, and especially for the virus of infectious hepatitis, leads one to believe the former to be the case. It is evident that the widespread dissemination of enteric viruses may be a serious hazard to the public’s health. Large-scale epidemics are not our only concern, however, for, should a community containing a large proportion of susceptible individuals be situated downstream from a community in which enteric infections are common, the release of even very low densities of enteric viruses might be suf€icient to cause an outbreak in the second community. Even when virus levels are below detectable levels and water treatment methods are adequate by the coliform standards, the possible introduction of viruses into a susceptible community may be significant. Once infection is initiated in that community, the virus may be spread by means other than water. In addition, because there is no proof that viruses of other-than-human origin cannot enter and infect human cells, we should be certain that our treatment removes not only some viruses from water but all viruses from water (Berg,
170
STEPHEN ALAN KOLLINS
1963a). A good deal of this responsibility lies with the sewage treatment plants.
V. Presence of Viruses in Sewage Before one can be assured that viruses are removed by sewage treatment processes, the occurrence of viruses in sewage and water under normal conditions must be known, as well as the efficiency of the various sewage treatment processes in removing the viruses. Although it was known for quite some time that human feces were a copious source of enteric viruses, a reliable quantitation of this virus content was not available until 1955. Because of the problems inherent in the suitable culturing of viruses, this problem required a sophisticated technique and the development of new methods. Examination of the feces of human volunteers who had ingested live chimpanzee-avirulent poliovirus types I and I11 showed as high as lo5 to loo TCBodoses of virus excreted per gram of feces. These were peak values, the average density of viruses being about lo4 TC50 doses per gram (Sabin, 1955). As has been mentioned, the excretion of viruses by healthy individuals is essentially limited to children. Using the above data and assuming that (1) only 10% of children on the average excrete virus at any given time (this is based upon the above figures and upon other work), and ( 2 ) 31.1% of the population falls within the under-15 age group ( 1960 census); Clarke et al. (1964) have calculated that there are about 200 virus units per gram of feces on a per capita basis. Work in their laboratory has also shown the density of coliform organisms to be about 13 X loo per gram of feces, a ratio of about 1 virus to 65,000 coliform organisms. Further and more recent data indicate mean densities of coliform organisms in domestic sewage to be 46 X106 per 100 ml. and in surface water polluted with fresh sewage between lo4 and los per 100ml. This results in an expected density of enteric viruses of about 700 virus units per 100 ml. of sewage and 0.15 to 1.5 virus units per 100 ml. of polluted surface water. These calculated values must be compared with the frequency of recovery from natural sources to have any meaning. In 1940 Paul et al. (1940a) reported on attempts made to isolate poliovirus from sewage during two rural and three urban epidemics. After their samples were treated for 24 hours with 15% ether, 20-120ml. was inoculated intraperitoneally into Macacus rhesus monkeys for virus isolation. Upon the development of experimental disease the medulla and spinal cord were examined for histological demonstration of characteristic lesions. Further passage to another monkey confirmed the presence of the poliovirus. They isolated the poliovirus in four samples from two cities. It was subsequently reported by Paul and Trask (1942) that many
REMOVAL OF HUMAN ENTERIC VIRUSES IN SEWAGE
171
positive sewage samples were obtainable in the vicinity of hospitals. Poliovirus was still present after considerable dilution and up to one-eighth of a mile downstream. The authors considered the virus not to be a normal inhabitant of sewage. Their reasoning was based upon the record of negative samples they obtained from sewage even during epidemic periods. This conclusion is questionable, for in a more recent 2-year study in which sewage was regularly examined for the presence of poliovirus by the more sensitive swab method, the virus was isolated in 21% of the samples (Kelly et al., 1957). It seems probable, then, that the crude methods available in 1942 were not capable of isolating the virus unless it was present in very high concentrations. Also at that time the virus had not yet even been isolated from river water or bathing beaches (Paul and Trask, 1942). The seasonal distribution of poliovirus is well documented, its presence being common in the late summer and fall (July-October), with the highest frequencies in August (Rhodes et aL, 1950a; Kelly et al., 1957; Melnick et al., 1954). Kelly et al. ( 1955) stated that “the rare appearance of these viruses out of season suggests that they may be present in sewage continuously in small amounts and are undetectable only because of limitations in sampling and isolation procedures.” It was recognized in 1946 that the lack of recoverable poliovirus in sewage during winters and nonepidemic summers did not entirely eliminate the possibility of its presence, but rather merely excluded the presence of widespread infection in the community (Gear and Measrock, 1946). Studies on the concentration of viruses in sewage and water have been severely limited by the lack of suitable quantitative methods. Although poliovirus was recovered from sewage as early as 1940 (Paul et al., 1939, 1940a, b; Paul and Trask, 1942; Trask and Paul, 1942) and repeatedly since then (Clarke and Kabler, 1964; Carlson et al., 1942; Gear and Measrock, 1946; Kelly, 1957; Melnick, 1947; Rhodes et al., 1950a; Kelly et al., 1957; Kelly and Sanderson, 1959a, 1960a, 1964; Bloom et al., 1959), accurate quantitative methods are not yet available. The inherent lack of sensitivity in the methods requires that large volumes of sewage or water be collected and subsequently concentrated before virus isolation is possible. The virus densities in water and sewage are so low that the data obtained in such a manner demonstrate only minimal levels, for some loss of virus must be assumed. Because physical and chemical methods are employed to concentrate the viruses, grab samples are considered no more than partially quantitative. Very often the viruses are present in concentrations below those readily detectable by this intermittent sampling method. These problems led to the development of the gauze pad (or swab) method of sampling.
172
STEPHEN ALAN KOLLINS
This method of obtaining integrated and concentrated samples is a modification of a method originally used in 1916 for the detection of the tubercle bacilli ( Kelly and Sanderson, 1959a) and subsequently modified by Moore (1948, 1950) for an epidemiological study of typhoid carriers. His technique was to fold a piece of gauze 4 ft. in length and 6 in. wide into a “pad of eight thicknesses . . , attaching it firmly by one end to a long piece of stout string. The gauze was then immersed in the flowing sewage, the string attached suitably just under the manhole cover, and the gauze left in position for 48 hours” (Moore, 1948). These gauze pads were then stored in sterile containers until they were ready for examination. It is obvious that this method is not at all quantitative. There is no way to determine how much sewage has flowed through the pad, nor how much of the virus flowing through the pad has been absorbed. The losses to the pad itself during elution may be inconsistent. Because the sample is taken over a period of time (usually several days), there is no way of characterizing the sewage positively, for its nature probably varies over the period during which the pads are suspended. In fact, the viruses probably are not deposited uniformly but rather during one or several short periods (Berg, 1963b). This feature may, however, be considered an advantage of this method. For if virus release occurs infrequently, a grab sample may miss it entirely. In addition, the increased sensitivity of this method of sampling over mere concentration of a grab sample may permit detection of lower levels of transmission. This increased recovery has been shown by a comparison of the Coxsackie virus content of the fluid expressed from 24 or 48 hour swab samples with that of grab samples taken at the beginning or end of these sampling periods (Table XI) (Kelly, 1953). TABLE XI VIRUSCONTENT OF GRABSAMPLES AND GAUZEPADSAMPLES~ Sample
No. Positive
No. Tested
Crab Gauze pad
2 4
16 8
a
% Positive 12.5 50
From Kelly (1953).
When these methods were combined with ion-exchange treatment of the expressed liquid the results shown in Table XI1 were obtained. An extensive study in 1961 of the various treatment methods for gauze pad eluates (Gravelle and Chin, 1961) gave the results shown in Table XIII. Although Table XI11 appears to contradict tables XI and XII, such variations may be “due to differences in the viruses encountered or in the sensitivities of the host systems used in the two studies” (Berg, 1963b).
173
REMOVAL OF HUMAN ENTERIC VIRUSES IN SEWAGE
VIRUS
TABLE XI1 CONTENT OF RESIN-TREATED GRABSAMPLES AND GAUZE PAD SAMPLESa
Sample
No. Positive
No. Tested
70Positive
Grab (resin treated) Gauze pad (resin treated)
11 10
19 14
58 71
a
From Kelly ( 1953).
TABLE XI11 COMPARISON O F THREEMETHODS OF TREATING GAUZE PAD SAMPLESa Sample
No. Positive
No. Tested
% Positive
Unconcentrated Resintreated Ultracentrifuged (39,000 r.p.m., 1 hour)
34 34
108 108 108
31 31 53
a
57
From Gravelle and Chin (1961 ).
Table XI11 suggests that ultracentrifugation may greatly increase the sensitivity of virus detection. Other workers using somewhat lower speeds and shorter times have also indicated the superiority of this method of analysis (Melnick et al., 1954). Coxsackie viruses were originally isolated in 1947 by the New York State Laboratories at Albany. The first strains were seen in stool extracts from two children in Coxsackie, N. Y. These children were suffering from clinical poliomyelitis and were also excreting poliovirus. Further studies have shown that the incidence of group A Coxsackie viruses in stool specimens is about the same as that of poliovirus during the same time of the year, and isolation of A viruses and poliovirus together is not uncommon. In contrast the isolation of group B viruses and poliovirus together is rare, This suggests than an interference mechanism may be operating, a theory supported by epidemiological evidence. In communities in which group B infections prevail the incidence of poliovirus infections may be very low. Also, in years when both types of infections are reported the peak incidence of poliomyelitis usually follows that of Coxsackie infections by 1to 2 months (Rhodes and VanRooyen, 1962). The presence of Coxsackie viruses in sewage has been demonstrated repeatedly (Clarke and Kabler, 1964; Kelly, 1953, 1957; Clarke et al., 1964; Berg, 1963a, 1963b; Kelly and Sanderson, 1959a; Melnick et d., 1949, 1954; Kelly et al., 1955; Gravelle and Chin, 1961; Clarke et d., 1961). Considerable work has been done in New York State on the occurrence of enteric viruses in sewage, and many enteroviruses have been found (Table XIV). Kelly and Sanderson ( 1959a) have found that over 90% of raw sewage samples collected during the summer months contain viruses. About 80% of these positive samples contain Coxsackie viruses, and one-third of them contain polio and ECHO viruses.
174
STEPHEN ALAN KOLLINS
Shortly after their discovery Coxsackie viruses were isolated from sewage samples and a seasonal incidence very similar to that of poliovirus was demonstrated (Kelly and Sanderson, 1959a). Kelly et al. (1955) recognized the typical peak in August, continuous presence from June and November, and only sporadic appearance the rest of the year. TABLE XIV VIRUSESISOLATED FROM SEWAGEIN ALBANY AREA,1951-1957a Coxsackie Polio ECHO Group A, types 1-10 Group B, types 1-5 a
Types I, 11, and I11
Types 4 , 6 , 7 , 9 , 10, 11,12
From Kelly and Sanderson ( 1959a).
They attributed minor fluctuations in virus density to (1) errors in sampling and subsequent treatment, ( 2 ) differences in susceptibility in the mice used to demonstrate their presence, ( 3 ) actual fluctuations in the amount of virus in sewage even during the peak season, and (4)daily variations with flow of sewage, higher in the morning, often absent in the afternoon (Kelly et al., 1955). At first it seems possible that Coxsackie viruses are excreted year round and the seasonal distribution in sewage is due to their inability to survive winter conditions. Considerable evidence indicates otherwise: ( 1 ) Samples containing Coxsackie viruses stored at about -55°C. are not reduced in infectivity for at least 5 months. ( 2 ) Temperature variations in sewage are not great. ( 3 ) The survival of other microorganisms, such as coliform bacteria, is relatively constant throughout the year (Kelley, 1953). Further advances in technique permitted quantitation by tissue culture. In 1957 Kelly reported the presence of poliovirus in sewage at the level of about 80 PFU (plaque-forming units) per 100 ml. or less in nonconcentrated sewage. The difficulty of isolating these viruses from potable water becomes evident. If the ratio of coliform organisms to enteric viruses in sewage is maintained, then in potable waters with a coliform density (MPN) of less than two per 100 ml. a concentration of about 10,000-fold would be necessary to isolate the virus as plaque-forming units. It should be noted that this concentration of PFU is much higher than the earlier-observed monkey infectious doses (Melnick, 1947). This demonstrates the variability of results which may be due to variations in host systems. Further work describing host systems indicates that the isolation of all the agents present in a single sample may require the use of several host systems (Kelly et al., 1957). The most definitive attempt to quantitate the enteroviruses in sewage was reported by Kelly and Sanderson in 1960 (1960a). This work utilized
REMOVAL OF HUMAN ENTERIC VIRUSES IN SEWAGE
175
the plaque-forming ability of the enteroviruses in tissue culture combined with the sensitive swab method of collecting samples. Swab expressions ( 100ml. ) were concentrated on Dowex 1resin, eluted, and then treated with antibiotics and diethyl ether before storage in a dry ice chest. Monolayers of monkey kidney epithelium were inoculated with 0.5-ml. samples and incubated at 33°C. Plaque formation was studied daily until the tissue degenerated. Confirmation of virus strains was done serologically as well as by testing for plaque-forming ability in another cell system. The calculations were based upon the number of plaques formed and corrected for sample volume. A concentration factor of 4 was assumed, to compensate for the efficiency in collection of the swab method. Maximum densities of 5 virus units per 100ml. during the cold months and of 100 virus units per 100ml. during the warm months were observed. Because of the inherent inefficiency in isolation of enteroviruses, these results should be examined carefully. The values compare favorably with the theoretical calculations described in preceding pages based upon colifonn density (700 virus units per 100 ml. ), the true values probably being about 500 virus units per 100 ml. (Clarke and Kabler, 1964).
VI. Removal of Viruses by Sewage Treatment Methods A. STORAGE As early as 1929 it was known that poliovirus was capable of surviving for more than 100 days in water. In 1942 it was shown that poliovirus was not removed completely from artificially heavily contaminated water by storage at refrigeration temperatures in the dark (Carlson et al., 1942). It has subsequently been seen that in river water contaminated with a 1:200 suspension of feces the virus was not removed completely at 4°C. by storage up to 188 days (Rhodes et al., 1950b). More recent work (Clarke and Chang, 1959; Clarke and Kabler, 1964) has shown poliovirus survival in stored sewage samples greatly exceeding the survival of enteric bacteria, especially at low ( 4°C. ) temperatures. Early work with Theiler's virus (Fair et al., 1947) has shown survival in water of longer than 315 days at 8"C., 132 days at 20"C., and 21 days at 30°C. It was noted here that survival appeared shorter in river water and sewage than in tap water at 23°C. and 30°C. Storage in 10% sewage at 10°C. for 440 days did not inactivate the virus, and it was thought then that the organic matter present protected the virus. In 1945 it was shown that the virus of infectious hepatitis could be stored at least 10 weeks and still infect human volunteers (Neefe and Stokes, 1945). The virus is also capable of remaining infective for several
176
STEPHEN ALAN KOLLINS
years when stored frozen or liquid at 4°C. or desiccated (in yellow fever vaccine) for more than a year (Neefe et aZ., 1947). In 1953 Kelly noted that samples of sewage and gauze pads could be stored frozen at -55°C. for up to 5 months with no loss in infectivity of Coxsackie virus. In 1955 Gilcreas and Kelly demonstrated that the storage of Coxsackie A5 at 8-10°C. for 280 days in water did not inactivate the virus. Later work by Clarke et aZ. (1956) produced similar results with another strain, as shown in Table SV. TABLE XV SUHVIVALOF COXSACKIE Vmus A2 UNDERVARIOUS CONDITIONS~ Medium
Temp., "C.
Sewage Distilled water Ohio River water Raw
Autoclaved a
Survival time, days
8 20 8 20
61 41 272 41
8 20 8 20
16 6 171 102
From Clarke et al. ( 1956).
Kelly (Kelly and Sanderson, 1959a) has also reported that long storage in the cold may inactivate coliform organisms without inactivating COXsackie viruses. At higher temperatures the converse is true, while at extremes of temperature survival of the two groups of agents is similar. Table XVI presents further data comparing survival times in sewage. TABLE XVI SURVIVAL TIMESOF VARIOUS MICROORGANISMS IN SEWAGE@ Survival time, days Microorganism
4" c.
20" c.
28" C.
Poliovirus I ECHO 7 ECHO 12 Aerohacter aerogenes Escherichia coli Streptococcus faecalis
110 130 60
23 41 32 21 20 26
17 28 20 10 12 14
a
56 48 48
From Clarke and Kabler ( 1964).
Finally, a laboratory study (Berg, 1963b) demonstrating the reduction of Coxsackie virus A2 in sewage as determined by mouse inoculation tests produced the results shown in Table XVII. It can be seen from Table XVII that the rate of loss of virus activity is a function of the temperature of storage, with the most rapid inactivation occurring at the higher tem-
REMOVAL OF HUMAN ENTERIC VIRUSES IN SEWAGE
177
peratures. At very low temperatures the virus remains active for great lengths of time. Even at higher temperatures the period needed to completely inactivate the virus particles is long. Although the application of heat would decrease the necessary storage time considerably, this method TABLE XVII EFFECTS OF STORAGE ON COXSACKIE VIRUSA2 AS DETERMINED BY MOUSEINOCULATION TEST@ 20” c.
8” C.
Days stored
5 20 41 61 117 a
No. dead/ no. inoculated
9/9 13/17 9/19 8/15 0/5 From Berg (196313).
No. dead/
70Dead 100 76 47 53 0
no. inoculated
14/15 4/11 1/19 018
-
% Dead 93 36 5 0
-
of treatment is obviously of limited practical value (Berg, 1963b; Bagdasaryan, 1961) . The mechanism of inactivation during storage remains to be discovered. Because of the increased survival times observed with storage in distilled water or in autoclaved river water, it seems that inactivation involves some action by the natural flora. The fact that the rate of kill almost doubles with a 10°C. rise in temperature is suggestive of a chemical reaction. The effect of organic matter is uncertain, for some work reports that survival is longer in clean or treated water than in raw sewage or polluted water, yet one would expect particulate matter to enhance survival by providing a place where the virus would be protected from the “natural elements.” Much more work needs to be done in this area before definite answers will be obtained. B. PRIMARY SEDIMENTATION Primary sedimentation is the most common and often the only treatment afforded sewage before it is discharged. It can be considered, in a sense, an abbreviated type of storage, measured in hours instead of days. From the discussion of storage it may be inferred that effluents from primary sedimentation tanks should frequently contain the viruses present in raw sewage. In practice this i s the case, for, as will be demonstrated, the virus-removal capacity of this method is slight. In an early field study poliovirus was recovered from both settled sewage and raw sludge (Gear and Measrock, 1946). Sedimentation in an Imhoff tank had no effect upon the virus, for it was isolated from the effluent just as frequently as from the influent to the tank (Kelly et al.,
178
STEPHEN ALAN KOLLINS
1955). Other work has produced similar results, with 93% recovery of viruses from the primary tank effluent compared with the raw sewage influent (Kelly et al., 1961). Similarly, another study showed 23.8% of settled sewage samples to be positive for enteric viruses when the raw sewage displayed only 32.6% positive samples ( a 73% recovery). The raw settled sludge, in this case, contained virus particles in 38% of the tests (Bloom et al., 1959). Many types of virus agents are isolated from primary treatment plants ( Table XVIII). TABLE XVIII FROM PRIMARY TREATMENT PLANTS~ VIRUSES ISOLATED Raw sewage
virus
Polio I I1 I11 Coxsackie A B 2 3 4
+ + + + + + + +
Primary effluent
+ + + + +
Outfall
+
+
Unknown a From Kelly et al. ( 1957).
Very often field observations show no decrease in the frequency of positive samples and also no change in the kinds of viruses isolated from primary treatment effluents, whether from Imhoff tanks or from mechanically cleaned sedimentation tanks. In some cases the concentration of viruses (PFU per milliliter) drops, probably owing to the virus content of the settleable solid (Kelly and Sanderson, 19591,). It has also been observed, however, that the virus content appears to increase with primary treatment ( Mack et al., 1962). With influent samples containing viruses 25% of the time and settled sewage showing viruses 24.3% of the time, one would assume there was essentially no change due to primary treatment. Quantitive examination showed that the raw sewage contained 34.9 PFU per milliliter while the settled sewage contained 52.1 PFU per milliliter. Not totally inexplicable, this is thought due to a breakup of the particulate matter with the release of virus particles therefrom. A rather complete study of the problems was completed in 1961 by Clarke et al. These investigators seeded 20 gallons of raw sewage with poliovirus type I and mixed it thoroughly. Portions were transferred to 4-liter bottles and allowed to settle. Samples were drawn carefully from the top and analyzed for virus titer, coliform count biochemical oxygen demand (BOD), and suspended solids. Although a 3-hour settling slowly reduced the BOD and suspended solids, it had little effect on enteric
179
REMOVAL OF HUMAN ENTERIC VIRUSES I N SEWAGE
virus content. At the end of 24 hours a considerable portion of the virus activity remained, while 75% of the suspended solids had been removed (Table XIX). The sharp decrease in virus content between the 3- and 6-hour readings indicates a removal by some method other than sedimenTABLE XIX EFFECTOF SETTLING ON RAW SEWAGE CONTAINING TYPEI POLIO VIRUS^
-Suspended Solids
Settling time, hrs.
PFU
% Virus
per ml.
remaining
p.p.m.
% Remaining
0 0.75 1.5 3.0 6.0 12.0 24.0
310 300 300 330 230 220 220
100 97 97
72 49 44 40 28 20 18
100 68 61 56 39 28 25
a
no loss
74 71 71
From Clarke et al. (1961).
tation, which one would expect to result in gradual removal of viruses. A repetition of this experiment gave very similar findings. A somewhat greater virus removal was seen, but again it did not begin until the sharp drop after 3 hours was seen. The final removal of suspended solids at 24 hours was almost identical with the previous results. Primary sedimentation, then, cannot be assumed to remove more than a slight portion of the enteric viruses present in raw sewage. Field studies have shown viruses to be present in the effluent of both primary sedimentation tanks and Imhoff tanks as well as in the settled sludge. One would expect to find virus in the settled sludge, for it represents a sample taken over a considerable period of time. The finding of virus in the sludge while the raw or settled sewage shows no viruses is not surprising, for the virus content of the sewage may change considerably in a period of hours. The sludge, consisting of material other than the influent, is retained for longer periods than the dentention time through the tank. C . TRICKLING FILTERS There have been few studies of the removal efficiency of trickling filters. All of these studies were field observations. As will be evident, they all suggest that trickling filters are of little value in the removal of enteric viruses. Viruses have been isolated as frequently from trickling filter effluents as from the raw sewage influents (Kelly and Sanderson, 1959b). Kelly has reported the recovery of Coxsackie viruses in two of four trickling filter effluents when the raw sewage inffuent contained the virus in seven of 13 samples (Kelly et d.,1955). Another study demonstrated a 70% recovery when 34 trickling filter effluents contained viruses
180
STEPHEN ALAN KOLLINS
as compared to the 49 virus-containing samples of raw sewage (Kelly et al., 1961). Further work confirmed that trickling filters are not effective in removing Coxsackie viruses. Other viruses were not isolated after filter treatment, while Coxsackie B2 appeared in both the trickling filter effluent and downstream from the outfall. This downstream recovery was thought to be due to drainage from sludge drying beds between the two sampling points ( Kelly et al., 1957). Reductions have been observed, however, in the concentration of viruses across the filter. As much as 40% reduction was seen in one instance, suggestive of some virus removal, but no virus could be recovered froin the matter deposited on the filter (Kelly and Sanderson, 1959b). Because only one sample of the slime was taken, this does not necessarily discount removal. In another study the virus content after trickling filter treatment varied from 12.5 to 53% of that found in raw sewage (Kelly and Sanderson, 1960a). The results of this study are shown in Table XX. TABLE XX EFFECTS OF PRIMARY SETTLING AND PASSAGE THROUGH A TRICKLING FILTERON VIRUSCONTENTOF SEWAGE& Virus Content (PFU per 100 ml. )
Sewage Sample
Observation Raw sewage Primary settling Trickling filter effluent a
1
2
124 90 68
160
-
less than 20
Kelly and Sanderson (1960a).
It has been noted that virus recovery was possible from trickling filter effluents when no virus was seen in the raw sewage (Kelly, 1953). Like settled sludge, the filter slime is maintained for periods much longer than the “detention time” of the sewage through the filter. Its nature is very different from that of raw sewage, the growths permitting the lodging of virus particles over a considerable area. Its virus content, therefore, is probably very different from that of the influent sewage.
D. ACTIVATEDSLUDGE This method of treatment has been studied far more than any other and appears to be the most effective means of removing enteric viruses from sewage. Qualitative field work (Bloom et aZ., 1959) has shown that viruses are much more easily demonstrated in raw sewage or primary sedimentation effluents than in the subsequent effluent from activated sludge treatment (Table XXI),
181
REMOVAL OF HUMAN ENTERIC VIRUSES I N SEWAGE
TABLE XXI EFFECTSOF PRIMARY SEDIMENTATION AND ACTIVATED SLUDGETREAT MEN^ Sewage sample Raw sewage Primary sedimentation Activated sludge treatment and secondary sedimentation a From Bloom et
% Samples containing virus 31 24
5
al. (1959).
After activated sludge treatment Kelly and Sanderson were able to isolate enteric viruses from only 4 of 41 sewage samples. They found that recoveries were even higher from samples of settled activated sludge than from raw sewage, indicating that the sludge floc may be important in removing the viruses (Mack et al., 1958). Examination of the mixed liquor, in another experiment, did not yield viruses; however, they could be recovered from the return sludge seed. This indicates that the removal process is not necessarily a destructive process. This work showed further that the sludge-virus complex is probably very stable, for, in a laboratory test, more than 99% of virus added to activated sludge was removed from the supernatant but not recoverable from the sludge (Kelly and Sanderson, 195913). In studying the virus density by plaque formation the same authors found that after activated sludge treatment and secondary sedimentation the effluent contained only 20% the number of viruses detected in the corresponding raw sewage (Kelly and Sanderson, 1960a). The qualitative nature of these determinations should be emphasized, for it is highly unlikely that all viruses present in a sample would be detectable using a single host system. The inherent problems in virus detection have been discussed above, but mention should be made of the often-confusing quantitative estimates of virus density. The measure of efficiency of virus removal is usually assumed to be a decrease in the number of virus-containing samples at each point through the plant as compared to the number of positive samples of raw sewage. Very often plaque formation in tissue culture is used to quantitate the number of viruses in each sample (PFU per milliliter). It is assumed here that removal of viruses should be accompanied by a decrease in the plaqueforming ability of the sample compared with that of raw sewage samples. Very often the study of this property produces confusing results owing to the inherent variability of the methods. This lack of adequate quantitative methods reflects upon the basic assumptions (Berg, 1963b). Early laboratory work in 1943 by Carlson et al. attempted to simulate removal by activated sludge treatment under field conditions. The sludge
182
STEPHEN ALAN KOLLINS
used contained 16% ash, and the mixed liquor was at a pH of 6.8. This sludge was found to remove 90% of the 5-day BOD and 95% of the suspended solids in 6 hours, at the same time producing 15 p.p.m. NO,nitrogen. Before use the sludge had been aerated in the laboratory for 24 hours. As will be seen shortly, this may have been a very important step in the procedure. Concentrations of volatile solids of 1100, 2200, and 3300 p p m . were used, one part of poliovirus being added for every 300 parts of activated sludge. The mixtures were aerated up to 9 hours, and samples were taken after 2, 3, 6, and 9 hours of aeration for mouse infectivity tests. It was found that 1100 p.p.m. activated sludge removed the virus from the mixed liquor in 6 hours of aeration. These samples were not infective for mice. Heavier sludges with longer aeration periods will largely eliminate the infectivity of the sewage, It was difficult to produce mixed liquor which was nontoxic for the mice, and nonspecific deaths were discounted. The mechanism of virus removal was as yet unclear. The authors suggested four possible mechanisms: ( 1) mechanical adsorption onto the floc, ( 2 ) partial precipitation by aeration, ( 3 ) partial flotation on the surface of virus-containing shreds of brain and cord protein used as inoculum, and (4)virucidal, oxidative, or enzymatic activity of the sludge itself. Two extensive studies of activated sludge treatment remain to be discussed. The first, by Clarke et al. (1961), utilized both batch and continuous-flow models. The batch method showed rapid removal of the Coxsackie virus A9; 99.26%was removed in 45 minutes, with an additional 90% of the remaining virus being removed with every 2-fold increase in time (Table XXII). In this phase of the study a linear log-log relationship between the ratio of PFU of virus removed to p.p.m. suspended solids present and the PFU of virus remaining in the supernatant was seen, Demonstrating conformity to a Freundlich isotherm, this suggests that an adsorption phenomenon is operating. At low temperatures the sludge appeared to be a poor adsorbant, and a significant increase in virus removal TABLE XXII
EFFECTOF BATCHMETHODOF ACTIVATED SLUDGE TREATMENT ON CONTENT OF COXSACKIE A9 VIRUS^ Virus concentration (PFU/lOO ml. ) Time, hr.
Initial
Remaining
"/o Virus reduction
0 0.75 1.5 3.0
1.08 x 105
-
-
800 80 20 3
99.26 99.93 99.98 99.998
6.0 0
Clarke et al. (1961).
~
REMOVAL OF HUMAN ENTERIC VIRUSES IN SEWAGE
183
at 4°C. was possible only with a very large increase in sludge concentration. Viruses were recovered from only a small number of samples of settled sludge, which implies either that a very stable complex is formed or that the virus is somehow inactivated. Using a bench model continuous flow unit, Clarke et al. (1961) obtained further evidence that removal was an adsorption phenomenon. Further conformity to a Freundlich isotherm was clearly evident. The unit had an average detention time of about 7 hours and was demonstrated to remove about 98% Coxsackie virus A9 and about 90% poliovirus type I when the concentration of volatile solids was at least 400 mg./liter. While higher concentrations of volatile solids ( 6OO-15OO mg./liter ) did not increase removals, concentrations below 400 mg./liter resulted in poor sludge formation, poor sedimentation, and lower removals of Coxsackie virus. Excellent sludge formation and good settling were evident at all levels above 400mg./liter of volatile solids, and removal of coliform organisms and fecal streptococci was substantial (greater than 95% in most cases). The mechanism of removal was assumed to be adsorption, but the nature of the adsorbant was not yet determined. Preliminary observations had shown virus removals as high as 75% with simple mixing of virus and sewage. An experiment was then performed in which the same continuous-flow unit was used, but no activated sludge seed was added. The mixture of virus and “fortified sewage was aerated for 6 to 7 hours and there was 60% virus removal. This indicated to the authors that the sewage-virus complex which is formed may play an important role in the removal of viruses by activated sludge, The sludge floc, then, must adsorb this complex, as well as “free” virus particles, to achieve the 99% removals observed. The importance of the suspended solids, colloidal material, or toxic substances contained in the sewage may well be associated with its complex-forming ability and subsequent virus removal. In the continuous-flow system it was noted that removal of Coxsackie virus A9 appeared greater than that of poliovirus type I. Because the Coxsackie virus is not as stable as the poliovirus, this difference may be due to a die away in the system. This hypothesis is in accord with the adsorption theory, for one would expect all members of the enterovirus group to be similarly adsorbed onto the floc. Kelly et al. (1961), in a study of the importance of aeration to the virus-removing ability of activated sludge, found that the physical state of the sludge is evidentIy unimportant in the removal of viruses (Table XXIII). Virus removal ceased when nitrogen was bubbled through the mixed liquors at a rate of from 12 to 14 liters per hour for periods up to 5 hours. An increase in the virus density was often noted in the mixed liquors so treated,
184
STEPHEN ALAN KOLLINS
a phenomenon not yet explained. The fact that aeration is required for removal, however, is not meant to imply that it is the mechanism of removal. Aeration of sewage-virus mixtures in the absence of activated sludge seed does not reduce the concentration of either poliovirus or bacteriophage. The lack of reduction of poliovirus is very surprising in TABLE XXIII EFFECT OF AEHATION AND PHYSICAL STATEOF SLUDGE ON VIRUS REMOVAL^ Aeration,
hrs.
Sludge intact PFU/ml.
1.1 x 106 2 3.7 x 105 4 2.4 x 105 a From Kelly et al. ( 1961). 0
Sludge dispersed
% ' Removed
PFU/ml.
% Removed
-
1.5 x 108 5.5 x 105 5.5 x 105
-
66 78
64 84
light of the 60-75% reductions of Coxsackie virus seen above. It is not, however, surprising that no loss of bacteriophage was observed, for the presence of coliform organisms in extremely high numbers provides an environment ideally suited for their proliferation. The experiments of Kelly et nl. (1961) demonstrate that about 50% of the virus removal accomplished by activated sludge occurs while the mixed liquors are settling, although a substantial portion is accomplished during aeration. This aeration procedure in the presence of nutrient is thought to put the floc in a state of endogenous metabolism which somehow makes it receptive to virus particles. The long period of time required for virus inactivation suggests a biological mechanism, for a chemical oxidation or inactivation would occur instantaneously. There was also no observed effect of redox potential. Two possible mechanisms were discussed, the first being an inactivation due to removal of some essential nutrient or stabilizer by the sludge. This type of phenomenon is manifested by Salmonella in its need for tryptophan in digested sludge. The theory is supported by the observation that phosphorus, a factor in virus survival in water, is rapidly taken up by the sludge floc. Another possibility is the inactivation of the virus by biological antagonists in the floc. The quality of the activated sludge-aeration, nutrients, microorganisms, foreign matter-is well suited for the development of many agents. At least four strains of bacteria have been isolated with antiviral activity. Should these antagonists play an important role, the fact that the physical state of the floc seems unimportant in the removal process is somewhat clarified. If the removal is first dependent upon an adsorption phenomenon, however, then the surface characteristics of both the floc and the virus must be very important. It is possible
REMOVAL OF HUMAN ENTERIC VIRUSES I N SEWAGE
185
that a biophysical adsorption occurs during the aeration process, followed by either inactivation of the virus or its incorporation into the sludge, resulting in a very stable complex. Perhaps a close examination of the surface properties of the viruses is in order, for this appears to control their behavior under a number of other conditions.
E. DISINFECTION It is surprising that little study has been made of the disinfection of secondary treated wastes, for the evidence above suggests that this may be the only means of inactivating the enteric viruses. The most clear cut studies of disinfectant action upon viruses have utilized water suspensions. No data on the disinfection of sewage other than recoveries from efFluents have been reported, as there have been no laboratory studies using sewage. The earliest studies of the action of disinfectants upon virus particles were reported in France in 1931 (Levaditi et al., 1931) The effects of chlorine residuals of 0.4 and 0.5 p.p.m. in water for 10-minute exposures were of particular interest, for one strain of poliovirus was inactivated at 0.4 p.p.m. while another was not inactivated at 0.5 p.p.m. Although Levaditi et al. concluded that normal chlorination practices were virucidal, they did not determine the minimum concentration required or the shortest effective exposure time, nor were temperature or pH recorded. In addition, too few monkeys were used for the conclusions to be statistically valid. Nevertheless, however unrefined their methods and techniques, their work was important in opening up this new area for study. Many of the early studies will not bear scrutiny. Even in water disinfection studies, the chlorine demands could not be closely controlled. Very often residuals dropped considerably by the end of the experiments. Initial doses as high as 120 p.p.m. have been reported to drop to 15 p.p.m. at the end of a 10-minute exposure (Kempf et al., 1942). Other studies (Lensen et al., 1947) reported that partially purified poliovirus suspensions in distilled water were inactivated within 30 minutes whenever free chlorine was present, although results were inconsistent when mixtures of free and combined forms were seen. Even in these purified suspensions it is difficult to achieve conditions where only free chlorine residuals exist. By attempting to reduce the need for chlorine, CIarke and Kabler (1954) hoped to examine the effects of disinfectant on Coxsackie virus more closely. Using varying exposure periods they found that the time of exposure, pH, and temperature all influence the inactivation of viruses. This would be expected, for these are the main controlling factors in the disinfection of bacteria. The results appear very similar to those of Butterfield and others with E. coli, but from 7 to 46 times as much free
186
STEPHEN ALAN KOLLIXS
chlorine is needed for equal kills (Table XXIV). Note that the residuals listed in Table XXIV are those required for purified suspensions of virus, which exert almost no chlorine demand at all! Gilcreas and Kelly (1955) also have reported that resistance of Coxsackie virus to disinfection is high. They warn that marginal levels of TABLE XXIV KILLS O F E . coli A N D COXSACKIE VIHUS CHLORINE REQUIHED FOR EQUAL UNDERVARIOUS CONDITIONS~
Organism
E . coli
Exposure, min. 3
5
Coxsackie virus a
3
5
PH
Temp., "C.
8.5 7.0 9.0 7.0
25 2-5 27-29 2-5
Residual Cl,, p.p.m. 0.14 0.03 1.0
1.4
70 Kill 99.9 99.9 99.6 99.6
From Clarke and Kabler (1954).
chlorination may well remove the coliform organisms but leave the viruses relatively unaffected. Nonlinear inactivations have been reported with three poliovirus strains (Kelly and Sanderson, 1957). The data suggest that types I and I11 are more resistant than type 11, for exposures of 15 to 30 minutes were needed to completely inactivate the former while only 4 minutes was required for type I1 at the levels studied. This may well be true, for it appears that the most frequent isolations of poliovirus from sewage are among groups I and 111. Later studies by the same workers noted greater sensitivity of group I1 to chlorination (Kelly and Sanderson, 1958, 1960b). Here again it appears that chlorine residuals in water sufficient to kill coliforms will not completely inactivate poliovirus. Strain type and pH were further demonstrated to affect the combination of exposure time and concentration required for inactivation. In water at pH 7, 25"C., exposure to residual chlorine concentrations of at least 0.3 p.p.m. for at least 30 minutes was necessary; at higher pH levels or lower temperatures, more intensive chlorination ( higher residual ) was required. Combined chlorine was found to react more slowly than the free forms. At p H 7, 25"C., inactivation of poliovirus required 30 minutes' exposure with at least 9 p.p.m. combined residual chlorine. With residuals of 0.5 p.p.m. more than 7 hours was required for virus inactivation! In contrast with the observations made above, a lower pH decreased the rate of inactivation. It is obvious, then, that under conditions where combined and free residuals are both present, a pH must be chosen which is optimum for the particular system. Because of the greater effectiveness of HOCl and the slow action of the chloramines, lower pH levels may be indicated.
REMOVAL OF HUMAN ENTERIC VIRUSES IN SEWAGE
187
As stated previously, the field observations of disinfectant action upon enteric viruses are not abundant. They do, however, indicate that current treatment methods may not be sufficient to inactivate the enteric viruses completely, although there has been no uniform opinion presented. Few practical applications have been suggested other than breakpoint and superchlorination. Standards for treatment probably should be increased to insure virus-free water. The observations which have been reported are very inconsistent. Kelly et al. (1961) detected enteric viruses in 19 samples of chlorinated effluent from a trickling filter. The raw sewage influent contained viruses in 49 samples, indicating a removal of about 60%. In another plant using essentially the same method of treatment, 80% removal was reported for the chlorination step. This plant was not always this effective, for overloading and mechanical difficulties often reduced the chlorine residual to zero (Kelly et al., 1955). Other reports state that poliovirus was not recoverable from the chlorinated effluent of a trickling filter plant, although it was recovered from other samples taken throughout the plant (Kelly et al., 1957). At another time, it was reported that this virus was not present in more than 12.5% of the samples from the same effluent! Activated sludge treatment must also be coupled with chlorination to produce virus-free effluents. The need for substantial free residuals as well as adequate contact times is clearly evident, for viruses have been recovered from activated sludge plant effluents chlorinated to 0.5 p.p.m. for a 15-minute contact period at pH 7.2-7.5 (Berg, 196313). Other evidence indicates that residuals of less than l p.p.m. in sewage are ineffective. Effluents from secondary treatment plants were shown to contain viruses about one third of the time even though they contained 0.5 p.p.m. residual chlorine. Free residuals of 0.1 to 0.3 p.p.m. in raw sewage show no effect on the virus content. Combined chlorine residuals of 0.5 p.p.m. require as much as 4 hours’ contact to be effective. Depending on pH and temperature, for 15-minute exposures to inactivate enterovirus at least 9 p.p.m. combined chlorine residual is required (Kelly and Sanderson, 1959b). The virus of infectious heptatitis has been reported by Neefe and Stokes ( 1945; Neefe et al., 1947) to be capable of withstanding 40 minutes’ contact with free residuals of 1.0 p.p.m. In volunteers there was no change in incidence rate, severity of cases, nor length of the incubation period after such treatment, and the virus was assumed to be completely unaffected. Superchlorination to residuals of 15-23 p.p.m. with contact periods of 30-60 minutes attenuated the virus to some degree, but subclinical hepatic disturbances in the human volunteers were still suspected. At another time breakpoint chlorination was thought effective, since not
188
STEPHEN ALAN KOLLINS
only was clinical disease absent in volunteers but no resistance to reinfection was demonstrable serologically.
F. SETTLEDSLUDGE The fate of viruses in settled sludge is highly dependent upon the manner in which the sludge is disposed of. Raw sludge contains the virus particles removed by both primary and secondary sedimentation (Kelly and Sanderson, 1959b). Viruses have not been found upon examination of either digested sludge (Kelly, 1957) or settled sludge in an Imhoff tank (Kelly and Sanderson, 1959b). Any viruses initially present may have been inactivated by the partial digestion of the sludge. It is also possible that no viruses were initially present in the sludge, for passage through an Imhoff tank with a 2-hour retention time has been shown to be ineffective in removing viruses, the same recoveries being obtained before and after treatment. It seems doubtful that thickening or other physical treatments such as elutriation or vacuum filtration will inactivate the enteric viruses. Most viruses are not extremely resistant to heat and incineration at high temperatures. Wet oxidation methods would probably result in a virus-free product. It is well known that sand filtration alone will not effectively remove viruses from water. For this reason, filtration of sludge (or sewage) over drying beds must be done carefully, for contamination of nearby ground water by the filtrate is possible. Such filtrates have found their way back into the streams, and enteric viruses have been seen well below outfalls when not present in the effluents. This observation has been discussed previously. G. OXIDATION LAGOONS Removal of viruses in stabilization ponds has been studied neither in the laboratory nor in the field. Generally involving no more than large excavations, usually lacking forced aeration, the treatment is essentially a form of storage combined with some biological action. We have seen that sedimentation does little to remove the viruses. Here if it were effective at all it would merely bring them to the bottom of the pond. In activated sludge treatment forced aeration appears essential for removal of viruses. In the lagoons not only is forced aeration lacking, but, in some cases, the lagoons may be completely devoid of oxygen! Also, if the surface properties of the sludge are important, it must be remembered that here we are dealing with a nonflocculated (dispersed) growth. The removal, if any is to be effected, must depend upon the biological activity which is occurring. If the observation cited with Imhoff tanks
REMOVAL OF HUMAN ENTERIC VIRUSES I N SEWAGE
189
truly indicates removal due to the digestive process, then perhaps some removal may be expected in an anaerobic lagoon. I doubt very much that complete virus removal will be found.
VII.
Summary
Aware of the demands being placed upon our country’s water resources, we have recognized the potential hazards of water-borne transmission of enteric virus disease. These diseases are many in number and vary greatly in their clinical manifestations, ranging in severity from very mild cases, which may escape the attention of a physician entirely, to the paralytic and fatal cases of poliomyelitis and aseptic meningitis. The presence of large numbers of these viruses in feces and sewage has been well documented. The catastrophic water-borne spread of infectious hepatitis in Delhi as well as numerous other outbreaks around the world speak for themselves. The possible transmission of poliomyelitis and other enteric diseases warrants careful consideration of the removal of their agents from our water supply. We have observed that during the summer and fall enteric viruses are present in sewage in large numbers. A significant step in their removal may be effected with careful treatment of our domestic wastes. Both laboratory experiments and field observations have been made. In most cases, these studies have been limited by lack of suitable methods, especially those of a quantitative nature. The obligate intracellular culture of these agents requires a biological host system. Only with the advent of tissue culture techniques and subsequent observations on the plaque-forming ability of these viruses have partially quantitative methods been developed. Limitations in sampling methods and the subsequent treatment of samples still render these methods only partially quantitative. Considerable work must be done in these areas before evaluation of our sewage treatment processes can become more refined. The possible transmission of these viruses at levels undetectable by current methods adds emphasis to this statement. The extent of removal of enteric viruses by a number of individual treatment methods has been discussed. The survival of these viruses greatly exceeds that of most bacteria, especially at low temperatures. The inactivation of coliform bacteria in much shorter time periods than those required for virus inactivation reflects unfavorably upon their use as a standard for water quality. In addition, the mechanism of virus removal with storage is as yet undiscovered. For this and other practical reasons, the use of storage is obviously of limited value. Because primary sedimentation is the most common and often the only
190
STEPHEN ALAN KOLLINS
treatment afforded sewage before its ultimate disposal, its virus-removal potential has been well examined, Unfortunately, neither mechanically cleaned sedimentation tanks nor Imhoff tanks are effective in virus removal. The problems inherent in treating the settled sludge are obvious but may be solved easily if methods such as wet oxidation or high-temperature incineration are employed. The action of sludge digestion is questionable, although virus-free products have been reported. The effectiveness of trickling filters is also questionable, and only a few studies have been made, The removal of viruses by the filter growth appears probable, but their subsequent release is uncontrolled. Viruses have even been recovered from filter effluents when the influent sewage was virus-free! The best-studied treatment method has been that of activated sludge. Although the absolute removal of viruses appears impossible by any single method, this appears the most effective means for removal available today. Considerable evidence in favor of removal by the sludge floc exists, although quantitative determinations have proved inconsistent. The most probable mechanism of removal appears to be biophysical adsorption. Subsequent virus inactivation or the formation of a very stable “complex” is also probable. Not only have removals been rapid, but in many cases they have been highly effective. The variables in this system have not yet been fully described. The surface properties of both virus and floc, as well as temperature, appear to influence the adsorption process. Conformity of the curve for removal to a Freundlich isotherm has been observed in both batch and continuous flow systems. The concentration of volatile solids appears relatively unimportant when it is above that level needed for good floc formation and subsequent settling. Although aeration is not the mechanism of removal or inactivation, it appears essential; possibly it renders the sludge receptive to the viruses. If this is true, a variation of the activated sludge process in which the return sludge is aerated prior to contact with fresh sewage is in order. This possibility should be examined, for the use of such altered systems is becoming very common. Although disinfection may be the only means for insuring virus-free effluents, the disinction of secondary effluents has been little studied. All of the evidence to date has indicated that high levels of disinfection are needed. The variables here appear to be the same as in the disinfection of bacteria: time of exposure, concentration, temperature, and pH. Great variations in resistance among the viruses have been observed, but it is generally assumed that substantial residuals of free chlorine must be present at the end of the exposure period for disinfection to be effective. It is clear, then, that no single treatment method is capable of removing
REMOVAL OF HUMAN ENTERIC VIRUSES I N SEWAGE
191
all enteric viruses from sewage. A biologically treated waste may still contain these viruses, and high levels of disinfection are in order. It seems doubtful that sewage treatment processes in themselves will be totally effective in removing enteric viruses. The importance of adequate treatment cannot be overemphasized, however, for even low-level transmission of these agents may be extremely significant. The total picture definitely involves water treatment facilities, and considerable attention should be placed upon their role in removal of enteric viruses from our water supplies. ACKNOWLEDGMENT This research was supported in part by training grant ES-T-8 of the National Institutes of Health, Bethesda, Md.
REFERENCES Anderson, G. W., Arnstein, M. G., and Lester, M. R. (1962). “Communicable Disease Control,” 4th ed. Macmillan, New York. Bagdasaryan, G. A. (1961). Zh. Mikrobiol., Epidemiol. i Immunobiol. 32, 292. Bancroft, P. M., Englehard, W. E., and Evans, C. A. (1957). I. Am. Med. ASSOC. 164, 836. Berg, G. (1963a). “Virus Transmission by the Water Route. I. Viruses.” U S . Department of Health, Education and Welfare, Bureau of State Services, Public Health Service. ( Mimeographed.) Berg, G. (1963b). “Virus Transmission by the Water Route. 11. Virus Removal by Sewage Treatment Procedures.” U S . Department of Health, Education and Welfare, Bureau of State Services, Public Health Service. (Mimeographed.) Berg, G. (1964). J. New Engl. Water Works Assoc. 78, 79. Bloom, H. H., Mack, W. N., Krueger, B. J., and Mallman, W. L. (1959). J. Infect. Diseases 105, 61. Carlson, H. J., Ridenour, G. M., and McKhann, C. F. (1942). Am. J. Public Health 32, 1256. Carlson, H. J., Ridenour, G. M., and McKhann, C. F. (1943). Am. J. Public Health 33, 1083. Clark, E. M., Knowles, D. S., Shimada, F. T., Rhodes. A. J., Ritchie, R. C., and Donohue, W. L. (1951). Can. J. Public Health 42, 103. Clarke, N. A., and Chang, S. L. (1959). J. Am. Water Works Assoc. 51, 1299. Clarke, N. A,, and Kabler, P. W. (1954). Am. J. Hyg. 59, 119. Clarke, N. A., and Kabler, P. W. (1964). Health Lab. Sci. 1, 44. Clarke, N. A., Stevenson, R. E., and Kabler, P. W. (1956). J. Am. Water Works Assoc. 48, 677. Clarke, N. A., Stevenson, R. E., Chang, S. L., and Kabler, P. W. (1961). Am. J. Public Health 51, 1118. Clarke, N. A., Berg, G., Kabler, P. W., and Chang, S. L. (1964). Intern. Conf. Water Pollution Res., London, 1962; pp. 523-542. Pergamon Press, Oxford. Dennis, J. M. (1959). 1. Am. Water Works Assoc. 51, 1288. Ellsworth, S. M. (1940). New En&. J. Med. 222, 55. Fair, G. M., Chang, S. L., and Moore, E. W. (1947). Annual Report on Germicides to the Quartermaster General, U. S. Army, 1946-1947.
192
STEPHEN ALAN KOLLINS
Gear, J., and Measrock, V. ( 1946). Poliomyelitis and Sewage. South African Institute for Medical Research, Dept. Environmental Sciences Library (mimeographed pamphlet ) . Gilcreas, F. W., and Kelly, S. M. (1955). 1. Am. Water Works Assoc. 47, 683. Gravelle, C. R., and Chin, T. D. Y. (1961). 1. Infect. Diseases 109, 205. Henderson, D. A. (1961). The Relationship of Infectious Hepatitis to the Consumption of Raw Clams from Raritan Bay. Paper presented at the Conference in the Matter of Pollution of the Interstate Waters of Raritan Bay, August 22-23, 1961 (mimeographed pamphlet). Karzon, D. T., Barron, A. L., Winkelstein, W., Jr., and Cohen, S. (1956). J. Am. Med. Assoc. 162, 1298. Kelly, S. M. ( 1953). Am. J. Public Health 43, 1532. Kelly, S. M. (1957). Acfa Med. Scand. 159, 63. Kelly, S. M., and Sanderson, W. W. (1957). Science 126, 560. Kelly, S. M., and Sanderson, W. W. (1958). Am. 1. Public Health 48, 1328. Kelly, S. M., and Sanderson, W. W. (1959a). Health News (Albany) 36. 14. Kelly, S. M., and Sanderson, W. W. (195913). Sewage Ind. Wastes 31, 683. Kelly, S. M., and Sanderson, W. W. (1960a). J. Water Pollution Control Federution 32, 1269. Kelly, S. M., and Sanderson, W. W. (1960b). Am. J. Public Health 50, 14. Kelly, S. M., and Sanderson, W. W. (1961). Public Health Rept. ( U . S . ) 76, 199. Kelly, S. M., and Sanderson, W. W. (1964). 1. Water Pollution Control Federation 36, 905. Kelly, S. M., Clark, M. E., and Coleman, M. B. (1955). Am. J . Public Health 45, 1438. Kelly, S . M., Winsser, J., and Winkelstein, W. (1957). Am. 1. Public Health 47, 72. Kelly, S . M., Sanderson, W. W., and Niedl, C. (1961). J. Water Pollution Control Federation 33, 1056. Kempf, J. E., Wilson, M. G., Pierce, M. E., and Soule, M. H. (1942). Am. 1. Public Health 32, 1366. Kessel, J. F., and Moore, F. J. (1945). Am. J. Hyg. 41, 25. Kramer, S. D. (1939). Public Health Rept. ( U . S . ) 54, 1914. Lensen, S. G., Rhian, M., and Stebbins, M. R. (1947). Am. I . Pul?lic Health 37, 869. Levaditi, C., Kling, C., and Lepine, P. (1931). Bull. Acad. Med. ( P a r k ) 105, 190. McLean, D. M. (1963). Pediatrics 31, 811. Mack, W. N., Mallman, W. L., Bloom, H., and Krueger, B. J. (1958). Sewage Ind. Wastes 30, 957. Mack, W. N., Frey, J. R., Riegle, B. J., and Mallman, W. L. (1962). I. Water Pollution Control Federation 34, 1133. Maxcy, K. F. (1943). Am. J. Public Health 33, 41. Maxcy, K. F. (1949). I . Am. Water Works Assoc. 41, 696. Melnick, J. L. (1947). Am. J . Hyg. 45, 240. Melnick, J. L., Shaw, E. W., and Curnen, E. C. (1949). Proc. SOC. Exptl. Biol. Med. 71. 344. Melnick, J. L., Emmons, J., Coffey, J. H., and Schoof, H. F. (1954). Am. 1. H y g . 59, 164. Moore, B. (1948). Monthly Bull. Min. Health Public Health Lab. Serv. 7, 241. Moore, B. (1950). Monthly Bull. iMin. Health Public Health Lab, Serv. 9, 72. Mosely, J. W., Schrack, W. D., and Matter, L. D. (1959). Am. I. Med. 26, 555. Murphy, W. J., and Petrie, L. M. (1946). Am. J. Public Health 36, 169.
REMOVAL OF HUMAN ENTERIC VIRUSES I N SEWAGE
193
Neefe, J. R., and Stokes, J., Jr. (1945). J. Am. Med. Assoc. 128, 1063. Neefe, J. R., Baty, J. B., Reinhold, J, G., and Stokes, J., Jr. (1947). Am. J. Public Health 37, 365. Ormsby, H. L., and Aitchison, W. S. (1955). J. Can. Med. Assoc. 73, 864. Paul, J. R., and Trask, J. D. (1942). Am. I . Public Wealth 32, 235. Paul, J. R., Trask, J. D., and Culatta, C. S. (1939). Science 90, 258. Paul, J. R., Trask, J. D., and Gard, S. (1940a). J. Bacteriol. 39, 63. Paul, J. R., Trask, J. D., and Gard, S. (1940b). J. Exptl. Med. 71, 765. Poczenik, A., Duttweikr, D., and Moser, R. H. (1956). J. Am. Public Health Assoc. 46, 1008. Poskanzer, D. C., and Beadenkopf, W. G. (1961). PubZic Health Rept. ( U S . ) 76, 745. Randel, H. W., and Bovee, C. W. (1962). Am. J. Public Health 52, 1438. Rhodes, A. J., and VanRooyen, C. E. (1962). “Textbook of Virology,” 4th ed. Williams & Wilkins, Baltimore, Maryland. Rhodes, A. J., Clark, E. M., Knowles, D. S., Shimada, F. T., Goodfellow, A. M., Ritchie, R. C., and Donohue, W. L. (1950a). Can. 3. Public Health 41, 248. Rhodes, A. J., Clark, E. M., Knowles. D. S., Goodfellow, A. M., and Donohue, W. L. (1950b). Can. J. Public Health 41, 146. Rivers, T. M., ed. (1948). “Viral and Rickettsia1 Infections of Man.” Lippincott, Philadelphia, Pennsylvania. Sabin, A. B. (1955). Am. J. Med. Sci. 230, 1. Smith, D. T., and Conant, N. F., eds. (1960). “Zinsser Microbiology,” 12th ed. Appleton, New York. Smith, W., ed. (1963). “Mechanisms of Virus Infection.” Academic Press, New York. Trask, J. D., and Paul, J. R. (1942). J. Ezptl. Med. 75, 1. Vogt, J. E. (1961). 1. Am. Water Works Assoc. 53, 1238. W. H. 0. Expert Committee on Hepatitis. (1964). World Health Organ., Tech. Rept. Ser. 285 (2nd Rept. ).
This Page Intentionally Left Blank
Oral Microbiology HEINERHOFFMAN Department of A.licrobiology, New York University, College of Dentistry, New York, New York
I. Introduction . . . . . . . . . . A.
First Observations
......
. . . . . . . . . . . . . 195
B. Nineteenth Century ..........................
111. Present State ........... A. Oral Microbiota of the Infant
B.
196
. . . . . . . . . . . . . . . . 199 ...........
Oral Microbiota of the Child and Adult . . .
D. Oral Immunology . . . . . . E. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . 343
1. Introduction Strictly conceived, oral microbiology is an excursion into microbial ecology. Unfortunately for the oral microbiologist, the ecological niche he is concerned with is the most complex of those which man harbors. Thorough elucidation demands studies in a wide range of biological phenomena involving the microorganisms, their host, and their interactions. Oral microbiologists are indistinguishable on the one hand from “pure” microbiologists concerned only with the biological characteristics of microbes in the laboratory, and on the other hand from pathologists who are concerned with the reaction of the body to these inhabitants of the oral cavity. Thus it is difficult for those not directly involved to obtain a coherent picture of a highly complex subject with a large and rapidly growing literature. It was hoped that this chapter would present a detailed as well as a comprehensive introduction, but the subject proved intractable under our present circumstances. Enough of the original plan survived, we hope, to have made the original concept apparent. 11.
History of Oral Microbiology
A. FIRSTOBSERVATIONS The first direct observations, approximately 300 years ago, of the microscopic organisms in the human mouth were, in fact, among the first observations of any microbial forms. Anthony van Leeuwenhoek‘s investigations into the curious “little animals” on his teeth were so clearly 195
196
HEINER HOFFMAN
drawn that it is possible to identify among them the three basic forms of the bacterial cell ( Dobell, 1932). This brilliant beginning, however, quickly faltered with the death of Leeuwenhoek, and for well over 100 years the science failed to develop. In large measure this was due to the inadequate microscopes available, but the inherent difficulties in studying the oral microbes played an important role as well. The magnitude of the problems is pointed up by the failure of one English microscopist as late as 1871 (F. G. Young, 1871a,b) to observe microbes in matter taken from between the teeth when examined at magnifications of 450 to 500 diameters. At this time, the American dentist Clark (1882) was so confused by the chaotic mixture of forms, living and dead, which he observed in saliva that he suspended his observations for a year or more before he regained enough confidence to renew them. The revival of Leeuwenhoek‘s studies on the oral microbiota began toward the end of the eighteenth century with the discovery of Trichomonas tenax by 0. F. Miiller in 1773 (Kofoid, 1929). B. NINETEENTH CENTURY
1. Revizjal of Interest The first half of the nineteenth century was marked by increasing interest in the microscopic study of body tissues and fluids, and it was not surprising, therefore, that a number of oral microorganisms were discovered. The fungus of thrush was first observed by Vogel in 1841, and by D. Gruby (1842) during the course of anatomical studies on infants. Robin (1853) gave a careful description of Leptothrix buccalis. Although A. Bassi in 1835 ( Andrewes et al., 1923) had established the role of Botrytis bassiana in muscardine disease of the silk worm, the wide acceptance of the idea of microbial pathogenicity had to wait for the work of Pasteur and his contemporaries (Bulloch, 1938), In regard to the oral microbiota, the typical attitude was that held by Bowditch (1850), who considered the presence of parasites in the mouth simply the result of uncleanliness, rather than evidence of disease. He advocated brushing the mouth thoroughly after every meal to maintain its cleanliness. The basis for Bowditchs recommendation appears to have been the widely popularized concepts of the Sanitary movement, which reached its first peak of support in England during the 1840s, and which eventually led to the modern public health movement. The “Sanitarians” believed that there was a simple progression from dirt to sickness-and by dirt was meant filth of every kind, especially decaying animal and vegetable matter which produced bad odors and “poisonous” effluvia. A
ORAL MICROBIOLOGY
197
prominent Sanitarian of this period, Sir Edwin Chadwick, frequently remarked that good drainage would cure toothache (Stevenson, 1955). The second half of the nineteenth century was dominated by the discoveries of Pasteur, Lister, Koch, and the other giants in the golden age of bacteriological discoveries. Oral microbiology fared well in these advances, and the record may be found in a number of monographs of the time (David, 1890; W. D. Miller, 1889; Rasmussen, 1883; Vignal, 1886). Two major problems which were of great concern then can still stimulate our interest; namely, the virulence of saliva, and the microbial causation of dental caries. 2. Virulence of Saliva The circumstances surrounding the transmission of rabies naturally threw suspicion upon the possible roIe of saliva. The infection of a normal dog by the saliva from a rabid dog was first demonstrated in 1804 by Zinke (1804) and in 1813 by Reifferscheidt (Johnson, 1948). Sanitary measures based upon these observations soon followed in Norway, Sweden, and Denmark. Destruction of stray dogs and quarantine of domestic dogs led to freedom from rabies in these countries by 1826. The disease, however, had become established in wild animals in some parts of Europe, where it continued to present a problem. The need for a satisfactory treatment of rabies eventually led to renewed attention to the role of saliva. Sternberg (1881), according to Welch (1892), was apparently the first to inoculate saliva into rabbits in an attempt to isolate the agent of rabies, but instead he obtained a diplococcus from the ensuing fatal septicemia. This work, however, was not published until after a similar study appeared by Pasteur and his associates [ Pasteur et al., 188l), who produced a hemorrhagic septicemia in rabbits by the intravenous injection of saliva from a human with rabies. In the blood of the animals which consequently sickend and died, Pasteur found a diplococcus surrounded by a clear halo. When subsequent inoculations, by other investigators, of saliva from patients with various illnesses and also from apparently healthy persons led to the same results, it became clear that the agent of rabies had not been isolated. Fraenkel (1886) later proved that the bacterium Pasteur and Sternberg had found was the pneumococcus. Subsequently Biondi (1887), working in Koch's laboratory, injected saliva into various sites of the animal body and demonstrated that a number of other pathogenic bacteria can be detected in the saliva of both healthy and ill persons. These early difficulties in the isolation of rabies virus from human saliva continued until fairly recently (Sabin and Ruchman, 1940), apparently because only during a convulsion is virus present in the saliva in sufficiently
198
HEINER HOFFMAN
large quantities to be detected by intracerebral inoculation of experimental animals (Swiss mice) ( Sulkin and Harford, 1943).
3. Dental Caries Dental caries has been detected in the teeth of prehistoric skulls (Driak, 1956), and theories attempting to explain its etiology may be found in records left by the Babylonians. Present theories implicating microbes have their origin in the mid-nineteenth century (Cox, 1952). Erdl ( 1843) was apparently the first to associate dental caries with microorganisms when he found filamentous parasites in a colorless membrane removed from the crowns of teeth by acids. Ficinus (1847) attributed dental decay to the action of microorganisms or of “denticolae” which formed colonies and proliferated on or in the enamel cuticle which they decomposed. Presumably, the denticolae attacked the enamel, destroyed the connection between the enamel prisms, and then penetrated into the dentine. Klencke ( 1850) described the parasite he labeled Protococcus dentalis, which presumably liquefied enamel and dentine in the same manner that the fungus Merulius lacrymans softens the wood of houses and furniture. Leber and Rottenstein (1867) advanced the theory that in the first stage of caries acids of the mouth act upon the enamel to make it porous. The bacteria then enter the enamel and work their way into the dentinal tubules, enlarging them and thus facilitating the more rapid penetration of the acid. Although Leber and Rottenstein did not believe that the acids responsible for the initial lesion were produced by microorganisms, they did implicate microorganisms in the progress of the carious lesion into the dentine. In their opinion, the microorganism responsible for the dentinal phase of caries was Leptothrix buccalis. However, they could not substantiate their claim as they were unable, by the cultural techniques then available, to separate microorganisms into distinct species and test their activities experimentally. Underwood and Milles (1881) were the first to express clearly the idea that decalcification of tooth substance was caused by bacteria growing on the carbohydrate residues in the mouth. That same year, W. D. Miller, working in Kochs laboratory, began a series of investigations summarized in “Die Mikroorganismen der Mundhohle” ( 1889), which developed the first solid scientific evidence for the predominant role of bacteria in the etiology of dental caries. A reading of the nineteenth century dental periodical literature gives the impression that a few alert dentists quickly incorporated into their practice the current significant developments in microbiology. However, formal recognition of the subject in the curriculum of dental schools and
199
ORAL MICROBIOLOGY
in the academic requirements for dental licensing did not progress at a comparable pace. The Royal College of Surgeons of England did not begin to require a course in dental bacteriology for students of dentistry until May 1, 1902 (Royal College, 1904). Among the American dental schools in 1915, 4 out of 47 still gave no instruction in bacteriology, while 23 had an “uncertain” number of hours scheduled (Semans, 1916).
111. Present State As Williams (1963) has described it, the oral cavity of man is the opening segment of the alimentary tract whose central space is nearly filled by the teeth, tongue, and projections of the maxilla and mandible. These structures present a series of complex surfaces which are roughly symmetrical in three planes. In the adult, the total surface area may be estimated to contain 4 x 10l2 p2 if one includes the film of saliva on the mucous membranes and teeth. If cocci with diameters of 0 . 5 ~were spread, one cell thick, over the surface, 16 X1Ol2 bacterial cells would be needed. The total number of microorganisms in the mouth, however, is actually much higher. The nature of this microbial population, the factors affecting it, and some aspects of the significance of this microbial population are the concern of the following discussion.
A. ORALMICROBIOTA OF
THE
INFANT
The oral cavity of the fetus in the healthy mother is sterile (Campo, l899), and normal birth or delivery by caesarian section usually does not introduce microbes into the neonatal infant’s mouth (Clauss, 1922; Kneeland, 1930; J. W. Smith and Bloomfield, 1950). When microbes do gain entrance during the intranatal period, they are typically coliforms and remain present only transiently (Hurst, 1957a). The extent of this initial contamination appears to depend mainly on the circumstances of the birth itself-e.g., the type of delivery and the degree of manual manipulation resorted to during parturition (Salomon, 1923). By the third day following birth, these contaminants have disappeared completely from the mouth (Schweitzer, 1919). A striking illustration of the effectiveness of this ability to reject transients is given by Torrey and Reese (1944). They describe a case of a Negro infant whose throat and nasopharynx yielded pure growth of gonococci 16 hours after birth, with the heavier growth from the throat. On the second day the gonococci had disappeared and were replaced by Staphylococcus albus. Two weeks later examination of the mother showed gonococci in her cervix and urethra. In the early days of this century, when ophthalmia neonatorum was still prevalent, gonococcal stomatitis of newborns was rare (Pfaundler and Schlossmann,
200
HEINER HOFFMAN
1912). Torrey and Reese (1945) suggested that some bacteria may fail to persist because the immature mucous surface does not provide the physical and nutritive conditions required by the bacteria. They thought it unlikely that passive immunity of the infant could account for the observations. The normal resident flora begins to colonize in the oral cavity at about the eighth to the twelfth hour of life, coincidently with the first feeding ( Bloomfield, 1922a). The numbers of microbes increase very rapidly after their first appearance. According to Torrey and Reese (1944), they seem to be derived principally from the upper respiratory tract and oral cavity of those coming near the infant. Transmission seems to occur mostly through saliva droplets expelled during talking and other respiratory activities. The organisms seeded within the infant’s mouth during the first day of life are gram-positive cocci ( Schweitzer, 1919). They included very low numbers of staphylococci in about half of the newborns examined by McCarthy and co-workers ( 1965), and much larger numbers of greening streptococci in most newborns (Dold et al., 1958; Bloomfield, 1922a; McCarthy et al., 1965). By the second day of life, the streptococci are quite numerous. The streptococci are the only organisms cultured consistently; quantities range on the average from 98% of culturable bacteria in the first day or two of life to 70% at the end of the first year (McCarthy et al., 1965). Thus, in comparison to adults, the initially established bacterial flora of the newborn infant’s mouth is dominated by fewer species, although large numbers of cells may be present. Since the total counts from swabs of the infant’s mouth do not change significantly through the end of the first year of life, it appears that the displacement of streptococci from their prominence in the oral population as the infant ages occurs by substitution of other bacteria (McCarthy et al., 1965) rather than by accretion as suggested by Kostecka (1924). From the second day, an increasing variety of forms may be found (Salomon, 1923). A small proportion of infants at this age show grampositive rods (Schweitzer, 1919), probably lactobacilli (Cox, 1952). By the third day, at which time the oral flora appears to have been temporarily stabilized (Schweitzer, 1919; Salomon, 1923), a varied microbial population is present. Hurst ( 1957a) found fusiform bacilli occasionally by the fifth day and regularly by the eighth month. She isolated fusiform bacilli from 58% of 18 babies without dentition, as well as from those with teeth. Moreover, Berger and co-workers (1959) found instances of Veillonella alcalescens occurring in the mouth on the first day of life, and occurring regularly from the end of the first week of life. The veillonella were present in the greatest numbers upon the pharyngeal mucosa and
ORAL MICROBIOLOGY
201
in the smallest numbers upon the mucosa of the oral cavity. According to Berger and co-workers (1959), spirochetes may be found only after the primary dentition begins to appear in the mouth, McCarthy and co-workers ( 1965) determined quantitatively the main groups of microorganisms that could be cultivated from swabs on a variety of selective media and found the following present during the first year of life: streptococci, nocardia, neisseria, staphylococci, actinomyces, fusobacteria, veillonella, bacteroides, leptotrichia, lactobacilli, coliforms, corynebacteria, and candida. These workers usually found a striking difference between the actual numbers of organisms identified and the total viable counts obtained from aerobic and anaerobic cultivations on sheep blood agar plates. It was thought that the discrepancy may have been a result of poor sensitivity of the selective media used, or possibly that the thirteen groups of microorganisms sought do not constitute the entire oral population. The occurrence of anaerobes in the mouths of infants was a disputed point for a number of decades. Anaerobes were first described by X. Lewkowicz in 1901, when he found Veillonella alcalescens. This observation, however, was subsequently cast under suspicion by the claim of KosteEka (1924) that the flora of the edentulous mouth is only aerobic. This did not seem unreasonable, since it is difficult to see at first glance where anaerobes might be harbored in the edentulous mouth of the infant. Hurst (1957a) has suggested that the presence of anaerobes in the baby’s mouth, in the absence of recognizable anaerobic niches such as the gingival crevice, may be possible because of symbiotic growth with some of the oral aerobes. Pratt (1927) had reported some decades earlier that oral fusiform bacilli grow in ordinary infusion broth incubated aerobically if other oral bacteria are present in the tube. Regarding colonization of staphylococci, Dancis and co-workers ( 1957) suggested that the early prominence of staphyIococci in the nasopharyngeal flora of infants may be related to the high chloride concentration in the saliva during early infancy. Hurst (1957b), on the other hand, pointed out that staphylococci probably have a better chance of becoming permanently established on the sterile mucosa of a newborn baby than on that of an adult, when other bacteria already exist in ecological balance. The infants Hurst studied each maintained his own individual strain of staphylococcus so consistently as to suggest that strains encountered neonatally become a part of the normal residential flora. Experiments on the implantation of bacteria indigenous to the mouth of man into the oral cavity of germfree mice (Gibbons et al., 1964c) appear to elucidate the dynamics of initial colonization of the mouth of
202
HEINER HOFFMAN
the human infant. Of thirteen strains of bacteria which were tested, six could be established as monocontaminants ( Streptococcus mitis, Staphylococcus albus, aerobic diphtheroid, Fusobacterium fusiforme, anaerobic diphtheroid, a Bacteroides) . Seven anaerobes could not be established as monocontaminants (two strains of Bacteroides melaninogenicus, Vibrio sputorum, Bacteroides orulis). Of a mixture of ten organisms representing the predominant groups of cultivable bacteria in the human gingival crevice, only B. melaninogenicus and Treponema microdentium failed to become established as polycontaminants. Dietary alfalfa appeared to have an inhibitory effect upon B. melaninogenicus. When mice were monocontaminated with a facultative diphtheroid and were maintained on an alfalfa-free diet, it was possible to establish B. melaninogenicus in their mouths. It was concluded that microbial interactions and diet are important in determining what organisms may establish themselves in the oral cavity and in what sequence.
B. ORALMICROBIOTA OF
THE
CHILDAND ADULT
According to Koste5ka (1924), there is a pronounced shift in the balance of the oral microbiota from aerobic forms toward anaerobic forms on the eruption of the teeth. Late in life, as the teeth are lost, the balance shifts back toward the aerobic forms. A number of studies have shown that the balance of the oral microbiota is specifically related to the oral environment. This close adaptation is evinced by the fact that the adjacent structures have distinctly different microbial populations, although the oral microbial forms are constantly contaminating them ( Scherp, 1956). The specificity of the oral flora extends even to demonstrable differences at various sites within the mouth. This is not surprising in view of the variations in the quantity and quality of the oral fluids bathing these sites, in the nature of the tissues constituting them, in the availability of oxygen, etc. 1. Constancy of the Oral hilicrobiota The complexity of the oral microbial population makes it difficult to assess its stability. Several investigations in recent years have led to the conclusion that considerable stability is present, but a recent study by Wagg (1965) led to qiiite the opposite conclusion. It is apparent that this question needs further investigation. Stained smears from the mouth ( Bibby, 1939) suggest that the morphological types tend to maintain the same relative frequency of occurrence. Lammers (1952) repeatedly examined resting saliva of three subjects over a period of 7 weeks and found that the counts for streptococci and
ORAL MICROBIOLOGY
203
lactobacilli remained somewhat constant. Total counts and the relative proportions of these two bacteria, however, were distinctively different for the three subjects. Williams et al. (1953) concluded from cultivations of human saliva that the relative proportions of the various microorganisms are characteristic for each person. When samples from children and adults were compared, however, marked differences were found in the relative numbers of colonies of the various organisms studied. Moreover, total counts (but not relative proportions) varied with different saliva samples from the same person on different days, and between samples from different persons. Kraus and Gaston (1956) found that the tendency toward higher or lower counts of aerobic organisms in unstimulated saliva appeared to be an individual characteristic. The same held true for certain genera of these bacteria, such as the streptococci, and also for certain species, such as Streptococcus salivarius. An increase of one kind of organism was accompanied by a corresponding increase in the other organisms studied. They concluded that ecological regulation affects both the numbers within and the proportion among species. The most detailed study supporting the concept of a certain degree of stability in the oral microbial microcosm is that of Richardson and Jones ( 1958), who carried out a bacteriologic census of the unstimulated saliva of 14 adults on the basis of 14 microbial groups. The veillonella, total anaerobes, and total aerobes were the categories in which the least fluctuation occurred from examination to examination for all the subjects. Population counts of salt-tolerant micrococci, Neisseria, Leptotrichia, fusobacteria, and hydrogen sulfide producers were found to provide an index of gingival health. Subjects with statistically significant high counts in one or more of these categories had varying degrees of gingivitis and calculus deposits, whereas subjects with significantly low counts had clinically healthy gingiva and little or no calculus deposits. Knighton ( 1965) followed the fluctuations in the incidence of coagulase-positive staphylococci in the saliva and on the nasal mucosa of seventy dental students over the course of their 4 years in dental school. The staphylococci were repeatedly isolated from both the oral and nasal areas of forty students in 1960, twenty-nine in 1961, and thirty-two in 1963. In seven cases a student carried a specific bacteriophage pattern of a staphylococcus for 1960, 1961, and 1963. Seventeen other students carried specific patterns for 1960 and 1961, and acquired a different strain by 1963. This stability appears to be characteristic of the alimentary tract in general, since the intestinal flora in animals and man remains approximately constant under stable conditions (Dubos et al., 1963). On the
204
HEINER HOFFMAN
other hand, lasting alterations in the flora can result from environmental and physiological disturbances. In contrast to the studies cited in the preceding paragraphs, Wagg (1965), on the basis of differential counts of microcultures of flora of dental plaque, noted wide proportional variations at different sites at the same time and at particular sites from day to day. His results, therefore, appear to support the concept that the microflora of the oral surfaces is in a state of continuous change. Studies of the yeastlike flora of the mouth by Lilienthal (1950) indicated that Candida albicans tends to be a constant inhabitant of some mouths and persistently absent from others. In only a few instances did it seem to be irregularly transient. No significant difference was found between C. albicans carriers who were edentulous and carriers who were partially edentulous. On the other hand, edentulous individuals who had not worn dentures after extraction of their teeth had a much lower incidence of C. albicans than persons with teeth or dentures. A similar relationship was found to exist for lactobacilli. 2. Tooth Surface, Tooth Form, Tooth Structure The integrity of the teeth is threatened by microorganisms in two basic ways: by assaults upon the hard tissues themselves, and by assaults upon the supporting tissues. The former is discussed in this section. Those smooth surfaces of the tooth crown that are exposed to the cleansing excursions of the food, cheeks, tongue, saliva, and toothbrush are relatively resistant to the bacterial action resulting in dental caries. The lingual surfaces of the teeth become cleansed more quickly than the buccal surfaces (Valtonen et al., 1960), since the cleansing action of the cheeks is apparently not as effective as that of the tongue. The surface of the crown just below the mesial and distal contact areas is frequently subject to decay. A wide contact area with an adjoining tooth, e.g., the distal contact area of the maxillary second bicuspid (Lu, 1965), or an unusually close contact between teeth promotes food impaction and thus leads to an increased incidence of caries (Muhler et al., 1954). The areas of greatest risk from dental caries are the pits, fissures, and grooves of the occlusal, buccal, and lingual surfaces of the teeth (D. A. Boyd, 1956). The lingual surfaces, however, have a lower incidence of caries than the buccal surfaces. Vertical cracks of the exposed dentine are known to develop at the incisal edge of the lower anterior teeth and may become populated with gram-positive bacteria. Nevertheless, these faults rarely undergo decay (Rushton, 1948). Apparently the dental plaque over these defects is either very thin or entirely missing, so that the microorganisms present
ORAL MICROBIOLOGY
205
are unable to obtain the nutrients required for sufficient acid production to initiate decalcification. Tooth form determines to a significant extent susceptibility to dental caries. This is why dogs have high resistance to dental decay: the shape of their teeth does not favor the retention of food (Gardner et al., 1962). Swartz and Phillips (1957) demonstrated in vitro that bacteria accumulate to a greater degree on a rough abraded enamel surface than upon a highly polished surface. Moreover, bacteria are retained in greater numbers by these rough surfaces even after vigorous brushing. The influence of the tooth surface on bacterial attachment was especially well demonstrated in vitro by Bowen and Gilmour (1961), who found that teeth immersed in cultures of actinomyces and leptotrichia had layers of organisms attached primarily to the cementum surfaces of the tooth roots. The organisms appeared on the crown only in pits in which brown stain had survived the original cleaning procedures. Ureolytic bacteria appear in greatest number on those tooth surfaces which lie opposite the duct openings of the major salivary glands, i.e., on the lingual surfaces of the lower anterior teeth and on the buccal surfaces of the upper first and second molars (Onisi et d., 1957). An adaptation of the Lederberg replicating technique to the oral cavity led Bahn and Quillman (1963) to the observation that in patients with dental caries the heaviest growths of lactobacilli in agar replicates of the mouth occur at the interproximal embrasures of the teeth and on the gingival margins adjoining the carious areas. If rampant caries was present, lactobacilli were also detected in large numbers on the palate. These observations seem to indicate a clear association of lactobacilli with the carious lesion-an association which cannot be detected from salivary lactobacillus counts (G. E. Green and Weinstein, 1959). G. E. Green and Weinstein (1959), however, found on the basis of cultural studies of plaque that the presence of lactobacilli in four or more areas of plaque during the year of their study was associated with higher caries increments (group average) than in persons not having plaque lactobacilli. Nevertheless, it was not possible to predict accurately the future caries rate in individuals. Bacteria placed upon exposed primary dentine will be forced through the tubules into the deep pulp tissue by dental procedures such as cavity preparation, impression taking, or other manipulations requiring pressure (Bender et al., 1959). The number of microorganisms which can be forced through the dentine into the pulp must be small, and apparently the bacteria are destroyed in the pulp before they can do any harm (Seltzer et al., 1961). To test the ability of bacteria invading odontoblastic processes of non-
206
HEINER HOFFMAN
vital dentine in the mouth, Chirnside (1961) examined crowns of extracted teeth which were worn in the mouth over a period of several weeks. He found that within 3 weeks bacteria had invaded the odontoblastic processes and reached the pulp chamber. Chirnside pointed out the possibility, in the case of intact teeth with dead p d p ~ of , bacteria penetrating the enamel through enamel lamellae, tufts, spindles, and basement membrane to the terminal ramifications of odontoblastic processes in the dentine. Bacterial breakdown of the hard dental tissues of the tooth crown in the course of dental caries appears to begin with acid decalcification followed by proteolysis of the enamel matrix ( Frisbie et al., 1944). Microorganisms penetrate the lysed surface matrix and advance beyond the general area of decomposition and occupy the core in the interior of individual enamel rods or lie within the inter-rod matrix. In such areas of penetration the microorganisms are progressively fewer in number. The heterogeneous admixture of morphological types seen in the surface cavity is no longer found; the microorganisms in the deeper zones of penetration are uniformly spheroidal and gram positive. Penetration through the enamel rod core is usuaIIy more extensive than through the inter-rod matrix. As the tract widens from extension of the process to neighboring rods, and with increasing proteolytic changes, the spheroidal organisms are followed and are almost wholly replaced by numerous and various gram-positive and gram-negative threadlike and bacillary forms. The process extends into the dentine first by an initial invasion of the odontoblast fibril in the dentinal tubule. This is followed by decalcification and a consequent softening of the tubule. An invasion of the tubule lumen then occurs, and further production of acids results in progressive decalcification of the intertubular dentine ( Prophet, 1955; MacGregor et aE.,1956). The deepest decalcified dentine of active lesions is almost always sterile, while the intermediate layers are occasionally infected ( Parikh et al., 1963). Practically speaking, the dentine may still contain bacteria, or the dentinal surface of a cavity ready for filling may still contain bacteria at the time the tooth is filled by the dentist. However, whether such microorganisms affect the patient’s dental health is still debated ( Seltzer, 1949). 3. Dental Pulp
Histological study of carious teeth ( MacGregor et al., 1956) strongly suggests that bacterial entry into the dental pulp occurs at a very late stage in the carious attack, when a deep cavity already exists. Bacteriological examination (L. R. Brown and Rudolph, 1957) of pulp tissue in
ORAL MICROBIOLOGY
207
unexposed canals of pulp-infected teeth revealed a mixture of microorganisms, with a high incidence of streptococci, diphtheroids, and micrococci. Ninety per cent of the teeth were positive for microbial forms when examined with the phase-contrast and dark-field microscopes, whereas stained smears revealed organisms in only 71% of the teeth. Spirochetes and fusobacteria were detected. The results indicated that infected unexposed pulp canals contain a different flora from that reported for exposed pulps. The flora of the latter pulps, open to the oral cavity because of carious exposure, resembles that of closed infected pulps, although it is more varied; in one reported case (Crawford and Shankle, 1961), trichomonads were found also. Bacteriological examination of intact teeth whose pulps were devitalized by trauma (Macdonald et al., 1957; Chirnside, 1957) revealed that the majority contained bacteria in the chambers of the devitalized pulps. The largest single group of bacteria isolated by Macdonald and coworkers (1957) consisted of aerobic streptococci; this was followed by micrococci, then by anaerobic cocci. Thirty-two per cent of the strains were anaerobic. Apparently most of the organisms in the pulp reached that site from the oral cavity via the lymphatics and blood vessels of the periodontium, and probably localized in the pulp because of an anachoretic effect (Robinson and Boling, 1941). According to Arnim (1959b), anachoretic pulpitis and death of the pulp can result from dental operative procedures, such as injudicious use of anesthetic, deep excision of living dentine, excessive heat during cavity preparation, injurious drugs, and irritating restorative materials. The dental pulp contains all the cellular elements necessary for defensive and reparative functions and reacts to infection with a vigorous inflammatory response (Orban, 1944). The very vigor of the response, ironically, probably reduces the circulation of blood within the hard casing of the dental pulp after swelling of the pulp from the inflammation. Infarction and necrosis follow (Murphy and Salviolo, 1949; A. Schroeder, 1962). If the pulpitis is a consequence of dental caries, removal of the carious dentine, local application of corticosteroid, and institution of antibiotic therapy (A. Schroeder, 1962) may save the pulp. Death of the pulp requires extraction of the tooth or, conservatively, removal of the dead pulp tissue and treatment of the root canals to remove or kill the microorganisms present. The root canals and pulp chamber are then hermetically filled and sealed with an inert material after sterility of the involved tissues has been verified by bacteriological culture. Certain microorganisms are difficult to remove from the pulp cavity. Enterococci have proved especially difficult, as have also yeasts
208
HEINER HOFFMAN
(Engstrom, 1964; Grossman and Stewart, 1949). If the root canal and pulp chamber are filled despite a persisting infection, the prognosis for the tooth is poor (Engstrom et al., 1964). 4. Dental Plaque
Dental plaque, the film of variable thickness that accumulates on erupted teeth, plays an important role in the development of dental caries and calculus, since it harbors the microbial forms involved in these processes ( Winkler and Backer Dirks, 1958). Considerable confusion is found in the dental literature concerning the definition and proper designation for this film (Stephan, 1953), and a large number of observations have been published which are difficult to draw into a coherent picture. Recent work, however, has gone far toward rectifying this confusion. The dental plaque is a complex coat which adheres so tenaciously to the tooth that it cannot be dislodged by a stream of water or by ordinary swabbing or brushing. The thickness of the plaque varies considerably from one site to another ( McDougall, 1963a). In protected areas, such as gingival to the contact point between adjoining teeth, the plaque frequently is at least 60 p thick. On self-cleansing areas, the plaque is often extremely thin (1 to 3 p) and completely or almost completely free of bacteria. The greatest plaque thickness is associated with increased numbers of microorganisms, but not necessarily or usually with food debris (Appleman et al., 1955). Overlyinp; this film, at the gingival sulcus on the necks of the teeth particularly, is a comparatively easily removed soft deposit referred to as materia alba. There are striking differences in the amounts of bacterial deposits in the mouths of different persons (King, 1951) and between persons in a “caries-active” group and those in a “caries-inactive” group (Frostell, 1960a). These differences may be of significance in the etiology of oral disease, since they entail large differences in the biochemical activity of these deposits. Frostell (1960a) found that the total amount of plaque material removable from the teeth of one of the subjects in his study produced approximately 70 times as much ammonia from urea per unit of time as did .the material taken from another person. The morphogenesis of the dental plaque has been studied recently by a number of investigators. Bjorn and Carlsson (1964) directly observed the early stages on tooth surfaces in man by means of a stereomicroscope. The first change on a cleaned tooth surface, after 24 hours, was the appearance of a membrane or pellicle almost free of bacteria. After 1 to 4 days, discrete hemispherical accumulations were detected which had diameters of about 30 to 40 p, These structures had a striking resemblance
ORAL MICROBIOLOGY
209
to bacterial colonies on an agar surface, and smears showed the presence of essentially coccoid microorganisms. At a later stage a substance was observed covering the plaque colonies which gradually extended beyond the colonies and coalesced with other such areas. A histologic study of early plaque formation based on stained sections of patients’ teeth extracted at varying intervals after a thorough cleansing (McDougall, 1963a) agrees in some respects with the observations of Bjorn and Carlsson (19G4), but differs in a very important point. It was found by McDougalI that the bacteria in the dome-shaped colonies, rather than being cocci, consisted mostly of clumps of parallel rods which originated from within the surface defects of the enamel, and which were arranged with their long axes at right angles to the enamel surface. Some areas of the pellicle or acquired cuticle were found to be covered by a thin layer of mainly gram-positive cocci, with a few rods also present. These organisms apparently were a random selection of bacteria usually found in the saliva. The microorganisms on the cuticle derived from the enamel defects lengthened and multiplied at right angles to the enamel surface with little or no lag phase, while the scattered bacteria seemingly derived from the saliva appeared to have slower rates of multiplication. A number of studies which followed the morphogenesis of the plaque on the surface of artificial plastic films held closely against the teeth gave substantially different results. These studies found that the early stages were dominated by coccal forms. In one study (Appleman et al., 1955), filaments never formed a significant proportion of the flora in the plaques, while in another (Miihleman and Schneider, 1959), the filaments eventually dominated the plaques but occurred first at the plaque periphery. The character of the results with plastic surfaces placed in the mouth is well illustrated by the study of Appleman and co-workers ( 1955).These investigators placed small Vinylite plastic slides in the mouth to serve as surfaces upon which plaque deposits would form. The early plaque usually seemed to consist of a matrix which included squamous epithelial cells and bacteria. For all mouths studied, the first and predominating group of organisms to be deposited appeared to be the genus Micrococcus, with Streptococcus next. When yeasts were found in the plaques they occurred as surface or superficial members of the flora, while the micrococci and streptocci were seen below these cells. Very fine filaments resembling Nocardia and Streptomyces were commonly noted, but neither constituted a significant proportion of the plaque. Fungal filaments were rarely encountered, but when they were found they had developed later than the other microbial forms, suggesting that they were not among the most important organisms. In certain mouths, Borrelia were present in
210
HEINER HOFFMAN
relatively large numbers sometimes associated with fusiform rods and long filamentous forms, but there was no significant difference in these in carious and noncarious mouths in the very small sample examined. Appleman and co-workers noted that rods resembling the genus Lactobacillus were not important members of the flora developing on the slides, either in the frequency with which they were found in various plaques or in their relative numbers compared to micrococci or streptococci. Fully mature dental plaque, on the basis of microscopic sections of teeth, appears to consist mainly of a mass of gram-positive filamentous microorganisms imbedded in an amorphous matrix containing a small amount of cellular and organic debris ( McDougall, 1963a). The closely packed filaments are more or less parallel to each other and usually run a fairly straight course at right angles to the enamel surface. As the filaments approach the surface, their courses become less regular and develop curvatures, Occasionally some filaments on reaching the surface curve over to lie flat upon the plaque, but more commonly they end more or less at right angles to the surface. Branching of the thread forms was rarely observed by McDougall (1963a). McDougall saw a greater variety of microorganisms toward the plaque surface, including cocci, rods, and occasionally the “fruiting heads” of Leptothrix racemosa. There is a problem in reconciling the microscopic structure of the inature dental plaque, which is dominated by closely packed filamentous forms in regular array at right angles to the tooth surfaces ( McDougall, 1963a), with the cultural studies which indicate that approximately half the forms are cocci. Although this point still appears to be unsettled, it seems possible that some of the filamentous forms are streptococci which are capable of continued growth in the oral environment, but which have impaired capacity for cell division (Hoffman, 1964). Furthermore McDougall ( 1963a), considering dental plaque, and Muhlemann and Schneider ( 1959), considering dental calculus, have pointed out that counts are rather deceptive since a long filament may have the same volume as a large number of cocci. An elegant solution to the problem of precise identification of the microorganisms of the dental plaque in situ is offered by the application of immunofluorescence. Hitz (1963) used this technique to show that Nocardia were restricted to the most superficial areas of plaque formed on Mylar film placed in the mouth. The organisms were found in large clumps at intervals along the length of the surface, although much less frequently small aggregates of 3 to 5 cells could be found in deeper areas of the plaque. With the use of nonselective enriched media followed by the selection
ORAL MICROBIOLOGY
211
of various colony types, Hemmens et al. (1946) found that dental plaques contained at least 27 varieties of microorganisms. More recently, Gibbons et at. (1964b) studied the cultivable bacteria present in high dilutions of dental plaque. The predominant cultivable forms were found to comprise the following groups, with their relative proportions given in percentages : facultative streptococci, 27%; facultative diphtheroids, 23%; anaerobic diphtheroids, 18%; peptostreptococci, 13%; Veillonella, 6%; Bacteroides, 4%; fusobacteria, 4%; Neisseria, 3%; and vibrios, 2%. Total microscopic counts of dental plaque averaged 2.5 x 10'l bacteria per gram, while total cultivable counts performed anaerobically and aerobically averaged 4.6 and 2.5 x 1O1O per gram respectively. The heavy concentration of microorganisms in the dental plaque is strikingly illustrated by the comparison Krasse (1954a) made between plaque material and the saliva. He found that 1 mg. of plaque material contained about twice as many lactobacilli and 50 times as many candidas as 1 mg. of saliva. For streptococci the difference was considerably greater: 1 mg. of plaque material contained about 300 times as many streptococci as 1mg. of paraffin-stimulated saliva. Krasse (1954b) investigated the relationship between the number of lactobacilli, candidas, and streptococci in saliva and plaque material in two groups of patients, one with active caries, the other with inactive caries. The group with active caries showed a larger number of lactobacilli and candidas in the saliva as well as in plaque material, and a large number of streptococci in plaque material. No difference was found, however, between the two groups with regard to the number of streptococci in the saliva or the incidence of Streptococcus salivarius. When the number of lactobacilli or candidas was high in the plaque material it was often also high in the paraffin-stimulated saliva. No such correlation was found in the number of streptococci. The saliva and plaque material exhibited wide differences in the quantitative interrelationships of the lactobacilli, candidas, and streptococci. Differences in pH levels produced in dental plaques from carbohydrates have been found to be related to caries activity (Stephan, 1940). It seems probable that the cariogenic activity of dental plaques is dependent on the prolonged retention or frequent renewal of fermentable carbohydrates on the tooth surface. This activity is modified by the flow and composition of the oral secretions and perhaps by many other factors. Among these is the one recently observed by Gibbons and Socransky (1962) that a large proportion of the microbiota of dental plaques produce intracellular polysaccharides in uiuo. Streptococci, diphtheroids, and fusobacteria were among the organisms capable of this. Pure cultures of
212
HEINER HOFFMAN
streptococci were shown to metabolize stored polysaccharide, forming lactic acid. Plaque from patients with active caries was shown to contain a higher proportion of microorganisms with a high level of stored polysaccharide than plaque from those with inactive caries. Gibbons and Socransky (1962) pointed out that this phenomenon may make possible production of acid and a low pH in the dental plaque over longer periods after environmental carbohydrate has been depleted.
5. Dental Calculus Dental calculus is not a normal deposit on the tooth surface but rather a calcification of dental plaque which usually occurs on certain areas of the teeth when cleansing is not adequate. Microorganisms have been held responsible for the calcification, but recently supragingival calculus-like deposits have been noted in germfree rats and mice (Fitzgerald, 1963). It is certain, therefore, that microorganisms are not the ultimate reason for the formation of calculus, although in the normal non-germfree state they contribute to the severity of the deposits. Clinically, persons with severe periodontitis have more calculus than those without periodontitis or with only slightly diseased gums (Frostell, 1960b). This appears to be a significant correlation, and there is now general agreement that gingival deposits, such as calculus and plaque, are the most important local etiologic agent for many periodontal diseases, ranging from simple gingivitis to severe periodontitis ( Leung, 1962). Considerable attention has been accorded to dental calculus in recent years and to the role played by the oral microorganisms. For clinical descriptive purposes, Berke ( 1935) distinguished three forms of dental calculus: ( 1 ) coronal, ( 2 ) cemental, and ( 3 ) inflammatory. Histologically ( Berke, 1935), mature coronal calculus (also referred to as supragingival calculus) is distinguished by the regularity of the microbial thread forms within it, arranged at right angles to the enamel surface, and by a pattern of incremental lines. Thus in many respects it resembles well-formed dental plaque in structure. A number of leukocytes, a few epithelial cells, and a large number of gram-positive cocci are dispersed throughout the deposit. Earlier in development, when mineralization begins, plaques developing into calculus consist primarily of coccoid forms (Mandel et al., 1957). Cementa1 calculus, deposited upon the exposed cemental root surface, differs from the coronal type in that the components are much more disorganized; the substance of the deposit is irregularly laminated and the lamellae can be seen distinctly in only a few areas. Inflammatory calculus is more cellular than the others. Leukocytes are the most prominent element, but thread forms are also present, This type lies within gingival pockets, is loose and soft in tex-
ORAL MICROBIOLOGY
213
ture, and is readily distinguished from the harder and denser cementa1 tYPea The attachment of subgingival calculus to the tooth has been described by Zander (1953) and by Mandel and Levy (1957). They found from microscopic sections that the calculus appears to be attached to the tooth surfaces by a cuticle-like material, but if the cuticle is absent the attachment seems to be by bacterial growth into areas formerly occupied by Sharpey’s fibers, or by penetration into defective areas in cementum and dentine. Voreadis and Zander ( 1958) have suggested that desquamated epithelial cells play a part in the formation of the cuticle-like material which attaches supragingival calculus to the enamel. This same structure is probably involved in attachment of plaque material. McDougall (1963b), in a histochemical study of the dental plaque and its attachments, concluded that the acquired cuticle contains little, if any, unaltered salivary mucoids. The evidence indicated that the cuticle may be derived from bacteria. An additional mode of calculus attachment was found to be through bacteria embedding themselves into enamel irregularities, cracks, and lamellae. The attachment of supragingival calculus usually is less firm than that of subgingival calculus (Forsberg et al., 1960). Plaque attachment appears to be similar ( McDougall, 196313). The role of microorganisms in the formation of dental calculus has been clarified in many respects within recent years. The prominent position of filaments in the mature structure of this deposit was emphasized several decades ago by Naeslund (1926). He concluded from extensive morphological and cultural studies that the metabolic activities of actinomyces and leptotrichia are the principal cause for the precipitation of the calcium hydroxyapatite in the calculus. Bowen and Gilmour (1961) recently incubated teeth in broth cultures of actinomyces, leptotrichia, or both in the presence of calcium and phosphate. Calculus formed in all three cultures. The deposits showed a layered pattern resembling the incremental lines of natural calculus, and they contained bacterial masses. Cultural studies of developing and mature human dental calculus plaques (Howell et al., 1965) are limited in their accuracy, but they have shown that streptococci predominate initially, although gram-negative rods and neisseriae are also present in large numbers. In older material, filamentous organisms, and particularly Actinomyces israeli and Actinomyces naeslundi, predominate, with streptococci still present in large numbers. The nature of the saliva must have considerable importance in the formation of calculus, since the saliva is probably the source of calcium. Richardson ( 1965), however, was unable to suppress calculus formation in cats by removal of the major salivary glands. Apparently, the minor
214
HEINER HOFFMAN
glands and the secretions from gingival crevices supplied sufficient calcium for the calcification. He found that brushing the cats’ teeth even only once a week prevented the accumulation of calculus. The mechanisms bringing about calcification are quite complex. The mineralized plaque initially consists of an organic noncellular matrix enmeshing a variety of microorganisms. The matrix itself seems to represent the bulk of the calcified deposits (Gonzales and Sognnaes, 1960). Ennever (1963) has suggested that the source of the matrix is microbial and that it is derived in part at least by an organic acid extraction of Bacterionema matruchotii, a prominent member of the soft plaque. The acid is probably lactic acid from bacterial glycolysis. Mandel and coworkers (1957), on the basis of histochemical studies, considered that the matrix consists of components derived from the saliva as well as from bacteria; from in vitro production of artificial calculus it appears that submaxillary saliva is probably of greater significance in this regard than is parotid saliva (Middleton, 1965). If a salivary mucin is a part of the plaque, it is likely to be present as only the protein core without any carbohydrate side chains ( Middleton, 1964), since the carbohydrates which are found in plaque are probably of bacterial origin. M. H. Smith (1964) found that the acquired pellicle of the tooth surface is mainly protein which differs significantly in its amino acid composition from the glycoprotein of saliva. He concluded that salivary glycoprotein forms only a small part of the pellicle if it is involved at all. It had been suggested early in this century by Kirk (1910) that in the formation of plaque salivary mucoid is precipitated upon tooth surfaces by lactic acid resulting from carbohydrate utilization by oral microorganisms. Dawes (1964) recently studied the problem experimentally and found that saliva adjusted with lactate or acetate buffer showed no obvious precipitation of protein unless the pH was 3.5 or less. He concluded that acid precipitation of salivary proteins is not likely to be a factor in plaque formation. Both matrix and microorganisms contain calcium hydroxyapatite ( Ennever, 1963). Rizzo and co-workers (1962) have shown that a variety of viable and nonviable bacteria become mineralized with hydroxyapatite when implanted in dialysis bags in the peritoneal cavity of rats. The microscopic pattern of mineral deposition appeared to be analogous to that in the formation of oral calculus. Since the nonviable organisms were mineralized at an accelerated rate, it was concluded that bacterial metabolic processes may not be essential for mineralization. The bacteria exhibiting calcification were isolated from human dental calculus and included Streptococcus salivarius, Actinomyces nueslundii, Bacterionema inatruchotii, and an unclassified oral diphtheroid. Calcification consists of
ORAL MICHOBIOLOGY
215
impregnation of the matrix and the microorganisms with microcrystals (H. E. Schroeder et al., 1964). The intermicrobial pattern results in inhomogeneously structured mineralization centers; the intramicrobial pattern results in homogeneously structured centers. 6. Gingival Tissue
Shadowed replicas of the gingival surface, upon examination with the phase-contrast microscope, reveal flourishing colonies of cocci which are covered by a granular coating (R. Frank, 1957). Swabs taken of the lower anterior gingiva of 850 normal young adults and cultured on aerobic blood agar plates yielded Streptococcus viridans as the dominant organism (96% of the cases examined) (Massler and Blum, 1949). In many cases it was the only organism present on the culture plates. y-Streptococci grew in relatively much smaller numbers and were found in only 27% of the cases. Staphylococcus albus was found in relatively small numbers in 20% of the cases. Pneumococci and micrococci were seen somewhat less often. (3-Hemolytic streptococci were isolated from 9% of the cases, while Staphylococcus aureus was obtained in 5%. Diabetics proved to have a much higher prevalence of Staphylococcus aureus and (3-hemolytic streptococci on the gingiva ( Massler, 1949). This appears to be a significant difference from the normal, but it has not been explained. Yeast counts from cultivations of swabbings of the gingiva are far lower than counts from the saliva of the same subject (37% versus 47.7% in a study by Lilienthal, 1950).
7 . Gingival Crevice The gingival crevice is a space surrounding the necks of the teeth. It is formed by a projection of the gingiva above the attachment of the gingival epithelial mucosa to the teeth. Histological study of the keratinization of human gingiva led Trott (1957) to the finding that crevicular epithelium shows no evidence of keratinization. This reduces its effectiveness as a barrier to infection, as evidenced by the frequency with which leukocytes are observed infiltrating this epithelium. The lack of keratinization, however, allows the operation of a distinctive protective mechanism which depends upon the increased permeability of the crevice. Experimental studies with the dog (Brill, 1959) indicate that tissue fluid flowing into gingival pockets is able to flush bacteria out into the mouth. In the dog, intravenous administration of tetracycline (Bader and Goldhaber, 1965) is followed by the gradual appearance of the antibiotic within the attached gingiva and its spread to the alveolar mucosa and the gingival margin. Except for the gingival sulcus area, the tetracycline does
216
HEINER HOFFMAN
not appear on the surface of the mucosa. No fluorescence is observed in the saliva of the dog, which suggests that a major pathway of systemically administered tetracycline into the oral cavity may be the gingival sulcus. Measurements of the rate of crevicular fluid flow in fifteen healthy dental students (Bissada et al., 1965) revealed that the rate of flow varies considerably between individuals and also between different crevices of the same individual. The rate of flow was found to be highest in the evening and lowest in the early morning. The gingival sulcus fluid does not react with antisaliva sera ( Weinstein et al., 1965). The gingival crevice, according to Waerhaug and Steen (1952), does not as a rule contain bacteria, but more recent studies (among others, W. S. Boyd and Rosenthal, 1958; Gavin and Collins, 1961; Wilkinson, 1962; de Castro and Going, 1964) have failed to support this judgment. The negative results obtained by Waerhaug and Steen apparently were due to sterilization of the gingival sulcus by the iodine-glycerine solution they used to free the gingival margin of the sulcus of bacteria (W. S. Boyd and Rosenthal, 1958). The microbial aggregations in the gingival crevice start from dental plaque along the gingival border. Further development of the plaque results in a growth of microorganisms into the gingival pocket (Krasse, 1963). The material contained within gingival pockets is made up almost entirely of bacteria ( Socransky et al., 1963). Total microscopic counts are approximately 1.7 x 10l1 organisms per gram wet weight. Total viable aerobic counts average 1.6 x lolo, while total anaerobic viable counts average 4 x lolo. Thus, the majority of the bacterial population appears to be obligately anaerobic. The gingival crevice microbiota has the following composition (Gibbons et d., 1963) : 28.8% gram-positive facultative cocci; 20.27% gram-positive anaerobic rods; 16.1% gram-negative anaerobic rods; 15.3% gram-positive facultative rods; 10.7% gram-negative anaerobic cocci; 7.4% gram-positive anaerobic cocci; 1.1% gramnegative facultative rods; and 0.4% gram-negative facultative cocci. Gram-positive organisms constituted 71.5% of the cultivable organisms and bacillary forms 52.8%. The microbiota of the gingival crevice of the preschool child with no erupted permanent teeth differs from that of adults only in the fact that two microorganisms, Bacteroides melaninogenicus and spirochetes, are observed in only a small proportion of the cases studied (de Araujo and Macdonald, 1964 ) . The prevalence of B . melaninogenicus increases sharply in the period of mixed dentition (Bailit et al., 1964), and by adolescence the organism is almost universally present. Courant and co-workers (1965), comparing the debris of the gingival crevice from normal individuals with that from persons with periodontitis
ORAL MICROBIOLOGY
217
or acute necrotizing ulcerative gingivitis, found no significant differences in the ability of the debris to produce transmissible localized infections in guinea pigs. A number of investigations have been concerned with whether the oral bacteria are able to penetrate into the gingival tissue, particularly from the gingival crevice, through the intact epithelial barrier. One view has maintained that active invasion of gingival tissue by bacteria does occur (Beckwith et al., 1927; Gutverg and Haberman, 1962), while others have considered that bacterial activity is limited to the periodontal pocket ( Fish, 1939; Bibby, 1953). Biopsy sections of normal gingival papillae examined by Schaffer (1953) showed bacteria only on the surface of the epithelium. The bacteria seemed unable to penetrate into the vital tissues underlying the surface even when a superficial pathological process was present. Gibson and Shannon (1963), in a recent study, found that approximately 10% of gingival strip biopsy specimens contained bacteria in the tissues if the subjects were not given an oral prophylaxis, while 60% of the specimens from subjects who received oral prophylaxis showed bacteria within the tissues. A later study (Gibson and Shannon, 1964) was able to simulate the tissue distribution of the bacteria with carbon black when the subject was given an oral scaling in the presence of lamp-black powder particles suspended in physiological saline solution. These findings were subsequently reenforced by Wertheimer ( 1964) on the basis of a study on intact periodontal tissues from autopsy specimens of the human jaw which alIowed examination of all periodontal tissues in undisturbed relation to teeth, bacterial plaques, and calculus. Wertheimer found a paucity of evidence of microbial penetration in connective tissue contiguous to massive accumulations of bacteria in gingival crevices and pockets. Microorganisms were found only in the superficial epithelial layers in a few sections in areas of disrupted epithelial cells. Wertheimer concluded that previous reports of microorganisms in the connective tissue of excised gingiva may have been based on mechanically induced artifacts or the presence of nuclear debris. 8. Oral Mucous Membrane and Desquamated Cells
The lining of the oral cavity covers a large area and therefore has an important role in the well-known resistance of the oral cavity to infection. A great deal of attention has been lavished upon this structure, but important microbiological questions nevertheless remain to be resolved. The multifaceted epithelial cells of the most superficial layer are continuously sloughed off into the saliva, where they are found usually as single cells but occasionally in groups ( Dreizen et al., 1956). In the saliva,
21s
HEINER HOFFMAN
the cells appear flat and the comers may be folded; the cytoplasm is large in comparison to the oval nucleus and seems quite granular (Bradley, 1948). At least some of the intracellular granules are capable of limited enzymatic activity, since desquamated epithelial cells tested cytochemically for esterase activity (Baer and Burstone, 1959) give a positive reaction limited to these granules. Since salivary bacteria which deposit upon mylar strips placed in the mouth also have esterase activity (Baer and Burstone, 1959), enzyme may be present in the dental plaque from two sources-a fact which may have significance in calculus formation. This possibility arises because the release of fatty acids may form calcium and magnesium soaps, and calculus is known to contain both fat and fatty acids (Glock and Murray, 1938; Mandel et al., 1957). That desquamated epithelial cells liberate phosphatase ( G. H. Smith, 1930) may also be of significance in this regard, as the soaps may undergo a transformation into the less soluble phosphates or perhaps carbonates (Baer and Burstone, 1959). Large numbers of desquamated epithelial cells are found in the saliva. A group of thirty-eight children ranging in age from 1 to 18 months were found to have approximately 800,000 clearly formed epithelial cells per milliliter of saliva (Klein, 1962). The number of epithelial cells in the saliva of ten adults ( Deakins et al., 1941) ranged between 2620 and 18,500 per milliliter. The higher counts in the very young are paralleled by increased cell counts in edentulous persons ( Calonius, 1958). These high counts may be due to the friction on the oral mucosa of foods, sucking, etc. (Klein, 1962), which rubs off the cells. The epithelial cells of the oral mucosal surface are constantly renewed while desquamation is progressing-especially during the mastication of food, when enormous quantities of cells are rubbed off (Hugenschmidt, 1896). At least some of the desquamated cells are retained in the mouth as part of the dental plaque (Appleman et al., 1955). Desquamated oral epithelial cells may carry a variable burden of closely adherent bacteria, which in some cases may completely cover the surface (Bradley, 1948) and in a few cases may be within the cytoplasm (Montgomery, 1951). Koste6ka (1924) detected, in the mouths of newborns and older persons, gram-negative diplococci which were almost exclusively within the epithelial cells. Intracellular bacteria, generally gram-positive cocci, of the epithelial cells in the dental plaque appear to multiply within these cells and eventually cause their disappearance ( McDougall, 1963a). The significance of the intracellular microorganisms is unknown, but it seems possible that these forms are more pathogenic than those confined to the surface. Labrec and co-workers (1964) found in a study of the pathogenesis of bacillary dysentery that a nonpathogenic
ORAL MICROBIOLOGY
219
variant of Shigella flexneri was not able to penetrate the intestinal epithelial cell, while the virulent strain could. Saliva from persons with immunity to dental caries contains a greater number of epithelial cells per milliliter than does saliva from caries-susceptible persons. In saliva from caries-immune persons most of the epithelial cells are covered with microorganisms, whereas in saliva from caries-susceptible persons the epithelial cells are virtually free from bacteria (Orban and Weinmann, 1939; Bradley, 1948). Dreizen and coworkers (1956) found that salivary samples from patients with periodontal disease contained substantially greater numbers of bacteria-covered epithelial cells than did those from patients free of periodontal disease. In 90% of the samples obtained from subjects with periodontal disease, a minimum of 70% of the epithelial cells were covered with bacteria. In contrast, in 88% of the samples from patients without periodontal disease, fewer than 50% of the epithelial cells were covered with bacteria. Morphologically, each of the samples from the patients with clinically active periodontal involvement contained an abundance of disintegrated epithelial cells, whereas those in the samples from the group without periodontal disease were mainly intact. In the healthy mouth, cells of the blastospore stage of Candida albicans lie upon the oral mucosa, but in the diseased state the blastospores apparently play an essential role in tissue penetration. Only after the fungus has penetrated the oral epithelium and begun to form pseudomycelia do clinical manifestations of thrush appear (Taschdjian and Kozinn, 1957). Although in experimental vaginal candidiasis of the mouse ( Taschdjian et al., 1960) parasitization is limited exclusively to the keratin of the horny layer during the estrous cycle, the process in the human oral cavity is more developed. The deeper layers of tongue epithelium (Lehner, 1964) are invaded by means of pseudohyphae which are oriented at right angles to the surface. The fungus does not extend down into the corium except in necrosis or ulceration of the mucosa, which is best seen in patients with leukemia. The parotid saliva of patients with mumps (viral parotitis) contains large numbers of epithelial cells cast off from the glandular acini (Sohier and Jaulmes, 1939). They often are united to each other in cylinders, and sometimes they may even end in a cul-de-sac. Cytologic smears from early lesions of herpes simplex, herpes zoster, and varicella show identical diagnostic features in the epithelial cells (Blank et al., 1951). These consist of tremendously enlarged giant epithelial cells with eight or more nuclei formed by amitotic division. The norma1 chromatin granules and nucleoli are absent. The principal loci of microbial growth on the epithelial cells of the
220
HEINER HOFFMAN
mucosal surface appear to be in the superficial layers, the many crevices between the epithelial cells, and gland openings ( Bloomfield, 1 9 2 2 ~ ) . The presence of taste buds and papillae on the dorsum of the tongue gives an extremely large surface which favors the retention of bacteria upon this area especially, so that it appears to be one of the three main sites for bacterial colonization in the oral cavity (Krasse, 1963; Bartels, 1954; Morris, 1949), the other two being the tooth surfaces and the gingival crevices. The organisms on the tongue, moreover, appear to be more readily shed and appear to be the principal source for the bacteria present in the saliva (Gibbons et al., 1964a). The high concentration of bacteria on the tongue is probably the cause for the sharp drop in the pH on the dorsum of the tongue after a glucose rinse (Stephan, 1944). This pH change is similar to that on the tooth surfaces. The mucous coating on the dorsum of the tongue characteristically shows the presence of desquamating epithelial cells, leukocytes, tissue fluids, and occasionally red blood cells ( Arnim, 1959a) in addition to the large number of bacteria. When the saliva and surface debris on the tongue dorsum are removed, the remaining microbiota as sampled by vigorous swabbing give a general picture similar to that presented by the microbiota of the saliva, but different in several respects from the microbiota indigenous to the gingival crevice area and dental plaque (D. F. Gordon et al., 1965). The predominant microorganisms upon anaerobic incubation are Streptococcus saliuarius, Streptococcus mitis, Veillonella, Bacteroides sp., gram-negative facultative rods, and anaerobic and facultative diphtheroids. Staphylococci, fusobacteria, peptostreptococci, vibrios, and Neisseria are present, but in lesser proportions. The oral mucosa near the entrance of Stenson’s duct and near the entrance of Wharton’s duct has a very low incidence of fusiform bacilli and spirochetes, while the lingual surface of the lower incisors and lower posterior molaxs in the same mouths show a quite high incidence of these organisms (Brooke, 1938). High percentages of gram-positive cocci and a reduction of all other organisms characterize the smears from buccal mucosa (Bibby, 1938b). After examination of the bacterial flora in the different parts of the mouth, Bibby (1938b) suggested that gram-positive cocci may proliferate on the buccal mucosa. Rabbits locally infected with herpes simplex virus in the oral mucosa adjacent to the submaxillary duct orifices continue to excrete virus in their saliva for an average of 40 days after inoculation (Ashe et al., 1965). Histological examination of the oral mucosa reveals that the lesions also involve the orifices of the salivary glands; the lining epithelium of the gland ducts becomes ulcerated by direct extension and the ducts show specific histological signs of herpes
ORAL MICROBIOLOGY
221
simplex virus infection in deeper segments. The intact oral mucosa appears to be very resistant to Mycobacterium tuberculosis. When infection does appear local trauma and weak immunological response may be important factors in overwhelming the natural resistance of this tissue ( O’Neil, 1963). Aerobic cultivation of mucosal scrapings ( Bloomfield, 1922c) and direct examination of desquamated cells reveal that only a limited number of microbial forms are in intimate relationship to the mucosal cells. Orban and Weinmann (1939) found that only coccal forms were present on the epithelial cells obtained from human saliva, while Schaffer (1953), examining sections of the interdental gingival papilla of normal subjects, found that cocci were present on the surface in six out of 12 cases and bacilli (other than fusiforms) in one case. No spirochetes, vibrios, or filaments were found. It seems unlikely, therefore, that Howitt and Fleming (1930) were correct in their suggestion that the remains of desquamated epitheIiaI cells furnish food for such fastidious forms as the spirochetes. A determinative study of the bacteria which are closely attached to the oral epithelial cells apparently has never been carried out. A very poorly studied but quite distinctive microbial group was most recently reexamined by Steed (1963) and found to have a close relationship to epithelial cells. After a 6-hour incubation of serum agar plates inoculated with oral swabs from sheep and rabbits, Steed obtained microcolonies of Simonsiella and Alysiella which had grown out mainly over deposited epithelial cells. Several decades earlier, Beust ( 1929) had described an organism in the mouth of the dog which appears to be very much like the organisms observed by Steed. It was often found by Beust embedded in epithelial cells or in transparent films; it averaged about 10 p in length and 4 p in diameter and contained transverse septa, in some cases as many as 16. His photomicrograph of the organism closely resembles the photographs presented by Steed. Beust never found evidence of the organism in any of his material from humans. Brooke (1938) found that bacteria on desquamated epithelial cells usually occupied the periphery of the cell, with but a few in the paranuclear region. This suggested to him that the intercellular cement substance affords better growth conditions for the bacteria than the cell itself. This also may expIain in some cases the route by which pathogens penetrate into the mucosa. In a study of the availability of bacterial growth factors in human gingival tissue (Nevin et al., 1958) it was found that while inflamed specimens generally had a considerably lower content of riboflavin, niacin, and biotin than did noninflamed tissue, negative assays were obtained in a certain number of cases, more frequently for
222
HEINER HOFFMAN
noninflamed tissue. Since the assays were conducted with oral streptococci, the authors suggested that these negative assays may reflect the presence of an inhibitory substance, perhaps antibody. The bacteria lodged on the mucosa are very firmly attached. Bloomfield ( 1 9 2 2 ~ )scrubbed the tongue with a stiff toothbrush and water for 15 minutes to the point at which the tongue began to bleed; cultures made from the area before and after scrubbing showed no demonstrable qualitative or quantitative differences in flora. Bloomfield suggested that the attachment of bacteria to the mucosal surface may be due to a reaction between the specific bacterial species and the tissue surface, rather than to the nutritive qualities of the saliva. There is some evidence that the attachment of bacteria to epithelial cells may be due to antigenically similar receptor sites on the two cell types interacting with agglutinins. Apropos of this possibility, it may be noted that Webb and Fedoroff (1963) found that natural antibodies present in human blood sera brought about mixed agglutination between L cells of mouse origin and Esclzerichia coli cells. Two decades earlier, Rosenthal (1943) had found that some strains of E. coli possess agglutinating properties against certain cellular elements such as leukocytes, thrombocytes, and spermatozoa. It would be of great interest to determine whether the few E. coli isolates obtained from the oral cavity of man exhibit such agglutinating properties. Very little is known concerning the antigenic composition of oral epithelium. It has been shown (Swinburne et al., 1961), however, that A antigen can be detected on the buccal epithelial cells of group Al and A, secretor and group A, nonsecretor persons. Moreover, bacteria are also known to contain blood group antigens ( Springer, 1958). Blood group-specific agglutinins are known to occur in plants (W. C. Boyd and Shapleigh, 1954), so that it is possible to consider that pIant agglutinins in food may operate in the attachment of bacterial cells to the epithelial cells of the oral mucosa. The firm attachment of bacteria to the oral mucosa, however, does not necessarily imply that a specific interaction must have occurred between the microbes and the mucosal surface. ZoBell (1943) found that when glass slides were immersed in suspensions of bacteria the bacteria which adhered to the glass surface were easily washed off only when the slides were kept in the bacterial suspension for a relatively short time. He found that bacteria attached to glass in largest numbers during the early logarithmic phase of the growth cycle. According to Berger (1952), the continued desquamation of the superficial layer of the oral mucosa may be properly regarded as a biological mechanism for clearing microorganisms from the oral cavity. At first glance, however, this idea does not appear to be very illuminating since
ORAL MICROBIOLOGY
223
the bacteria would be cleared from the mouth through the flow of saliva in any case. Some advantage might accrue to the body by a reduction in the number of infective particles if bacteria, and particularly pathogenic forms, are agglutinated upon the epithelial cell surface during the exposure of the epithelial cell to saliva while in situ or after it has been dislodged and is flowing through the mouth in the saliva. Shedding of the epithelial cell may conceivably be hastened by enzymes produced by the streptococci lying upon its surface. The remarkable ability of the oral mucosa to heal after operative or accidental trauma is a well-known phenomenon which has been variously described as the result of certain structural characteristics of the tissues or as due to the rapid elimination of oral pathogens from the mouth (Lammers, 1952). In the opinion of Lammers (1952), however, the oral mucosa owes its health to the particular ecological structure of the oral microbiota. Tomaszewski ( 1951) had suggested shortly before that the normal oral flora is necessary for the maintenance of health of the mucous membranes. This view was based on his study of 126 patients during antibiotic treatment: the oral flora was changed, and then the normal whitish coating of the tongue disappeared; denudation of the dorsal surface and sometimes atrophy of the filiform papillae occurred. However, Hine (1956) points out that it seems more probable that the antibiotic therapy interfered with the metabolism of the vitamin B complex, since Tomaszewski also reported that the tongue changes could be minimized by the administration of vitamin preparations.
9. Salivary Leukocytes The formed elements of saliva consist of bacteria, epithelial cells, and leukocytes. Of these last, the polymorphonuclear leukocytes are of particular interest since they form the overwhelming majority of the leukocytes and possibly may act as phagocytes in the defense of the oral cavity against microorganisms (Wright, 1964). The gingival sulcus is the major site of entry of leukocytes into the oral cavity (Sharry and Krasse, 1960). The number of leukocytes in the saliva (Calonius, 1958) of the toothless, healthy mouth is considerably lower (1000 to 143,000 per milliliter) than in the healthy mouth with teeth ( 110,000 to 1,364,000 cells per milliliter); in mouths with inflammation, the count increases quite remarkably (770,000 to 11,896,000 per milliliter). The number of leukocytes in the saliva appears to vary independently of the number of epithelial cells in the same specimen (Isaacs and Danielian, 1927). Leukocytes which appear in the mouth under normal conditions have a distinctive morphology which has led to their designation as “salivary
224
HEINER HOFFMAN
corpuscles.” These are spherical cells measuring 12 to 16 p in diameter which contain an eccentrically located nucleus of 1, 2, or 3 lobes. The cytoplasm is granular and shows considerable activity ( Rovelstad, 1964). When examined in fresh saliva, the salivary corpuscles give clear evidence that they are dying cells, but to Kovelstad (1964) it seems reasonable to consider that as salivary corpuscles leave the epithelial tissue they find a favorable environment in the mucous layer covering the gingiva, and are capable of functioning as phagocytes there. This would only be at times of rest of the major salivary glands, since stimulated saliva from these glands has an average pH value between 6.35 and 6.85, which is below the values for survival of polymorphonuclear leukocytes. Rapidly fixed salivary corpuscles examined in salivary smears frequently give evidence that phagocytosis occurs in the oral cavity, especially in gingival crevices. In such preparations numerous cells are seen to be ballooned with many ingested microorganisms. It is possible that the bacteria are ingested within the tissue before the leukocytes migrate through the epithelium, but this does not seem probable (Menkin, 1955). Eichel and Lisanti (1964) have proposed that intact, viable oral leukocytes are a main line of defense in the mouth, keeping the oral microorganisms under control in the person who is free of oral disease. It has been suggested that leukocytes in saliva may release proteolytic enzymes into the saliva upon disintegration, but it is not quite clear what the effect of these enzymes may be (Wright and Jenkins, 1953). In view of the dominant role of oral leukocytes in the metabolism of saliva, as found by Eichel and Lisanti (1964), these investigators have suggested that tooth decay and periodontal disease may result from a shift in the leukocyte-microorganism balance in the oral cavity in the direction of the disrupted leukocyte state. The uncontrolled metabolism of these homogenate-like fragments of cells and their by-products may be damaging to the teeth and gingivae.
10. Salivary Glands The parotid gland, its ducts, and the fluid it excretes are normally sterile (Nash and Morrison, 1949). The continued secretion of saliva and the corkscrew configuration of Stenson’s duct, which hinders the retrograde flow of fluid or organisms, are thought to be responsible for the maintenance of sterility, although the antibacterial action of parotid saliva (see Section 111, C, 2 ) may also possibly play a protective role. The sublingual gland for some unknown reason does not seem to be subject to inflammations ( McEachen et al., 1958). Acute sialadenitis of the submaxillary gland is secondary to obstruction of the duct by a calculus
ORAL MICROBIOLOGY
225
(Lathrop, 1956)) and removal of the stone is followed by complete resolution of the infection (Rose, 1954). When bacterial infection of the parotid gland develops, the person is usually seriously ill, in poor general condition, dehydrated, undernourished, and with poor oral hygiene. A dry mouth and decreased salivary secretion seem to be the principal precursors to onset of the disease (GilChrist and McAndrew, 1958). In one large series of cases ( Spratt, 1961), the principal situation leading to parotid infection was illness following major operative procedures. Abdominal surgery was the primary offender, while dental and gynecologic procedures each contributed about half as many cases. Acute parotitis is a consequence of such pathological dental conditions as pericoronitis, fracture of the jaws, infection of the mucous membrane, cellulitis, or phlegmon, or such operations as the extraction of a tooth (Schroff, 1939). Primary acute pyogenic parotitis, generally in elderly people with dry mouth, is becoming increasingly common (Liu and Page, 1960). The low incidence of sublingual and submaxillary gland infections when the mouth is dry is probably due to protection by the tongue against desiccation of the gland duct openings (Anthony and Fisher, 1948). Bacteriological study of exudate from the ducts of infected parotid glands has implicated Staphylococcus aureus as the organism most frequently involved, although a- and P-streptococci, staphylococci other than S. aureus, pneumococci, mixed gram-positive cocci, and actinomyces have all been found (Spratt, 1961). It is pertinent to note that when staphylococci are present in the mouth they attain their highest concentration on the buccal mucosa in the region of the molars adjacent to the orifice of Stenson’s duct ( Seifert, 1930); less commonly they occur sublingually (Talbot, 1934). In recurrent pyogenic parotitis, 65% of the cases in one series (Rose, 1954) showed Streptococcus viridans as the infecting organism, often in pure culture but occasionally combined with S. aureus. Other infecting organisms were the pneumococcus, Streptococcus haemolyticus, Bacillus proteus, Bacillus fusiformis, and Escherichia coli. Those with pneumococci showed much greater gland destruction than those with S . viridans. The parotitis which may develop with a serious infection in another part of the body, e.g., typhoid fever, typhus fever, and lobar pneumonia, does not necessarily arise from the microorganism causing the primary infection but appears rather to be due to an oral inhabitant that takes advantage of the effects of the earlier illness upon systemic and local resistance ( Custer, 1931). Bacteriological and histopathological studies of infected Stenson’s ducts
226
HEINER HOFFMAN
and parotid glands have shown that the infection usually takes an ascending route through the duct into the gland (Custer, 1931). Only rarely is parotitis blood borne, and septicemia rarely leads to involvement of the parotid gland (Diamont, 1959; Liu and Page, 1960). Two additional routes for gland infection are possible (Robinson, 1955) : the lymphatics, and direct extension from contiguous tissue. The former does not seem to have been responsible for clinically observed cases, and the latter occurs only occasionally from a suppurative process in the pharyngomaxillary space ( Lathrop, 1956). Experimental studies with dogs on the pathogenesis of acute suppurative parotitis have lent support to the clinical, histological, and bacteriological observations concerning the route of gland infection. Injection of a hemolytic strain of Staphylococcus aureus into the Stenson’s duct of dogs led to marked suppuration in seven out of ten glands, while only three out of fifteen injections into the superficial temporal artery led to recognizable inflammation of the gland (Berndt et al., 1931). 11. Saliva Total salivary microbial counts have been reported to be anywhere from 43 million to 5,500 million per milliliter of saliva, with an average of 750 million (Scherp, 1956). Although this would be regarded as very good growth in a broth culture, the oral microorganisms together with mammalian subcellular particles contribute only 1 6 4 4 % of the total metabolic activity of the whole saliva (Eichel and Lisanti, 1964). This low activity appears to reflect the fact that the total bacterial protoplasm in whole saliva is a very small portion of its total protoplasm. The metabolic activity of saliva appears to be accounted for largely (7648% ) by the oral leukocytes, which enter the human oral cavity at a rate of 103,000 per second ( Klinkhamer, 1963). Among the bacteria present in human saliva (Scherp, 1956), the overwhelming majority of the population consists of Streptococcus mitis, Streptococcus salivarius, and Veillonella gazogenes. At least 26 other species of microorganisms may occur. Prominent among these are spirochetes, fusiform bacilli, diphtheroid bacilli, actinomyces and other filamentous forms, and vibrios. Also found consistently in minor proportions are P-hemolytic streptococci, enterococci, micrococci, lactobacilli, yeasts, and PPLO. The protozoan Endamoeba gingivalis is commonly found in the oral cavity in some parts of the world. It is unlikely that the microbial population of saliva is derived from multiplication within the saliva (see next section). The flora of the saliva does not give a satisfactory picture of the bacteria of the plaque and gingival crevice (Krasse, 1954b; D. F.
ORAL MICROBIOLOGY
227
Gordon et al., 1965), but it does reflect the microbiota of the tongue (D. F. Gordon et al., 1965). Qualitative studies (Bowen, 1965) of the salivary flora of monkeys indicate that it is very similar to that of humans. Bacterial populations are higher in monkey saliva, perhaps in part because of the reported negligible resting flow of saliva into the monkey’s mouth. C. BIOLOGICAL CONTROLOF ORALMICROBIOTA 1. Rinsing Effect of Saliva The adventitious or exogenous microorganisms of the oral cavity constantly gain entrance with air, dust, food, and water but are unable to establish themselves permanently in the mouth. W. D. Miller (1889) was one of the first to point out how rapidly the adventitious flora are removed. He rinsed his mouth with highly concentrated suspensions of lactic acid bacilli and was able to show practically none of these organisms by the next morning. Bloomfield (1919) swabbed Sarcinu lutea on selected sites in the mouth and found that it disappeared at varying rates, depending upon the site. The organism was practically gone from the tongue after 1 hour. In contrast it persisted on the nasal mucosa somewhat longer, although after 24 hours it was largely gone from there, too. Appleton and co-workers (1938) found with Serratia marcescens that the heavier the initial oral contamination the longer it took to attain minimal or negative cultures with test rinsings. The time required to reach a low level of retained bacteria varied from one test subject to another. Bloomfield (1919) considered that this rapid clearing capacity of the oral cavity is dependent principally upon the saliva. The magnitude of the bacterial count attained by the saliva is significantly influenced by the rate of flow of the saliva and by the passage of fluids or food through the mouth. E. A. Brown and Cruickshank (1947) found that, if no food, water, or medication is taken during a test period of 3 hours, the salivary bacterial count increases 445.5% over the initial count by the end of that period. If food is ingested at the end of 3 hours the count after the meal is approximately 107% over the count just before the initial meal. With a 5-minute water rinse after 3 hours the increase is 338%. The oral fluid itself normally has an effective rinsing action. Stimulation of salivary flow with tablets containing malic acid may be of prophylactic value, since a small but statistically significant reduction in dental caries has been observed (Slack et al., 1964) when these tablets were used as an adjunct to toothbrushing. Human saliva is actually a mixture of fluids from three pairs of major
228
HEINER HOFFMAN
glands together with smaller mucous glands distributed in the mucous membrane. The normal young adult secretes about 12OOml. of saliva in 24 hours (Hill, 1939), but the rate at which the saliva is secreted varies significantly over the 24-hour period. Salivation temporarily slows or even ceases during sleep (Lear et al., 1965), while it is at a maximum during eating. Since saliva is promptly cleared from the mouth as it is secreted during the hours a person is awake, it is apparent that microorganisms do not have enough time to multiply in the flowing saliva during its progress from the salivary glands through the oral cavity into the pharynx (Macdonald, 1962). The large bacterial counts obtained from saliva must therefore result from its rinsing effect upon the various structures of the oral cavity. Bloomfield (1922b) had shown that the rinsing effect of saliva is apparently limited to the pathway of suction currents toward the esophagus. Bacteria introduced into the mouth were found to be drawn rapidly and directly backwards toward the esophagus, avoiding the tonsils and posterior pharyngeal wall. No forward movement of the bacteria was detected by Bloomfield from the tonsils or pharynx, nor did he find that bacteria became uniformly spread over the mouth from inoculation sites. A more precise description of the salivary flow patterns was recently formulated in an important study by Kleinberg and Jenkins (1964). They considered the oral cavity to be composed of two compartments, with the teeth providing some separation between the two. The oral vestibule is one compartment, bounded by the labial and buccal tooth surfaces and the buccal and labial mucosae, and contains the orifices of the ducts from the parotid glands and the minor glands of the buccal mucosa. The second compartment is the oral cavity proper, which has in it the lingual tooth surfaces and the orifices from the submaxillary and sublingual glands. This latter compartment is supplied with more saliva, mainly because of the higher secretory rate of submaxillary relative to parotid saliva, and because the saliva from the vestibule moves into this compartment before being swallowed. The anterior location of the submaxillary duct in the oral cavity results in a greater volume anteriorly than posteriorily in this compartment, while the posterior location of the parotids in the vestibule has the reverse effect. The approximal dental areas are considered to be intermediate between the two compartments. Since higher salivary flow rates were observed to favor higher plaque pH levels, it was possible to check the model of salivary compartmentalization against plaque pH values on the teeth throughout the mouth. A close correspondence was found. Significantly, the regional plaque pH levels showed a distribution pattern similar to the patterns previously reported for the intraoral incidence of caries and periodontal disease.
ORAL MICROBIOLOGY
229
Regions in which plaques had a greater tendency for lower pH values had a greater tendency for dental caries. The areas of increased incidence of gingivitis, subgingival calculus, and pathological pockets corresponded exceptionally well with the areas of the most alkaline pH levels. The rinsing effect of the saliva is appreciably influenced by the viscosity of the fluids which are contributed to it by the different salivary glands. The parotid gland, which accounts for 26% of the resting salivary secretions (Schneyer, 1956), produces a thin and serous fluid which is devoid of mucin; the sublingual, palatal, and buccal gland secretions are thicker because of a high mucin content. The secretion of the submaxillary gland, which accounts for 69% of resting salivary secretion, may be either thin and watery, thick and viscid, or a mixture of both, depending upon the nature of the secretory stimulus. Thick, ropy saliva that can be drawn out in long threads is thought to promote dental caries by allowing stagnation about the teeth (Macphee, 1935). If the flow of saliva is completely checked, all the teeth will be destroyed by caries unless some other means is resorted to promptly for removal of food debris (Prinz, 1919). Individuals with a unilateral deficiency in salivary secretion show much greater caries activity on the side with inadequate salivary flow than on the normal side (Gurley, 1939). 2. Antimicrobial Eflects of Saliva Intimations of salivary antimicrobial activity may have led in ancient times to the attribution of therapeutic activity to human saliva (Anonymous, 1913). Saliva was used externally especially for skin diseases, and notably for eczema, impetigo, and herpes, for simple wounds and superficial uIcers, for smallpox pustules, and even for some mild manifestations of leprosy. It was used chiefly, however, as a valuable remedy for the bites of venomous snakes. Avicenna taught that the saliva of a fasting man made worms come out of the ears. Pliny believed that inflammation of the eyelids was cured by the application of saliva, while Tacitus affirmed that blindness can be cured by wetting the eyes and cheeks of the blind man with saliva. The antimicrobial action of saliva has been studied intensively in recent years and has been found to be due to a series of complex phenomena. These conveniently may be separated into two groups for discussion: ( a ) activity against bacteria, and ( b ) activity against viruses.
a. Antibacterial Eflects Kletzinsky (1852) appears to have been among the first to apply the scientific approach to the problem of salivary antibacterial effects. His studies led him to the suggestion that thiocyanate in the saliva has an
230
HEINEH HOFFMAN
antiseptic action. Valude (1888) believed that saliva may have a special chemical power for killing the tubercle bacillus, since tuberculosis of the oral mucosa and salivary glands rarely occurs. An early study of Triolo (1897) failed to show any bactericidal action with filtered saliva. It was subsequently shown (Bibby et al., 1938) that in vitro inhibition occurs with resting saliva which has been passed through a Berkefeld V or N filter and tested against Lactobacillus acidophilus, staphylococcus, and streptococcus, but no activity was found against Micrococcus Zysodeikticus. Adams and Creamer ( 1961) found that the antilactobacillus factor was active against a group of strains resembling the subgenus Streptobacterium, but paradoxically it stimulated a group of lactobacilli in the subgenus Betabacterium. Zeldow (1955) found inhibition of L. acidophilus growth with saliva collected directly from the parotid gland; this saliva had a greatly reduced bacterial count (at least 99% lower than for mixed saliva). Since Zeldow also found that mixed saliva with high bacterial counts did not exhibit consistently different inhibitory activity, it appeared that the agent is not bacterial in origin. Clough and co-workers (1938) found that the salivary agent which was capable of preventing the growth of L. acidophilus and even of bringing about its death is inactivated by ultraviolet radiation, by repeated freezing and thawing, and by heating for 5 minutes at 75°C. These properties suggested that the antilactobacillus factor is an enzyme, and recent work has borne this out. Zeldow (1959) was abIe to separate the factor in parotid saliva into two components by dialysis. The nondialysable component is heat-inactivated at 75°C. and is probably salivary peroxidase (Klebanoff and Luebke, 1965), while the dialysable component is relatively heat stable (Dogon et al., 1962) and is probably thiocyanate. The mechanism by which the lactobacillus growth is inhibited, however, is not known. The lactobacillus inhibitory factor does not appear in the saliva of premature newborns, and no activity is found in the saliva during the first 4 days of life in the majority of full-term newborns (Austin and Zeldow, 1961). During the first decade of life there is a systematic increase in the titer of lactobacillus inhibitory factor until approximately the adult range of activity is reached. The antilactobacillus potency of the whole saliva cannot be correlated with the oral health of the individual from whom it is obtained. In 77 children, for example (Clough et al., 1938), no correlation could be established between the extent of dental caries and the degree of L. acidophilus inhibition demonstrated in test plates. The presence of lysozyme in saliva is well established. Lysozyme activity in human saliva was first reported by Fleming (1922). Bibby et al.
ORAL MICROBIOLOGY
231
(1938) found that, in contrast to the antibacterial factor active against lactobacilli, the agent active against Micrococcus lysodeikticus is unable to pass a Berkefeld filter. Submaxillary and sublingual salivary gland secretions show substantially higher lysozyme titers than parotid gland secretion (Hoerman, 1956). Berger (1952), in an assessment of the significance of lysozyme in the saliva, pointed out that since this enzyme is most active against saprophytic bacteria normally encountered in the air and since it has only moderate activity against pathogens, its role in the saliva and oral cavity can be considered at best to be that of an accessory factor for the destruction of contaminating saprophytes. In a recent reexamination of lysozyme sensitivity of 112 pure cultures representing 13 groups of bacteria occurring in the oral cavity, de Stoppelaar and Gibbons ( 1965b) found that 500 pg./ml. of lysozyme did not prevent the growth of any of them on blood agar plates. Thus the antimicrobial activity of saliva may well play an important role in the selection and maintenance of the characteristic members of the oral microbiota. On the basis of the extensive work which has been done on this problem Bibby (1949) has advanced the general proposition that “saliva wiII prevent the growth in the mouth of all except those types of organisms which are peculiar to the mouth. In other words, the oral streptococci, fusiform bacilli, certain diphtheroids, leptotrichia, and other specific mouth types can survive in the mouth, but pathogenic organisms are rapidly overcome.” There are many additional bacteria on which such antimicrobial effects have been reported on the basis of in vitro studies (Bibby et al., 1938; Bartels et al., 1962): staphylococci, streptococci, sarcinae, micrococci, pneumococci, Nocardia, tubercle bacilli, diphtheria bacilli, pseudodiphtheria bacilli, influenza bacilli, typhoid bacilli, colon bacilli, cholera vibrios, Proteus vulgaris, Serratia marcescens, Pseudomonas pyocyaneus, and Brevibacterium. Probably all human saliva possesses antibacterial properties effective against one or more species of microorganisms ( Bibby et al., 1938), although the activity varies during the day (Bonicke et al., 1953), from day to day, and from subject to subject ( Hine, 1936).
b. Antiviral E f e c t s Antiviral effects may also be demonstrated with human saliva. Influenza virus hemagglutination is inhibited by whole saliva (Francis and Minuse, 1948; Rolla, 1965), but the amount of inhibition varies with the individual source of saliva, and even from day to day in the same individual. The combined secretions of the submaxillary and sublingual glands have a greater inhibitory effect upon influenza virus hemagglutination than the parotid secretion. The inhibition seems to be due to a
232
HEINER HOFFMAN
reaction between inhibitor and virus, and the agent is probably a mucoprotein ( Seltsam et al., 1949; Rolla, 1965). Blood group A substance, which occurs in the saliva of group A secretors, has considerable inhibitory effect upon influenza virus hemagglutination ( R. H. Green and Woolley, 1947 ) . Some samples of saliva may inhibit hemagglutination by Columbia SK virus but not by influenza virus (Jungeblut et al., 1952). In this case it appears that the inhibitor acts upon the red blood cells rather than the virus, and additional evidence (Jungeblut and Knox, 1954) indicates that it is an enzyme which is closely related to receptor-destroying enzyme of bacterial origin. Receptor-destroying enzyme, however, cannot be isolated from mixed oral flora. This active principle is found more frequently and more consistently in the saliva of acutely ill and convalescent patients with poliomyelitis than in the saliva of healthy persons. Brody et al. (1936) found an association between blood group type and incidence of viral or bacterial infections.
3. Microbial Interactions in the Saliva Both in vivo and in vitro studies have yielded considerable evidence concerning the role of microbial interactions in the balance of microorganisms characteristic of the oral cavity. Holman (1928) has enumerated the possible interactions as follows: ( a ) the microorganisms in a simple microbial mixture have no demonstrable effect upon each other, referred to as commensalism; ( b ) one microbe favors the growth and activity of another, or probiosis; ( c ) two or more organisms are mutually beneficial, referred to as mutualism; ( d ) one action follows upon another, or metabiosis; and ( e ) one organism has a demonstrably harmful effect upon another, or antibiosis. [Rosebury (1962) suggests that the term commensalism be discarded in favor of a word with less objectionable connotations, namely amphibiosis, signifying a spectral position between probiosis and antibiosis and merging with both.] Another possible mode of interaction would be symbiosis, which is defined (Dougherty, 1953) as an intimate relationship between two (or rarely more) organisms such that one, the symbiont, gains nutrition or protection, or both, from another, the host. Not all of these relationships may be operating in the mouth, and the relative importance of those which do is not known with certainty. Moreover, a pair of interacting microbial species may simultaneously exert both probiotic and antibiotic effects upon each other. In some cases of bacterial association occurring in the oral cavity, the precise nature of the interaction has not yet been established, although at least one member of the association may obviously benefit.
ORAL MICROBIOLOGY
233
a. Probiosis Among the biological associations which may be operating is probiosis, in which one member only of a pair derives ecological advantages from the association. To detect this relationship, Scrivener et al. (1950) used a double pour-plate technique in which the diluted saliva was first incubated in the bottom agar layer and then incubated a second time with an overlay of agar inoculated with lactobacillus. Eighty-nine of ninety-eight saliva samples examined contained colonies that produced areas of larger lactobacillus colonies-stimulation or sateIlitism. The number of organisms per milliliter of saliva which had stimulatory activity ranged from zero to 3,700,000, with most samples below 1,OOO,OOO. Rosebury et al. (1954) found a stimulation of growth of Neisseria catarrhalis by several other species, and stimulation of Escherichia coli growth by Candida nlbicans. Both phenomena were observed only on media on which the test organism failed to grow alone or grew poorly. Combined probiotic and antibiotic effects were uncovered when G. Young and co-workers (1956) found that oral strains of lactobacilli and Candida albicans grown in mixed culture showed a decrease in candidas and an increase in lactobacilli when compared to pure cultures. It appeared that the inhibitory effect probably was due to lactic acid. In vitamin-deficient media unable to support the growth of lactobacilli alone, G. Young and co-workers (1956) found that these organisms grew well in the presence of C. albicans. The growth usually took the form of colonies, containing large numbers of both organisms mixed together centrally, and a margin of lactobacillus cells around the central mixture. They suggested that in the normal mouth a counterbalance exists between these two organisms, with the candidas providing nutritional stimulation for the lactobacilli, and the latter producing lactic acid which prevents the excessive development of the yeasts. GuiIlot (1958) later demonstrated in filtrates of Lactobacillus acidophilus cultures a substance which inhibited Candida albicans development. It was destroyed by heating for 20 minutes at llO"C., was water soluble, and could be absorbed by activated charcoal in neutral or acid medium. Koser and co-workers (1960) examined the in vitro lactobacillus-candida system and were able to define the nutritional factors involved. They found that Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus fermenti were capable of growing in association with Candidu albicans in a medium originally deficient in vitamins for the lactobacillus but satisfactory for the yeast. Of the three lactobacilli species studied, substantial growth of L. fermenti was secured only when an additional reducing agent and pentoses were added to the medium. The hazards of attempting to extrapolate such i n vitro observations to
234
HEINER HOFFMAN
the oral cavity is well illustrated by the observation of Krasse (1954b) that the number of candidas was usually greater than the number of lactobacilli from the same dilution of plaque material, whereas in the saliva more lactobacilli were present than candidas. Nevertheless, the presence of lactobacilli appears to be associated with the presence of C . ulbicans and vice versa ( Lilienthal, 1950). The finding of fusobacteria and other anaerobes in the dental plaque suggests that the associated aerobic bacteria may be exerting a protective effect (Hemmens et al., 1941). b. Antibiosis
Antimicrobial properties of the saliva may conceivably have their origin in sources other than the salivary glands. Among these possible sources are the oral leukocytes, the desquamated epithelial cells, and the oral bacteria, all of which are present in large numbers in saliva (Kerr and Wedderburn, 1958). The bacterial sources might be of considerable significance if the great number of antibiotic effects demonstrated in vitro with oral microorganisms actually operate in the mouth. This is a very difficult point indeed to establish (C. P. Miller, 1959). The vast number of in vitro studies of antagonisms exhibited by oral microorganisms can be touched upon only briefly in the following survey. M. H. Gordon (1916) accidently observed that meningococci failed to grow out on plates after the culture had been mixed with saliva. Upon further examination of the phenomenon he found that the inhibitory effect was chiefly due to the mixed salivary streptococci. Cultures of single streptococcic strains had a less marked effect, and a staphylococcus was found to have no inhibitory effect at all. The reason for the more vigorous action of the mixed streptococcic culture was not determined. Recent observations on streptococci, however, suggest that this may be due to increased acid production through stimulation of growth of one species by the other by means of excreted adenine (Dahiya and Speck, 1963). Some decades after Gordon’s observations, the problem of effects of oral bacteria upon pathogens was taken up again by Thompson and Shibuya ( 1946),who used Corynebacterium diphtheriae as the indicator organism. Staphylococci, sarcinae, and diphtheroids were found to be without effect; inhibitory activity was shown only by streptococci. Thompson and Shibuya demonstrated that the inhibitory action of saliva on the diphtheria bacillus is due to Streptococcus mitis. The mechanism involved in the in vitro action of streptococci has been unraveled through a series of investigations. Arvidson et al. (1949) reported that the inhibitory action of a-streptococci upon diphtheria bacilli can be attributed to a diffusible factor, the action of which is
ORAL MICROBIOLOGY
235
blocked by red blood cells. Hegemann (1950) and Thompson and Johnson (1951) subsequently found that the diffusible factor is hydrogen peroxide. Wheater et al. (1952) observed an antibiotic effect against Staphylococcus aureus by a strain of lactobacillus isolated from Gruykre cheese which subsequently was also attributed to hydrogen peroxide ( Wheater et al., 1952). Other organisms occasionally found in the saliva, particularly diphtheroids and staphylococci ( Thompson and Johnson, 1951), inhibit the diphtheria bacillus by mechanisms other than hydrogen peroxide. There is reason to question the regulatory importance of biogenic hydrogen peroxide in the oral cavity. Berger (1952) came to the conclusion after careful consideration of the evidence he had obtained that biogenic hydrogen peroxide, because of bacterial catalase, does not have a role in controlling or influencing the biological equilibrium in the oral cavity. Bonicke et al. (1953) failed to find hydrogen peroxide present in effective concentrations in the saliva and also considered that this was probably because of rapid breakdown of the compound by the available catalase, largely bacterial in origin (Nickerson et al., 1957). The observation that Escherichia coli is not commonly present in the oral cavity correlates well with the in vitro observations of the inhibitory effect of saliva upon E . coli growth (Appleton and Dietz, 1937; Clough, 1933). Knighton (1953) later suggested, on the basis of studies with filtered and unfiltered saliva supplemented with glucose or peptone, that the limitation of growth of E. coli in saliva is due to a competition for nutrients among the numerous microorganisms naturally occurring in the saliva. More recently, however, de Stoppelaar and Gibbons (1965a) found that the concentrations of volatile fatty acids in untreated oral debris are sufficient to inhibit growth of E . coli at p H 6 and 5. Addition of any of several amino acids was found to partially overcome fatty acid inhibition in a minimal medium. Since oral debris placed on plates of trypticase soy-glucose agar seeded with E . coli inhibited growth of the E. coli, it appears that the mechanism for inhibition of E . coli growth in the oral cavity depends upon a low p H combined with low concentrations of free amino acids and peptides. That this may be an important mechanism in the resistance of the oral cavity against pathogenic gram-negative forms is suggested by the investigations of C. P. Miller and Bohnhoff (1963) and Bohnhoff and co-workers (1964) on the role of Bacteroides in the resistance of the mouse intestinal tract to experimental Salmonella infection. C. P. Miller and Bohnhoff (1963) found that the resistance of the normal mouse to infection by oral inoculation with Salmonella enteritidis could be ascribed to the presence in its enteric flora of members of the
236
HEINER HOFFMAN
genus Bacteroides. These obligate anaerobes, which are among the most numerous inhabitants of the cecum and upper colon of normal mice, were found to release a potent inhibitor of Salmonella in vitro. Separation of fractions from broth cultures of Bacteroides (Bohnhoff et al., 1964) gave two compounds, acetic acid and butyric acid, both of which inhibited S. enteritidis. Further study demonstrated that under relatively anaerobic conditions and at the pH of colon contents of most normal mice ( p H 6.2 or lower), the concentrations of acetic and butyric acids present in the colon quite adequately account for its resistance to infection by moderate inocula of S. enteritidis. The possibility that inhibitory organisms of the “normal” microflora of superficial human tissues may be significant in resistance to or recovery from infections in these areas was suggested many years ago (Nissle, 1916; Dujardin-Beaumetz, 1932) , This possibility is now being explored in an interesting attempt to control dental caries. Scrivener et al. (1950) observed that gram-positive spore-forming bacilli isolated from saliva inhibited the growth of lactobacilli on streak plates. Since attempts to inoculate antagonists to oral lactobacilli into the mouth failed, Scrivener (1955) attempted to adapt to saliva a strain of Bacillus hrevis and of Staphylococcus albus, both of which were antagonistic to oral lactobacilli. These saliva-adapted strains survived in the mouth for only 30 days in a few cases and for as long as 7 months in others (Rutter et al., 1961). Nevertheless, the incidence of dental caries in the group receiving the bacterium was reduced by 50% over the control group. 4 . Saliva as a Microbial Nutrient Medium
a. p H Effects From an investigation into the forces acting upon the microorganisms of the oral mucosa, Lammers (1952) concluded that the biological equilibrium in the mouth is determined primarily by the growth characteristics of the oral bacteria. His evidence indicated that in vitro tests demonstrating antagonistic effects did not clarify the actual situation in the oral cavity since the antagonisms depend upon the inherent vigor of growth, for which the alkaline earth compounds are of significance. The alkaline earths, according to Lammers, inhibit the growth of the acidophilic organisms and shift the pH toward the optimum for proteolytic organisms. This conclusion appears to restrict the role of saliva unnecessarily, since oral microbes must have a source for nutrition, and the saliva seems to be an obvious one. If a certain nutrient in saliva were at a very low level relative to the nutritional needs of a particular microorganism this then might become the limiting factor in the growth of that species.
ORAL MICROBIOLOGY
237
Lammer’s conclusion, however, has received support from a subsequent study by Lightfoot and Coolidge (1959) on the role of acidity in the nutrition of oral acidogens. Their in vitro experiments indicated that, in the absence of inhibitors, the production of amino acids from saliva is sufficient for the growth of lactobacilli and streptococci only when the saliva is acid. This suggested to them that the production of acid from ingested carbohydrate greatly enhances the production of amino acids in places in which the dilution of locally retained saliva by freshly secreted saliva is low. This could well explain the positive relationship between dietary carbohydrate and the level of oral lactobacilli (Jay et al., 1936; Becks et al., 1944). Sylvester and co-workers (1963), on the other hand, believe that the buffering capacity of saliva accounts for increased growth of streptococci in saliva. These investigators found that the total growth of rat oral streptococci was decidedly stimulated when this organism was cultured in broth supplemented with saliva from rats, especially rats susceptible to dental caries. They suggested that the greater buffering capacity of saliva from susceptible rats compared with resistant rats would permit larger populations of acidogenic microorganisms to grow before the pH of the surrounding medium would become inhibitory.
b. Saliva as Medium Supplement
A number of investigators have studied the suitability of human parotid or whole saliva as a nutrient supplement in artificial culture media. Parotid saliva collected aseptically and found not to contain viable microbes was used by Umemoto and co-workers (1950a,b) as a supplement in culture media. The optimal proportions of saliva to medium differed for the several microbes studied. Williams and Powlen (1959) found parotid saliva alone to be a less than optimal medium for the 10 microbes they studied, inchding a Lactobacillus species, a viridans streptococcus, Streptococcus faecalis, and Candida albicans. The viable counts for eight bacteria, each cultivated in a different saliva sample, indicated that three of the bacteria ( a lactobacillus, a diphtheroid, and a P-hemolytic streptococcus) tended to die in the saliva, whereas all others ( a yeast, an aerobacter, an enterococcus, a staphylococcus, and a viridans streptococcus) tended to survive, although they rarely showed significant increases compared to the control. Since none of these bacteria was inhibited by parotid saliva when tested by the cup technique, it would seem that lack of growth in parotid saliva was due to insufficient essential nutrients rather than to antibacterial activity. Hammond ( 1959) found that growth in sterile submaxillary-sublingual secretion did not differ significantly from growth in parotid saliva. The
238
HEINER HOFFMAN
adequacy of saliva as a nutrient may hinge upon the concentration of microbial cells competing for the available growth substrates ( Stephan and Heinmens, 1947). In the dental plaque, where the microorganisms are in very high concentration, a strong competition for nutrients would be expected, whereas on the buccal mucosa, where the bacterial burden of the epithelial cells is relatively light, competition for limited substrates in the saliva would be expected to be relatively unimportant,
Salivary Vitamins Weisberger (1946) found that whole saliva could replace the mineral salts required in a synthetic medium for an oral lactobacillus, but that it was necessary to concentrate the saliva before it could be substituted for thiamin hydrochloride, calcium pantothenate, or nicotinic acid. Even then the growth was not optimal. Koser et al. (1951; Koser and Fisher, 1950) have studied the vitamin requirements of oral lactobacilli and the vitamin levels required to attain growth and acid production to p H 5.0, the point at which enamel decalcification may occur. This group of investigators ( K a u h a n et al., 1953) later found that saliva contains enough folic acid to support maximal or near maximal growth and acid production by oral lactobacilli. Salivary levels of vitamin Be (pyridoxine) in many cases appeared to be adequate for definite growth and acid production, and in some cases high enough to allow maximal or almost maximal growth and acid production. Paradoxically, several species of oral bacteria were found by Balogh et al. (1960) to show significantly less growth if they were cultured with 0.02% thiamin hydrochloride, 0.04% riboflavin, and 0.02% pyridoxine hydrochloride (singly or in mixture) added to saliva prior to incubation. The effect was not obtained when the vitamins were placed in wells in blood agar plates. Disraely and co-workers (1959) found that certain of the vitamins present in saliva can be formed as the result of microbial action. Incubation of saliva resulted, they found, in the increase not only of free folic acid but also of bound folic acid compounds. Furthermore, chromatography of incubated saliva indicated the presence of an unidentified folic acid-like compound and a compound suggestive of citrovorum factor. Microbial populations in the incubated salivas were studied. Of the four species determined, fusobacteria and veillonellae increased in numbers, streptococci decreased, and lactobacilli remained almost unchanged from the initial counts at the start of incubation. When the saliva samples were incubated with glucose, all four species showed increases ranging from a doubling for the veillonellae to more than a 25-fold increase for the lactobacilli. c.
ORAL MICROBIOLOGY
239
d. Salivary Carbohydrates and Amino Acids Salivary carbohydrates and amino acids may serve as nutrients for the oral microbiota. Glucosamine disappears from raw unstimulated saliva which is incubated from 1 to 3 days (Matt, 1954; Rogers, 1948). A large increase in bacterial count occurs after 24 hours, but the greening streptococci undergo both a relative and absolute drop in numbers. After 2 days’ incubation, the greening streptococci constitute a tenth of the total aerobic flora, in contrast to the initial two-thirds (Berger, 1952). Salivary mucin may serve as an effective source of essential amino acids for Lactobacillus acidophilus upon hydrolysis by proteolytic or amylolytic enzymes ( Dreizen et al., 1950). Autoclaved whole saliva supported the production of toxin by diphtheria bacilli (Tasman and Smith, 1953), which is in harmony with the earlier observation that the diphtheria bacillus on inoculation into fresh saliva will show a slight increase in numbers over 7 days (Berger, 1952). In an evaluation of his own considerable investigations of the problem of saliva as a source of microbial nutrients, Williams (1963) came to the conclusion that nutrients for the large bacterial populations existing in the mouth must come from residual food from meals and from synergistic and symbiotic relationships among microbes. Moreover, certain mechanisms may be present in the microorganisms which provide reserve nutrient during periods when the host is not eating. It has been suggested that oral streptococci (Gibbons and Socransky, 1962) and lactobacilli ( Williams, 1963) store carbohydrates intracellularly. D. ORALIMMUNOLOGY The immune responses of the human oral cavity have been poorly studied, but there is some evidence that they play an important role in the maintenance of oral health. 1. Passive Oral Immunity of the Infant The first immune globulins active against oral infections in the infant probably are maternal antibodies transferred to the fetus by way of the placenta, since it is known that gamma globulins increase in the fetal blood during gestation, although they decrease greatly during the first few months after birth (Moore et al., 1949). A good measure of the degree of placental antibody transfer is the ratio of maternal antiserum titer to cord blood titer. The maternal-cord ratio for Streptococcus MG antibodies has been found to be greater than 1:l (Florman et a,!., 1951), a fact of some interest since Streptococcus
240
HEINER HOFFMAN
salivarius is unable to colonize in the infant’s throat even when contamination of the mucosa occurs ( Bloomfield, 1922a). Herpes simplex-neutralizing antibodies are transferred through the placental barrier to the fetus, and the infant shows detectable antibodies in comparatively large quantity for the first 4 months of life. Nevertheless, very few cases of primary herpetic stomatitis appear before the age of 1 year. Both complement-fixing and agglutination-inhibiting antibodies for mumps are transmitted via the placenta with little loss in titer, but these are usually gone from the infant’s circulation after the seventh week of life (Florman and Karelitz, 1953). The rare occurrence of mumps among infants even after loss of demonstrable antibodies may be due to a number of factors, such as innate resistance of immature tissues, or limited opportunity for exposure to the virus. Gamma globulin obtained from umbilical cord blood was found by Kolesnikov and Kallings ( 1962) to have neutralizing titers against Coxsackie virus types A5 and B1 which were identical with the titers obtained from regular human gamma globulin (12%) obtained from adult blood. It appears, therefore, that transplacental transfer of neutralizing antibodies to Coxsackie virus types A5 and B1 does occur. This is of some interest since Coxsackie type A5 virus has been isolated from vesicles in the mouths of children from 1 to 5 years of age with “hand, foot, and mouth disease” (Flewett et al., 1963). The transfer of antibodies from mother to infant via milk is not a significant mechanism for humans, although breast milk has been shown (Hummeler et aE., 1953) to contain an inhibitor for influenza and mumps virus multiplication. It is possible that colostrum exerts a small effect against streptococci and staphylococci during the first 3 days of life, as it contains low titers of immune globulins in some 40 to 50% of cases studied ( Nordbring, 1957). There is some evidence of a hiatus in the immunological protection of the oral tissues against certain pathogenic microorganisms between the decline of maternal antibody and the establishment of active immunity. In two series of cases of Hemophilus influenzae cellulitis occurring largely in the cheek ( M . Green and Fousek, 1957; Feingold and Gellis, 1965) all of the patients were between 6 months and 2 years of age. It is of interest that Fothergill and Wright (1933) found that 80% of cases of H . influenme type b meningitis occurred in children between the ages of 2 months and 3 years. They established the fact that defibrinated blood from subjects under 2 months and over 3 years of age had considerable bactericidal effect on cultures of H . inflmnzae type b, while that from children in the intervening age group lacked this ability almost without exception.
ORAL MICROBIOLOGY
24 1
2. Active Oral Immunity Kraus and Konno (1963), in a recent review of antibodies in saliva, concluded that the evidence indicates the occurrence of salivary antibodies which are directed against exogenous pathogens, including agglutinins to brucella and to the typhoid-paratyphoid group of bacilli; antitoxin against diphtheria; complement-binding antibodies t o brucella; and syphilitic antigen. It was pointed out by Kraus and Konno that in all these older reports it was assumed that these reacting substances in the saliva were antibodies and sometimes examination of the subject’s serum was omitted. Studies of serological reactions of saliva with microorganisms commonly occurring in the oral cavity have been more limited, since only studies on agglutination of lactobacilli and opsonization of Lactobacillus acidophilus, Sarcina Eutea, and one unspecified streptococcus have been reported ( Kraus and Konno, 1963). Serological reactions with nonbacterial antigens have also been described. Kraus and Konno (1963) studied the occurrence of salivary antibodies to 13 bacteria and found that antibody appeared in saliva only when it was present in serum. Bacteria indigenous to the oral cavity seemed capable of inducing antibodies in man. Specific antibodies obtained by multiple cross-adsorptions of serum and saliva to remove common antibodies were found by Kraus and Konno to be excreted in saliva to a greater extent than were the common antibodies. These investigators suggested, therefore, that there may be a selective and individually regulated transfer of serum proteins into saliva. 3. Hypersensitivity
An early account of oral symptoms in a naturally occurring allergic syndrome was given by Wyman (1872) in his classic book on hay fever: “There is itching of the roof of the mouth and the parts beyond. The velum is relaxed and thickened, and the uvula often so much elongated and swollen, that it gives the sensation of a foreign body hanging there; and falling backward toward the throat, compels to frequent hawking to throw it forward upon the tongue.” Wyman also noted the difficulty in breathing through the nose and the consequent need for mouth breathing, which dries the oral mucosa. A number of clinicians have subsequently held that allergic rhinitis may affect the oral and dental hard structures. Criep (1948), for example, considered that in young children the bony structure of the hard palate is affected in allergic rhinitis, resulting in a high arched palate, the so-called “Gothic arch,” and in dental deformities. H. I. Miller
242
HEINER HOFFMAN
(1949), however, in an examination of the dental occlusion of students at the University of Michigan, could find no support for the hypothesis that allergy is a causative or a contributing etiologic factor in the maldevelopment of dental arch dimensions or occlusal relationships. Ragweed-sensitive individuals have recently been found to exhibit reaginic activity in their saliva (Ishizaka et al., 1964). These salivas, as well as specimens from normal individuals, contained ylA-globulin, possibly a trace of albumin, b u t no other serum proteins. I t appears likely, therefore, that the reaginic activity of the saliva is due to the ylAglobulin fraction. From the few studies of the oral cavity which have been carried out, it appears that, like other parts of the gastrointestinal tract (Kirsner and Goldgraber, 1960), the oral cavity is capable of exhibiting experimentally induced hyperimmune reactions. Hypersensitivity of the mucous membranes (I. Frank et d.,1942) has been shown in rabbits sensitized with egg white by a variety of routes. In highly sensitized animals ;in injection of the antigen into the lip gave massive swelling of the lip tissues in addition to a bleblike area at the test site. Brandtzaeg and Kraus (1965) have carried out a careful study on the possible role of autoimmunity in periodontal disease. No unequivocal support was obtained for the hypothesis of autoantibody production in periodontal disease, but the possibility could not be excluded. The active Arthus phenomenon was obtained by Terner (1965) in the tongue, attached gingiva, and buccal and palatal mucous membrane of the rabbit, guinea pig, and rat with normal equine serum as the antigen. The local Shwartzman reaction was elicited by Rizzo and Mergenhagen (1960) in the rabbit oral cavity and skin with endotoxin prepared from a strain of oral Veillonellu. Extracts from viridans streptococci failed to produce this reaction in either tissue.
E. CONCLUSION It is quite apparent, in retrospect, that a full survey of oral microbiology could not be made within the limits of this review. Moreover, our own special interests have led to a disproportionate emphasis of some topics over others. The problem of dental caries, fortunately, has been reviewed frequently by well-qualified investigators. Some recent summaries of various aspects of this centraI problem in dentistry are available in the volume edited by Sognnaes (1962). A somewhat older monograph still of interest is that of Lammers and Hafer (1956). A great deal of important work has been done within the past decade on the biology of the microbial species which occur in the oral cavity, but no comprehensive survey is available. The volume by Rosebury (1962)
ORAL MICROBIOLOGY
243
on microorganisms indigenous to man partially fills this need. A brief introduction to the oral bacteria and fungi is given by Bisset and Davis (1960), but this volume must be read with the awareness that Bisset’s special views on taxonomy permeate the text. It is now possible to obtain a meaningful picture of the oral cavity as a microbial habitat largely because of work done in recent years. This to very great degree is the fruit of well-informed support by the United States Public Health Service and the National Institutes of Health. Both the extramural program of the U. S. Public Health Service and the intramural program of the National Institute of Dental Research have been admirable. Without them it is difficult to see how the indolent tempo of progress characteristic of oral microbiology in the past could have been surmounted. ACKNOWLEDGMENTS Our studies in oral microbiology for the past 9 years were carried out with the aid of grants from the National Institute of Dental Research. The present study was supported in part by Public Health Service grant DE 01462-04 from the National Institute of Dental Research. REFERENCES Adams, A. B., and Creamer, H. R. (1961). J. Dental Res. 40, 1292. Andrewes, F. W., Bulloch, W., Douglas, S. R., Dreyer, G., Gardner, A. D., Fildes, P., Ledingham, J. C. G., and Wolf, C. G. L. (1923). “Diphtheria, Its Bacteriology, Pathology and Immunology,” p. 44. H. M. Stationery Office, London. Anonymous. (1913). Brit. Med. J. 11, 1444. Anthony, D. H., and Fisher, D. F. (1948). J. Tennessee State Med. Assoc. 41, 362377. Appleman, M. D., Freese, J. A., and Riera, M. (1955). Brit. Dental J. 99, 331-334. Appleton, J. L. T., and Dietz, A. K. ( 1937). J. Dental Res. 16, 325. Appleton, J. L. T., Klein, H., and Palmer, C. E. (1938). Am. J. H y g . 28, 213-231. Arnim, S. S. (1959a). J. Tennessee State Dental Assoc. 39, 1-28. Amim, S. S. (1959b). J. Prosthetic Dentistry 9, 1017-1036. Arvidson, M., Ericsson, H., Ouchterlony, O., and Vallquist, B. (1949). Acta Pathol. Microbiol. Scand. 27, 263-269. Ashe, W. K., Rizzo, A. A., and Mitchell, C. T. (1965). Intern. Assoc. Dental Res., Program Abstr., 43rd Gen. Meeting, Toronto, p. 85. Austin, L. B., and Zeldow, B. J. (1961). Proc. SOC. Exptl. Biol. Med. 107, 406-408. Bader, H. I., and Goldhaber, P. (1965). Intern. Assoc. Dental Res., Program Abstr., 43rd Gen. Meeting, Toronto, p. 70. Baer, P. N., and Burstone, M. S. (1959). OraZ Surg., Oral Med., Oral Pathol. 12, 1147-1 152. Bahn, A. N., and Quillman, P. (1963). Dental Progr. 3, 94-99. Bailit, H. L., Baldwin, D. C., and Hunt, E. E., Jr. (1964). Arch. Oral BioE. 9, 435-438. Balogh, K., Petrucz, K., and Angyal, J. (1960). J. Dental Res. 39, 886-891. Bartels, H. A. (1954). Oral Surg., Oral Med., Oral Pathol. 7, 559-564.
244
HEINER HOFFMAN
Bartels, H. A., Blechman, H., and Wasserman, R. H. (1962). J. Dental Med. 17, 8590. Becks, H., Jensen, A. L., and Millarr, C. (1944). J. Am. Dental Assoc. 31, 1189-1200. Beckwith, T. D., Simonton, G. W., and Rose, E. J. (1927). Dental Cosmos 69, 164171. Bender, I. B., Seltzer, S., and Kaufman, I. J. (1959). I. Am. Dental Assoc. 59, 466471. Berger, U. (1952). Z . Hyg. Infektionskrankh. 133, 371-397. Berger, U., Kapavits, M., and Pfeifer, G. (1959). Z . Hyg. Infektionskrankh. 145, 564-573. Berke, J. D. (1935). Dental Cosmos 77. 134-139. Berndt, A. L., Buck, R., and von L. Buxton, R. (1931). Am. J. Med. Sci. 182, 639649. Beust, T. B. (1929). J. Dental Res. 9, 333-341. Bibby, B. G. (1938a). 1. Dental Res. 17, 423-429. Bibby, B. G. (193813). I. Dental Res. 17, 471-476. Bibby, B. G. (1939). I. Am. Dental Assoc. 26, 629-636. Bibby, B. G. (1949). Oral Surg., Oral Med., Oral Pathol. 2, 72-81. Bibby, B. G. (1953). Oral Surg., Oral Med., Oral Pathol. 6, 318-327. Bibby, B. G., Hine, M. K., and Clough, 0. W. (1938). J. Am. Dental Assoc. 25, 1290-1302. Biondi, D. ( 1887). Z. Hyg. Infektionskrankh. 2, 194-238. Bissada, N. F., Schaffer, E. M., and Haus, E. (1965). Intern. Assoc. Dental Res., Program Abstr., 43rd Gen. Meeting, Toronto, p. 72. Bisset, K. A., and Davis, G. H. G. (1960). “The Microbial Flora of the Mouth.” Heywood, London. Bjorn, H., and Carlsson, J. (1964). Odont. Revy 15, 23-28. Blank, H., Burgoon, C. F., Baldridge, G. D., McCarthy, P. L., and Urbach, F. (1951). J. Am. hled. Assoc. 146, 1410-1412. Bloomfield, A. L. (1919). Bull. Johns Hopkins Hosp. 30, 317-322. Bloomfield, A. L. (1922a). Bull. Johns Hopkins Hosp. 33, 61-66. Bloomfield, A. L. (1922b). Bull. Johns Hopkins Hosp. 33, 145-149. Bloomfield, A. L. ( 1 9 2 2 ~ )Bull. . Johns Hopkins Hosp. 33, 252-256. Bonicke, R., Reif, W., and Arndt, J. (1953). 2. Hyg. Infektionskrankh. 136, 252-264. Bohnhoff, M., Miller, C. P., and Martin, W. R. (1964). J. Exptl. Med. 120, 805-816. Bowditch, H. I. (1850). Am. J. Med. Sci. [N.S.] 19, 363-364. Bowen, W. H. T. (1965). Intern. Dental J. 15, 12-53. Bowen, W. H. T., and Gilmour, M. N. (1961). Arch. Oral Biol. 5, 145-148. Boyd, D. A. (1956). In “A Symposium on Preventive Dentistry” (J. C. Muhler and M. K. Hine, eds.), pp. 154-162. Mosby, St. Louis, Missouri. Boyd, W. C., and Shapleigh, E. (1954). Science 119, 419. Boyd, W. S., and Rosenthal, S. L. (1958). J. Dental Res. 37, 288-291. Bradley, J. L. (1948). Oral Surg., Oral Med., Oral Pathol. 1, 423-426. Brandtzaeg, P., and Kraus, F. W. (1965). Odont. Tidskr. 73, 281-393. Brill, N. (1959). Acta Odontol. Scand. 17, 431-440. Brody, H., Smith, L. W., and Wolff, W. I. (1936). J. Lab. Clin. Med. 21, 705-710. Brooke, J. W. (1938). J. Dental Res. 17, 57-67. Brown, E. A., and Cruickshank, G. A. (1947). J. Dental Res. 26, 83-90. Brown, L. R., Jr., and Rudolph, C. E., Jr. (1957). Oral Surg., Oral Med., Oral Pathol. 10, 1094-1099.
ORAL MICROBIOLOGY
245
Bulloch, W. (1938). “The History of Bacteriology.” Oxford Univ. Press, London and New York. Calonius, P. E. B. (1958). Oral Surg., Oral Med., Oral Pathol. 11, 43-46. Campo, G. (1899). Pediatria (Naples) 7, 229 [abstr. in Brit. Med. J. 11, 68 (1899)l. Chirnside, I. M. (1957). New Zealand Dental J. 53, 176-191. Chirnside, I. M. (1961). J. Dental Res. 40, 134-140. Clark, F. Y. (1882). Trans. N . Y. Odontol. Soc. pp. 41-46 and discussion, pp. 46-52. Clauss, E. (1922). 2. Geburtshilfe Gynaekol. 84, 385-403. Clough, 0. W. (1933). J. Dental Res. 13, 183-184. Clough, 0. W., Bibby, B. G., and Berry, G. P. (1938). 1. Dental Res. 17, 493-498. Courant, P. R., Paunio, I., and Gibbons, R. J. (1965). Arch. Oral Biol. 10, 119-125. Cox, G. J. (1952). Natl. Acad. Sci.-Natl. Res. Council, Publ. 225, 243-324. Crawford, J. J., and Shankle, R. J. ( 1961). Oral Surg., Oral Med., Oral Pathol. 14, 1109-1123. Criep, L. H. (1948). 3. Am. Med. Assoc. 136, 601-604. Custer, R. P. (1931). Am. J. Med. Sci. 182, 649-661. Dahiya, R. S., and Speck, M. L. (1963). J. Bacteriol. 85, 585-589. Dancis, J., Grobow, E., and Boyer, A. (1957). J. Pediat. 50, 459-462. David, T. (1890). “Les microbes de la bouche.” Felix Alcan, Paris. Dawes, C. ( 1964). Arch. Oral Biol. 9, 375-376. Deakins, M., Cheyne, V. D., Bihhy, B. G., and Van Kesteren, M. (1941). J. Dental Res. 20, 161-170. de Araujo, W. C., and Macdonald, J. B. (1964). J. Periodontol. 35, 285-289. de Castro, C., and Going, D. H. (1964). J. Periodontol. 35, 216-221. de Stoppelaar, J. D., and Gibbons, R. J. (1965a). Intern. Assoc. Dental Res., Program Abstr., 43rd Gen. Meeting, Toronto, p. 64. de Stoppelaar, J. D., and Gibbons, R. J. (196513). Intern. Assoc. Dental Res., Program Abstr., 43rd Gen. Meeting, Toronto, p . 65. Dianiant, H. (1959). Actu Oto-Laryngol. 49, 375-380. Disraely, M. N., Shiota, T., and Caplow, M. (1959). Arch. Oral Biol. 1, 233-240. Dobell, C. ( 1932). “Anthony van Leeuwenhoek and His ‘Little Animals.’ ” Harcourt, Brace, New York. Dogon, I. L., Kerr, A. C., and Amdur, B. H. (1962). Arch. Oral Biol. 7, 81-90. Dold, H., Reimold, G., and Damniinger, R. (1958). Zentr. Bakteriol., Parmitenk., Abt. I . Orig. 173, 69-76. Dongherty, E. C. (1953). Parmitology 42, 259-261. Dreizen, S., Reed, A. I., and Spies, T. D. (1950). J. Dental Res. 29. 774-778. Dreizen, S., Gilley, E. J., and Spies, T. D. (1956). 0raZ Surg., Oral Med., Oral Pathol. 9, 278-283. Driak, F. (1956). Intern. Dental J. 6, 537-555. Dubos, R., Schaedler, R. W., and Costello, R. (1963). Federation Proc. 22, 13221329. Dujardin-Beaumetz, E. (1932). Compt. Rend. Soc. Biol. 110, 1210-1213. Eichel, B., and Lisanti, V. F. (1964). Arch. Oral Biol. 9, 299-314. Engstroni, B. (1964). Odont. Tidskr. 72, 249-301. Engstrom, B., Segerstad, L. H. A., Ramstrom, G., and FrosteU, G . (1964). Odont. Reuy 15, 257-270. Ennever, J. (1963). Ann. N. Y. Acad. Sci. 109, 4-13. Erdl (1843). Allgem. Z. Chir. uon Rohatzsch. 3, No. 19, 159. Feingold, M., and Gellis, S. S. (1965). New Engl. J. Med. 272, 788-789.
246
HEINER HOFFMAN
Ficinus, R. (1847). J. Chir. Augenheilk. [N. S.] 36, 1-43. Fish, E. W. (1939). 3. Am. Dental Assoc. 26, 691-712. Fitzgerald, R. J. (1963). J. Dental Res. 42, 549-552. Fleming, A. (1922). PTOC. Roy. Soc. B93, 306-317. Flewett, T. H., Warin, R. P., and Clarke, S. K. R. (1963). J. Clin. Pathol. 16, 53-55. Florman, A. L., and Karelitz, S. (1953). J. Immunol. 71, 55-57. Florman, A. L., Schick, B., and Scalettar, E. (1951). Proc. Soc Exptl. B i d . Med. 78, 126. Forsberg, A., Lagergren, C., and Lonnerblad, T. (1960). Oral Surg., Oral A4ed., Oral Pathol. 13, 1051-1060. Fothergill, L. D., and Wright, J. (1933). J. Immunol. 24, 273-284. Fraenkel, A. (1886). 2. Klin. Med. 11, 437-458. Francis, T., Jr., and Minuse, E. (1948). Proc. Soc. Exptl. Biol. Med. 69, 291-294. Frank, I., Blahd, M., and Howell, K. M. (1942). Arch. Otolaryngol. 35, 918-921. Frank, R. (1957). Intern. Dental J . 7, 1-25. Frisbie, H. E., Nuckolls, J., and Saunders, J. B. de C. M. (1944). J. Am. Coll. Dentists 11, 243-279. Frostell, G. (1960a). Acta Odontol. Scand. 18, 29-65. Frostell, G. (1960b). Actu Odontol. Scand. 18, Suppl. 29, 19. Gardner, A. F., Darke, B. H., and Keary, G. T. (1962). J. Am. Vet. Med. Assoc. 140, 433-436. Gavin, J. B., and Collins, A. A. (1961). 3. Periodontol. 32, 198-202. Gibbons, R. J., and Socransky, S . S. (1962). Arch. Oral Biol. 7, 73-80. Gibbons, R. J., Socransky, S . S., Sawyer, S . , Kapsimalis, B., and Macdonald, J. B. (1963). Arch. Oral Biol. 8, 281-289. Gibbons, R. J., Kapsimalis, B., and Socransky, S. S. (1964a). Arch. Oral Biol. 9, 101-103. Gibbons, R. J., Socransky, S. S., de Araujo, W. C., and van Houte, J. (1964b). Arch. Oral Biol. 9, 365-370. Gibbons, R. J., Socransky, S . S., and Kapsimalis, B. ( 1 9 6 4 ~ )J. . Bacteriol. 88, 13161323. Gibson, W. A., and Shannon, I. L. (1963). U. S. Air Force, School of Aerospace Med., Tech. Doc. Rept. No. SAM-TDR-63-88. Gibson, W. A., and Shannon, I. L. (1964). U.S. Air Force, School of Aerospace Med., Tech. Doc. R e p . No. SAM-TDR-64-4. Gilchrist, R. K., and McAndrew, J. R. (1958). A. M . A . Arch. Surg. 76, 863-867. Glock, G . E., and Murray, M. M. (1938). 3. Dental Res. 17, 257-264. Gonzales, F., and Sognnaes, R. F. (1960). Science 131, 156-158. Gordon, D. F., Jr., Socransky, S. S . , and Gibbons, R. J. (1965). Intern. Assoc. Dental Res., Program Abstr., 43rd Gen. Meeting, Toronto, p. 103. Gordon, M. H. (1916). Brit. Med. J. I, 849-851. Green, G. E., and Weinstein, P. R. (1959). J. Dental Res. 38, 951-960. Green, M., and Fousek, M. D. (1957). Pediatrics 19, 80-83. Green, R. H., and Woolley, D. W. (1947). 3. Exptl. Med. 86, 55-64. Crossman, L. I., and Stewart, G. G . (1949). Oral Surg., Oral Med., Oral Pathol. 2, 374-378. Gruby, D. (1842). Compt. Rend. 14, 634-636. Guillot, N. (1958). Ann. Inst. Pusteur 95, 194-207. Gurley, W. B. (1939). 1. Am. Dental Assoc. 26, 163-165. Gutverg, M., and Haberman, S . (1962). J. Periodontol. 33, 105-115.
ORAL MICROBIOLOGY
247
Hammond, B. F. (1959). J. Dental Res. 38, 701-702. Hegemann, F. (1950). 2. Hyg. Infektionskrankh. 131, 355-363. Hemmens, E. S., Blayney, J. -R., and Harrison, R. W. (1941). J. Dental Res. 20, 29-38. Hemmens, E. S., Blayney, J. R., Bradel, S. F., and Harrison, R. W. (1946). J. Dental Res. 25, 195-205. Hill, T. J. (1939). J. Dental Res. 18, 214-224. Hine, M. K. (1936). J. Dental Res. 15, 305-306. Hine, M. K. (1956). Oral Surg., Oral Med., Oral Pathol. 9, 316-327. Hoerman, K. C. (1956). PTOC.Soc. Exptl. Biol. Med. 92, 875-878. Hoffman, H. (1964). Ann. Rev. Microbial. 18, 111-130. Holman, W. L. (1928). In “The Newer Knowledge of Bacteriology and Immunology” (E. 0. Jordan and I. S. Falk, eds.), pp. 102-119. Univ. of Chicago Press, Chicago, Illinois. Howell, A,, Jr., Rizzo, A., and Paul, F. (1965). Arch. Oral Biol. 10, 307-313. Howitt, B. F., and Fleming, W. C. (1930). J. Dental Res. 10, 33-95. Hugenschmidt, ( 1896). Ann. Inst. Pasteur 10, 545-566. Hummeler, K., Gyorgy, P., Hoover, J. R. E., and Kunn, R. (1953). Science 118, 781782, Hurst, V. (1957a). J. Dental Res. 36, 513-515. Hurst, V. (195713). J. Hyg. 55, 299-312. Isaacs, R., and Danielian, A. (1927). Am. J. Med. Sci. 174, 70-87. Ishizaka, K., Dennis, E. G., and Hornbrook, M. (1964). J. Allergy 35, 143-148. Jay, P., Hadley, F. P., and Bunting, R. W. (1936). J. Am. Dental Assoc. 23, 846-851. Johnson, H. N. (1948). In “Viral and Rickettsia1 Infections of Man” (T. M. Rivers, ed. ), p. 213. Lippincott, Philadelphia, Pennsylvania. Jungeblut, C. W., and Knox, A. W. (1954). J . Immunol. 73, 264-272. Jungeblut, C. W., Horvath, B., and Knox, A. W. (1952). Arch. Pediat. 69, 321-324. Kauffman, S. L., Kasai, G. J., and Koser, S. A. (1953). J.Dental Res. 32, 840-849. Kerr, A. C., and Wedderburn, D. L. (1958). Brit. Dental J. 105, 321-326. King, R. M. (1951). J. Dental Res. 30, 399-402. Kirk, E. C. (1910). Dental Cosmos 52, 729-737. Kirsner, J. B., and Goldgraber, M. B. (1960). Gastroenterology 38, 536-562. Klebanoff, S. J,, and Luebke, R. G. (1965). Proc. Sac. Erptl. Biol. Med. 118, 483-486. Klein, H. (1962). J. Dental Res. 41, 1017-1020. Kleinberg, I., and Jenkins, G. N. (1964). Arch. Oral Biol. 9, 493-516. Klencke, H. ( 1850). “Die Verderbniss der Zahne.” Weber, Leipzig. Kletzinsky, (1852). Arch. Physiol. Pathol. Chem. 5, 172. Klinkhamer, J. M. (1963). Periodontics 1, 109-117. Kneeland, Y., Jr. (1930). J. Exptl. Med. 51, 617-624. Knighton, H. T. ( 1953). J. Dental Res. 32, 660. Knighton, H. T. ( 1965). J. Dental Res. 44, 467-470. Kofoid, C. A. (1929). J. Parasitol. 15, 151-174. Kolesuikov, G. P., and Kallings, L. 0, (1962). Actu Pathol. Microbiol. Scand. Suppl. 154, 340. Koser, S. A., and Fisher, B. J. (1950). J. Dental Res. 29, 760-773. Koser, S. A., Fisher, B. J., and Kauffman, S. L. (1951). J . Dental Res. 30, 532-541. Koser, S. A., Hodges, E., Tribby, I., and Stuedell, J. T. (1960). J. Infect. Diseases 106, 60-68. KosteEka, F. ( 1924). Dental Cosmos 66, 927-935.
248
HEINER HOFFMAN
Krasse, B. (1954a). Acta Odontol. Scand. 12, Suppl. 14, 1-32. Krasse, B. (1954b). Odontol. Revy 5, 241-261. Krasse, B. (1963). J. Dental Res. 42, 521-528. Kraus, F.W., and Gaston, C. (1956). J. Bacteriol. 71, 703-707. Kraus, F. W., and Konno, J. (1963). Ann. N . Y. Acad. Sci. 106, 311-329. Labrec, E. H., Schneider, H., Magnani, T. J., and Formal, S. B. (1964). J. Bacteriol. 88, 1503-1518. Lammers, T. ( 1952). Z . Hyg. Infektionskrankh. 135, 365-387. Lammers, T., and Hafer, H. (1956). “Biologie der Zahnkaries.” Dr. Alfred Huthig Verlag, Heidelberg. Lathrop, F. D. (1956). ~QryngOSCope66, 251-268. Lear, C. S. C., Flanagan, J. B., Jr., and Moorrees, C. F. A. (1965). Arch. Oral Biol. 10,83-99. Leber, T., and Rottenstein, J. B. (1867). “Untersuchungen iiber die Caries der Ziihne.” Hirschwald, Berlin. Lehner, T. (1964). Oral Surg., Oral Med., Oral Pathol. 18, 27-37. Leung, S. W. (1962). J. Dental Res. 41, 306-311. Lightfoot, L. H., and Coolidge, T. B. (1959). J. Dental Res. 38, 96-100. Lilienthal, B. (1950). Australian J. Exptl. Biol. Med. Sci. 28, 279-286. Liu, S. F., and Page, N. A. (1960). Geriatrics 15, 115-123. Lu, K. H. (1965). Intern. Assoc. Dental Res., Program Abstr., 43rd Gen. Meeting, Toronto, p. 48. McCarthy, C., Snyder, M. L., and Parker, R. B. (1965). Arch. Oral Biol. 10, 61-70. Macdonald, J. B. (1962). In “Chemistry and Prevention of Dental Caries” ( R . F. Sognnaes, ed. ), pp. 89-125. Thomas, Springfield, Illinois. Macdonald, J. B., Hare, G. C., and Wood, A. W. S. (1957). Oral Surg., Oral Med., Oral Pathol. 10, 318-322. McDougall, W. A. (1963a). Australian Dental J. 8, 261-273. McDougall, W. A. (1963b). Australian Dental J. 8, 398-407. McEachen, D. G., Moore, D. F., Manson, E. M., and Watson, T. A. (1958). Surg., Gynecol. Obstet. 106, 655-666. MacGregor, A., Marsland, E. A., and Batty, I. (1956). Brit. Dental J. 101, 230-235. Macphee, G. G. (1935). “Studies in the Aetiology of Caries,” p. 93. Bale & Danielsson, London. Mandel, I. D., and Levy, B. M. (1957). Oral Surg., Oral Med., Oral Pathol. 10, 874884. Mandel, I. D., Levy, B. M., and Wasserman, B. H. (1957). J. Periodontol. 28, 132137. Massler, M. (1949). J. Dental Res. 28, 674. Massler, M., and Blum, H. L. (1949). J. Dental Res. 28, 674. Matt, M. M. (1954). J. Dental Res. 33,673-674. Menkin, V. (1955). Ann. N . Y. Acad. Sci. 59, 956-985. Middleton, J. D. ( 1964). Nature 202,392-393. Middleton, J. D. (1965). Arch. Oral B i d . 10,227-235. Miller, C. P. (1959). Unio. Michigan Med. Bull. 25, 271-279. Miller, C. P., and Bohnhoff, M. (1963). J. Infect. Diseases 113, 59-66. Miller, H. I. (1949). Am. J. Orthodont. 35, 780-789. Miller, W. D. ( 1889). “Die Mikroorganismen der Mundhohle.” Thieme, Leipzig. Montgomery, P. W. ( 1951 ). J. Dental Res. 30, 12-18. Moore, D. H., du Pan, R. M., and Buxton, C. L. (1949). Am. J. Obstet. Gynecol. 57, 312-322.
ORAL MICROBIOLOGY
249
Morris, R. G. (1949). Australian 1. Dentistry 53, 79-92. Muhlemann, H. R., and Schneider, U. K. (1959). Helv. Odontol. Acta 3, 22-26. Muhler, J. C., Hine, M. K., and Day, H. G. (1954). “Preventive Dentistry,” p. 30. Mosby, St. Louis, Missouri. Murphy, J. M., and Salviolo, J. A. (1949). N. Y. State Dental J . 15, 83-89. Naeslund, C. ( 1928). Acta Pathol. Microbiol. Scand. 3, 637-677. Nash, L., and Morrison, L. F. (1949). Ann. Otol., Rhinol., Laryngol. 58, 976-987. Nevin, T. A., Appleman, M. D., and Kurtz, H. M. (1958). 1. Dental Res. 37, 427-431. Nickerson, J. F., Kraus, F. W., and Perry, W. I. (1957). Proc. SOC. Exptl. Biol. Med. 95, 405-408. Nissle, A. (1916). Deut. Med. Wochschr. 42, 1181-1184. Nordbring, P. W. (1957). Acta Paediat. 46, 481-496. O’Neil, R. (1963). Brit. Dental J. 115, 330-332. Onisi, M., Tachibana, Y., Nakamura, T., Takakura, S., and Ishioka, K. (1957). Bull. Tokyo Med. Dental Vniv. 4, 253-257. Orban, B. (1944). J. Dental Res. 23, 193. Orban, B., and Weinmann, J. P. (1939). J. Am. Dental Assoc. 26, 2008-2017. Parikh, S. R., Massler, M., and Bahn, A. ( 1963). N. Y. State Dental J. 29, 347-355. Pasteur, L., Chamberland, and Roux (1881). Compt. Rend. 92, 159-165. Pfaundler, M., and Schlossmann, A. (1912). “The Diseases of Children,” 2nd ed., Vol. 11, pp. 57-58. Lippincott, Philadelphia, Pennsylvania. Pratt, J. S. (1927). J. Infect. Diseases 41, 461-466. Prinz, H. (1919).“Dental Materia Medica and Therapeutics,” 5th ed., p. 282. Mosby, St. Louis, Missouri. Prophet, A. S. (1955). Brit. Dental J. 99, 225-228. Rasmussen, A. F. (1883). “Om Dyrkning af Mikroorganismer fra spyt af sunde Mennsker.” Lund, Copenhagen. Richardson, R. L. (1965). Arch. Oral B i d . 10, 245-253. Richardson, R. L., and Jones, M. (1958). 1. Dental Res. 37, 697-709. Ritz, H. L. (1963). Proc. SOC.Exptl. Biol. Med. 113, 925-929. Rizzo, A. A., and Mergenhagen, S. E. (1960). Proc. SOC. Exptl. Biol. Med. 104, 579582. Rizzo, A. A., Martin, G. R., Scott, D. B., and Mergenhagen, S. E. (1962). Science 135, 439-441. Robin, C. (1853). “Histoire naturelle des vbgbtaux parasites qui croissent sur l’homme et sur les animaux vivants,” p. 493. BailliAre et Fils, Paris. Robinson, J. R. ( 1955). Surgery 38, 703-707. Robinson, H. B. G., and Boling, L. R. ( 1941). J. Am. Dental Assoc. 28, 268-282. Rolla, G. ( 1965). Acta Pathol. Microbiol. Scand. 63, 291-298. Rogers, H. J. (1948). Nuture 161, 815-816. Rose, S. S. (1954). Ann. Roy. C o k Surg. Engl. 15, 374-401. Rosebury, T. ( 1962). “Microorganisms Indigenous to Man.” McCraw-Hill, New York. Rosebury, T., Gale, D., and Taylor, D. F. (1954). J. Bacteriol. 67, 135-152. Rosenthal, L. (1943). 1. Bacteriol. 45, 545-550. Rovelstad, G. H. ( 1964). J. Am. Dental Assoc. 68, 364-373. Royal College of Surgeons of England. (1904). Dental Record 24, 427-430. Rushton, M. A. (1948). Brit. Dental J. 84, 91-96. Rutter, R. R., Ruefenacht, W. G., Chamberlain, C. R., Thomasseu, P. R., Rose, M., and Scrivener, C. A. (1961). J. Dental Res. 40, 1112-115. Sabin, A. B., and Ruchman, I. (1940). PTOC.SOC.Exptl. Biol. Med. 44, 572-577. Salomon, R. ( 1923). Z. Geburtshilfe Gynaekol. 85, 306-323.
250
HEINER HOFFMAN
Schaffer, E. M. (1953). J. Periodontol. 24, 22-25. Scherp, H. W. (1956). Norske Tandlaegeforen. Tid. 66, 193-202. Schneyer, L. H. (1956). J. Appl. Physiol. 9, 79-81. Schroeder, A. (1962). Intern. Dental J. 12, 356-373. Schroeder, H. E., Lenz, H., and Miihlemann, H. R. (1964). Odontol. Acta 8, 1-16. Schroff, J. (1939). 1. Am. Dental Assoc. 26, 861-870. Schweitzer, B. (1919). Z. Gynaekol. 43, 641-653. Scrivener, C. A. (1955). J. Dental Res. 34, 726-727. Scrivener, C. A., Myers, H. I., Moore, N. A., and Warner, B. W. (1950). J. Dental Res. 29, 784-790. Seifert, E. (1930). Deut. Z. Chir. 222. 345-356. Seltsam, J. H., Lanni, F., and Beard, J. W. (1949). J. Irnnmnol. 63, 261-271. Seltzer, S. (1949). J. Am. Dental Assoc. 39, 148-157. Seltzer, S., Bender, I. B., and Kaufman, I. J. (1961). Orul Surg., Oral Med., Oral Pathol. 14, 856-867. Semans, H. M. (1916). PTOC.23rd Ann. Meeting Am. Inst. Dental Teachers, Minneapolis, pp. 12-20. Sharry, J. J., and Krasse, B. (1960). Acta Odontol. Scand. 18, 347-358. Slack, G. L., Millward, E., and Martin, W. J. (1964). Brit. Dental J. 116, 105-108. Smith, G. H. (1930). Australian J. Exptl. Biol. Med. Sci. 7, 45-77. Smith, J. W., and Bloomfield, A. L. (1950). J. Pediat. 36, 51-60. Smith, M. H. (1964). I . Dental Res. 43, 302. Socransky, S. S., Gibbons, R. J., Dale, A. C., Bortnick, L., Rosenthal, E., and Macdonald, J. B. (1963). Arch. Oral Biol. 8, 275-280. Sognnaes, R. F., ed. ( 1962). “Chemistry and Prevention of Dental Caries.” Thomas, Springfield, Illinois. Sohier, R., and Jaulmes, C. (1939). Compt. Rend. SOC. Biol. 131, 1000-1005. Spratt, J. S., Jr. (1961). Surg., Gynecol. Obstet. 112, 391-405. Springer, G . F. (1958). Acta Haematol. 20, 147-155. Steed, P. D. M. (1963). J. Gen. Microbiol. 29, 615-624. Stephan, R. M. (1940). J. Am. Dental Assoc. 27, 718-723. Stephan, R. M. (1944). J. Dental Res. 23, 257-266. Stephan, R. M. (1953). Intern. Dental J. 4, 180-195. Stephan, R. M., and Hemmens, E. S. (1947). J. Dental Res. 26, 35-41. Stemberg, G. M. (1881). Natl. Board Health Bull. 2, 781-783. Stevenson, L. G. (1955). Bull. Hist. Med. 29, 1-26. Sulkin, S. E., and Harford, C. G. (1943). Ann. Internal Med. [N.S.] 19, 256-262. Swartz, M. L., and Phillips, R. W. (1957). J. Periodontol. 28, 304-307. Swinburne, L. M., Frank, B. B., and Coombs, R. R. A. (1961). Vox Sangitinis [N.S] 6, 274-286. Sylvester, C. J., Rosen, S., Hunt, H. R., and Hoppert, C. A. (1963). J. Dental Res. 42, 803-810. Talbot, H. S. (1934). Am. J. Surg. 25, 267-276. Taschdjian, C. L., and Kozinn, P. J. (1957). J. Pediat. 50, 426-433. Taschdjian, C. L., Reiss, F., and Kozinn, P. J. (1960). J. Inaest. Dermatol. 34, 89-94. Tasman, A,, and Smith, L. (1953). Antonie oan Leeuwenhoek, 1. Microbiol. Scrol. 19, 135-148. Terner, C. (1965). Periodontics 3 , 18-22. Thompson, R., and Johnson, A. (1951). J. Infect. Diseases 88, 81-85. Thompson, R., and Shibuya, M. (1946). J. Bacterial. 51, 671-684.
ORAL MICROBIOLOGY
251
Tomaszewski, W. (1951). J. Clin. Pathol. 4, 393-401. Torrey, J. C., and Reese, M. K. (1944). Am. 1. Diseases Children 67, 89-99. Torrey, J. C., and Reese, M. K. (1945). Am. J. Diseases Children 69, 208-214. Triolo, G. (1897). U@c. Sun. (Napoli) 10, 529 [ahstr. in Centr. Bakteriol. 24, 596 (1898)l. Trott, J. R. (1957). Brit. Dental J. 103, 421-427. Umemoto, Y., Mori, M., Kakudo, Y., Kuwagata, M., and Hidaka, Y. (1950a). Shika Igaku 14, 14-21. Umemoto, Y., Mori, M., Sugimoto, S., Itami, T., Oe, M., Mashimo, A., and Henomatsu, K. (1950b). Shika Igaku 14, 22-31. Underwood, A. S., and Milles, W. T. (1881). Trans. 7th Intern. Med. Congr., London, Vol. 3, pp. 523-529. Valtonen, V., Calonius, P. E. B., and Renkonen, K. 0. (1960). Ann. Med. Exptl. Fenniae 38, 59-64. Valude, (1888). Compt. Rend. Congr. Tuberc. (Paris) p. 258. Vignal, G. (1886). Arch. Physiol. Norm. Pathol. 8, 325-391. Voreadis, E. G., and Zander, H. A. (1958). Oral Surg., Oral Med., Oral Pathol. 11, 1120-1125. Waerhaug, J., and Steen, E. (1952). Odont. Tidskr. 60, 1-24. Wagg, B. J. (1965). J. Dental Res. 44, 526-532. Webb, S. J., and Fedoroff, S. (1963). Can. J. Microbiol. 9, 155-162. Weinstein, E., Pappas, G. D., Mandel, I. D., Salkind, A., and Oshrain, H. (1965). Intern. Assoc. Dental Res., Program Abstr., 43rd Gen. Meeting, Toronto, p. 72. Weisberger, D. (1946). J . Dental Res. 25, 83-87. Welch, W. H. (1892). Bull. Johns Hopkins Hosp. 3, 125-139. Wertheimer, F. W. (1964). J. Periodontol. 35, 406-409. Wheater, D. M., Hirsch, A,, and Mattick, A. T. R. (1952). Nature 168, 659. Wilkinson, E. G. (1962). J. Periodontol. 33, 115-119. Williams, N. B. (1963). J. Dental Res. 42, 509-520. Williams, N. B., and Powlen, D. 0. (1959). Arch. Oral Biol. 1, 48-61. Williams, N. B., Eickenberg, C. F., and Florey, B. M. (1953). J. Dental Res. 32, 691-692. Winkler, K. C., and Backer Dirks, 0. (1958). Intern. Dental J. 8, 561-585. Wright, D. E. (1964). Arch. Oral Biol. 9, 321-329. Wright, D. E., and Jenkins, G. N. (1953). I. Dental Res. 32, 511-523. Wyman, M. (1872). “Autumnal Catarrh (Hay Fever),” p. 17. Hurd & Houghton, New York. Young, F. G. (1871a). Brit. I. Dental Sci. 14, 354-355. Young, F. G. (1871b). Brit. J. Dental Sci. 14, 427-428. Young, G., Krasner, R. I., and Yudkofsky, P. L. (1956). J. Bacteriol. 72, 525-529. Zander, H. (1953). J. Periodontol. 24, 16-19. Zeldow, B. J. (1955). J. Dental Res. 34, 737. Zeldow, B. J. (1959). J. Dental Res. 38, 798-804. Zinke, G. (1804). “Neue Ansichten der Hundswuth.” Gabler, Jena. [Cited by Hogyes, A. ( 1897). In “Specielle Pathologie und Therapie” (H. Nothnagel, ed. 1, Vol. V, Part V, Sect. 11, p. 32. Holder, Vienna]. ZoBell, C. E. (1943). J. Bacteriol. 46, 39-56.
This Page Intentionally Left Blank
Media and Methods for Isolation and Enumeration of the Enterococci’ PAULA . HARTMAN. GEORGE W. REINBOLD.AND DEVIS. SARA SWAT^ Departments of Bacteriology and Dairy and Food Industry. Iowa State Uniuersity. Ames. Iowa
I . Introduction ..................................... I1. Media Available ................................. A . General Information .......................... B. Crystal Violet (Gentian Violet) . . . . . . . . . . . . C . Sodium Azide ................................ D . Azide Plus Dyes .................... E . Azide Plus Elevated Incubation Tempera F. Azide Plus Esculin . . . . . . . . . . G . Precautions when Using AzideH . Thallium Salts .............................. I . Tetrazolium Salts and Thallous Acetate . . . . . . . . . . J . Thallium Salts Plus Other Ingredients . . . . . . . . . . . K . Citrate ............................. .... L . Sodium Chloride ..................... .... M . Tellurite .................................... N . Penicillin and Other Antibiotics . . . . . . . . . ... 0. Sodium Taurocholate and Bile Salts . . . . . . . . . . . . . P . Phenethyl Alcohol ............................ Q. pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R . Selenite ..................................... S . Tetrathionate ................................ I11. Comparative Studies on Media and Methods . . . . . . . . . . A. General Discussion ........................... B. Enterococci in Water ......................... C . Enterococci in Nondairy Foods . . . . . . . . . . . . . . . . D . Enterococci in Dairy Products . . . . . . . . . . . . . . . . . E . Enterococci in Intestinal Contents . . . . . . . . . . . F . Future Needs . . . . . . . . . . . . . . ............. References ..................... .............
253 254 254 260 261 264 265 266 266 267 269 271 272 273 274 276 276 277 277 278 278 279 279 280 281 282 282 282 283
I. Introduction Thiercelin (1899) first used the term “enterococcus” to describe a gram-positive diplococcus of intestinal origin. Thiercelin’s enterococcus 1 Journal Paper No . J-5257 of the Iowa Agricultural and Home Economics Experiment Station. Ames. Iowa . Projects No . 1050 and 1379. This work was supported in part by U . S. Public Health Service grant EF-112 from the Division of Environmental Engineering and Food Protection . 2 Rockefeller Foundation scholar . Present address: Rajasthan College of Agriculture. Udaipur. India .
253
254
HARTMAN, REINBOLD,
AND SARASWAT
has since gained world-wide reknown because of its ubiquity and versatility. Enterococci have assumed a prominent role in studies on the following subjects: infectious diseases of man and other animals ( McCarty, 1958; Krantz and Dunne, 1965); dental caries and oral microbiology (Keyes, 1962; Burnett and Scherp, 1962); food poisoning (Hartman et d., 1965); food fermentations (Niven, 1963; Hartman et al., 1965); silage fermentations (Langston and Bouma, 1960); plant epiphytes (Mundt, 1964); intestinal flora (Rosebury, 1965; Schaedler et d., 1965); pollution of food and water (Niven, 1963; Deibel, 1964); spoilage of foods (Frazier, 1958; Niven, 1963); air and environmental microbiology (R. E. 0. Williams and Hirch, 1950; Rosebury, 1965); enzyme production (Rainbow and Rose, 1963); microbial reduction of vitamin B2 (A. H. Rose, 1961); amino acid and vitamin assay (Gunsalus and Stanier, 1962b; Kavanagh, 1963); nutrition, metabolism, and physiology ( Gunsalus and Stanier, 1961, 1962a; Deibel, 1964); structure and function (Gunsalus and Stanier, 1960; Hartman et al., 1966); serology (Shattock, 1962; Hartman et d., 1966); genetics (Gunsalus and Stanier, 1964; Braun, 1965); taxonomy (Deibel, 1964; Hartman et al., 1966). A large volume of literature has accumulated on media and methods for isolation and enumeration of enterococci. Although the present essay is directed toward determination of enterococci in foods and allied products, the principles discussed are almost equally applicable to other areas of microbial ecology as well as the more distantly related fields of study.
II. Media Available A. GENERALINFORMATION All the agar and broth media known to us to have past or potential use for seZective isolation or enumeration of the fecal streptococci are shown in Tables I and 11. Each medium has been assigned a letter ( A = agar, Table I; B = broth, Table 11) and a number to facilitate subsequent discussion. The agent( s ) and conditions of incubation that impart primary selectivity to the media also are listed, as are the concentrations of each agent. Some common names are given, and a reference designates the origin of each medium. The media are arranged more or less into groups according to the selective agents used. This departure from a purely historical presentation will, we hope, enable better discussion of the comparative properties of each type of medium. Background information relative to each group of media has been included in the discussion so that trends in development can be ascertained. Usually the first use of a selective agent is followed by a flurry of activity in which new media and various formula modifications are made.
ISOLATION AND ENUMERATION OF ENTEROCOCCI
255
TABLE I SELECTIVE AGAR MEDIAFOR ISOLATION OF FECAL STREPTOCOCCI Medium A-1 A-2 A-3
A-4 A-5 A-6
A-7 A-8
A-9 A-10 A-11 A-12 A-13 A-14
A-15
A-16
A-17
A-18
Selective and differential agent( s ) , a conditions, comments, and synonyms 0.006% NaN,, 0.0001% crystal violet 0.01% NaN, 0.01% NaN,, 2% sodium citrate, 0.001% tetrazolium blue (Citrate Azide agar) 0.01% NaN,, 0.0002% basic fuchsin, anaerobic 0.02% NaN, ( Azide Blood Agar Base) 0.02% NaN,, 0.0002% crystal violet, 0.1% sodium citrate (Streptocel agar) 0.02% NaN,, 5% sucrose 0.029% NaN,, 0.002% acridine orange, 0.93% sodium glutamate, 0.0015% TTCb 0.03% NaN,, 3.25 IU/ml. penicillin 0.03% NaN,, 6.5 IU/ml. penicillin, 0.001% methylene blue 0.04% NaN, 0.04% NaN,, 0.001% methylene blue (after enrichment in medium B-20) 0.04% NaN,, 0.01% TTC (M-enterococcus agar) 0.04% NaN,, 0.0005% ethyl violet, 0.01% TTC (Ethyl Violet Azide or EVA agar) 0.0470 NaN,, 0.05% Tween 80, 0.2% sodium carbonate, 0.01% TTC (Tween-carbonate agar) 0.04% NaN,, 0.05% Tween 80, 0.2% sodium bicarbonate, 0.01% TTC (TC agar) 0.04% NaN,, 0.0015% bromcresol purple, 1.0% sodium glycerophosphate, 0.0636% sodium cmbonate, 0.01% TTC (KF agar) 0.04% NaN,, 2% sodium citrate, 0.001% tetrazolium blue (Citrate Azide agar)
Reference Chapman (1944) Snyder and Lichstein (1940)
Reinbold et al. (1953) M. Rogosa (Fitzgerald and Keyes, 1960) Anonymous (1953, 1956)
BBL (1963) Sherman et al. (1943)
Raibaud et al. (1961) J. C. White and Sherman (1944) Winter and Sandholzer (1946a) A. P. Harrison and Hansen (1950) Fujiwara et a,?. (1956) Slanetz and Bartley (1957) Mallmann and Kereluk (1957); Kereluk ( 1960)
Burkwall and Hartman (1964)
Lachica and Hartman (1965)
Kenner et al. (1961)
Saraswat et al. (1963)
256
HARTMAN, REINBOLD, AND SARASWAT
TABLE I (Continued) Medium
Selective and differential agent( s ) , a conditions, comments, and synonyms
Reference
~
A-19
A-20
A-21 A-22 A-23
A-24
A-25
A-26 A-27
A-28 A-29 A-30 A-31 A-32 A-33
A-34 A-35
0.04% NaN,, 0.001% methylene blue (Enterococcus Confirmatory slant); 0.04% NaN,, 0.001% methylene blue, 6.5% NaCI, 6.5 IU/ml. penicillin, pH 8.0 (Enterococcus Confirmatory broth) 0.04% NaN,, 4.5% NaCl, 0.003% water-soluble aniline blue, pH 8.3, 45°C. (for confirmation only; see also medium B-13) 0.05% NaN,, 0.0002'37 crystal violet 0.05% NaN,, 0.0002% crystal violet, 39.5"C. 0.05% NaN,, 0.000125% crystal violet, 0.008% brom thymol blue, 0.5% sorbitol 0.225% NaN,, 0.5% glycine, 0.1% esculin, pH 9.0 0.225% NaN,, 0.5% glycine, 0.1% esculin, 1.0% lactose, pH 9.0 (ELA agar 1 Hydrazoic acid vapor 0.0033% thallium sulfate, 0.000133% crystal violet, 0.1% esculin (TKT agar) 0.08% thallium acetate or thallium nitrate 0.1% thallium acetate, 0.01% TTC (TLTG agar) 0.1% thallium acetate, 0.01% TTC, 45°C. 0.1% thallium acetate, 0.01% TTC, 2% sodium citrate 0.1% thallium acetate, 0.00005% crystal violet 0.1% thallium acetate, 0.01% TTC, 0.5% tyrosine, 0.27% sorbitol, 37" and 45°C. 0.024% sodium selenite, 0.071% aniline blue (see A-20) 1%sodium taurocholate, 0.05% aniline blue, 0.1% esculin, pH 8.0
Winter and Sandholzer (1946a,b)
Guthof and Dammann (1958) Packer (1943) hlossel et al. (1957)
Kjellander (1960) Colobert and Morelis ( 1958); Morelis and Colobert (1958)
Horie and Saheki (1960) Gerencser and Weaver (1959)
Mieth (1960) Rantasalo ( 1947) Barnes (1956b,c) Franklin and Sharpe (1963) Lachica and Hartman (1965) McKenzie ( 1941)
Mead (1963) Guthof (1952) Koch ( 1935)
ISOLATION AND ENUMERATION OF ENTEROCOCCI
257
TABLE I (Continued) Medium A-36
A-37 A-38
A-39 A-40
A-41 A42 A-43
Selective and differential agent( s ) , a conditions, comments, and synonyms 1% sodium taurocholate, 0.035% potassium tellurite, 0.1% esculin, 2.5% NaCl, pH 8.0-9.0 0.001% potassium tellurite, 0.00025% crystal violet (S-1 ) 0.002% potassium tellurite, 0.00008% crystal violet, 0.0075% trypan blue Mitis-Salivarius agar ) 0.0067% potassium tellurite 0.00005% neomycin sulfate, 0.0025% phenol red, 1% mannitol (MN Trypticase Soy agar) 0.0014% neomycin sulfate, 0.003% polymyxin B sulfate (MSDH agar) 0.25% phenylethyl alcohol 4% NaCl
Reference
Schafer ( 1953) R. E. 0. Williams and Hirch (1950)
Chapman (1944) C. H. H. Harold (1936), cited in Kjellander ( 1960) Greer and Britt (1959); BBL (1963) Vera (1963) Anonymous ( 1956) Snyder (1940)
Blood (usually 5%) was included in some of these media to serve as an indicator of the type of hemolysis, and, in one medium, to serve as a source of F e + + + . TTC = 2,3,5-triphenyltetrazolium chloride. @
258
HARTMAN, REINBOLD, AND SARASWAT
TABLE I1 SELECTIVE BHOTH MEDIA FOH Medium
-~
...
ISOLATION OF h C A L STHEPTOCOCCI
Selective and differential agents( s ) ,a conditions, comments, and synonyms ~~
~
-
~~
~
0.006% NaN,, 0.0001% crystal violet (confirmation on medium of Bierkowski, 1956) 0.006-0.0077, NaN,, 0.0002% crystal B-2 violet B-3 0.01 NaN,, 0.0001% crystal violet B -4 0.01% NaN,, 0.0025% brom cresol purple B-5 0.02% NaN, B-6 0.02% NaN, (Azide Dextrose or AD broth) B-7 0.02% NaN, (AD broth, 45"C., presumptive test, see also B-33) B-8 0.02% NaN, (presumptive test, see also B-17) B-9 0.02% NaN,, 0.0002% crystal violet, 0.1% sodium citrate (Streptocel broth for enrichment) B-10 0.02% NaN,, 0.0005% crystal violet B-11 0.02% NaN,, 0.003% brom thymol blue B-12 0.02% NaN,, 1% sodium taurocholate, 3% NaCl (N-N broth) B-13 0.025% NaN,, 0.0032% brom cresol purple, 45°C. B-14 0.025% NaN,, 0.0032% brom cresol purple, 37°C. followed by 45°C. B-15 0.025% NaN,, 0.00025% water-soluble aniline blue, 3% NaCI, pH 8.3 (presumptive medium, see medium A-20) B-15a 0.04% NaN,, 0.0032% brom thymol blue, pH 8.0, 45°C. (Enterococcus Presumptive broth; see A-19) B-16 0.04% NaN,, 0.0032% brom thymol blue, 45°C. B-17 0.04% NaN,, 0.0005% ethyl violet (Ethyl Violet Azide or EVA broth) B-18 0.04% NaN,, 1% sucrose, 0.01% TTC,a 0.000012% ethyl violet optional (for membrane filter)
Reference -
B-1
Mastromatteo and Pisu (1959 Pike (1944, 1945a) Edwards (1938) McKenzie ( 1941) Mallmann (1940) Rothe (1948) Splittstoesser et al. (1961) Litsky et al. (1953)
BBL (1963) Ritter et d. (1956) Raj et ul. ( 1961)
Micth (1960) Hannay and Norton (1947) Childs and Allen (1953)
Guthof and Dammann (1958
Winter and Sandholzer (1964b Wang and Dunlop (1951) Litsky et al. (1953)
Slanetz et ul. (1955)
ISOLATION AND ENUMERATION OF ENTEROCOCCI
259
TABLE 11 (Continued) Medium B-19
B-20 B-21
B-22 B-23 B-24 B-25 B-26 B-27 B-28 B-29 B-30 B-31 B-32 B-33 a
Selective and differential agents( s ) , a conditions, comments, and aynonyms 0.04% NaN,, 1% sodium glycerophosphate, 0.0015% brom cresol purple, 0.0636% sodium carbonate, 0.01% TTC (KF broth) 0.05% NaN,, 0.0002% crystal violet 0.05% NaN,, 0.0015% brom cresol purple, 0.5% glycerol (BAGG broth) 0.05% NaN,, 0.0032% brom cresol purple, 45.5"C. ( SF medium) 0.05% NaN,, 6.5% NaCI, 45°C. 0.01% potassium tellurite, 0.00005% crystal violet 0.02% potassium tellurite 0.05% thallium acetate 0.1% thallium acetate 0.1% thallium acetate, 0.00005% crystal violet tetrathionate 10% ox bile 0.5% sodium taurocholate, 1.0% mannitol, 44°C. (confirmatory only) 0.05% Tween 80, 0.53% sodium carbonate, pH 10.0
6.5% NaCI, 45°C. (confirmatory test; see B-7)
TTC = 2,3,5-triphenyltetrazolium chloride.
Reference
Kenner et ol. (1961) Packer ( 1943)
Hajna ( 1951 ) Hajna and Perry (1943) Ostrolenk and Hunter (1946) Pike (194570) Cooper and Ramadan (1955) Cooper and Ramadan ( 1955) Barnes ( 1 9 5 6 ~ ) McKenzie (1941) Cooper and Ramadan (1955); Cooper et al. ( 1942) Weissenbach (1918) Rycroft (1956) Chesbro and Evans (1959) Splittstoesser et al. ( 1961 )
260
HARTMAN, REINBOLD, AND SARASWAT
After this initial burst of activity, there is little further refinement of the media. Applications of media parallel to a great extent the development of taxonomy of the streptococci. Many early media were probably fairly selective for the enterococci, but the nomenclatural status of the group was not defined sufficiently to recognize the importance of the discovery. Most early works were aimed primarily at isolation of streptococci associated with mastitis and human infections. Subsequent refinements in formula were made to adapt these media to studies on entercocci. Several media that have been used to isolate streptococci, including the enterococci, do not appear in the tables. For example, many nonselective media, such as blood agar or Standard Methods agar, have been omitted. Likewise, a few media, such as the modified Dorsett egg medium of Crowe ( 1913), have been omitted because the extensive manipulations needed for their preparation indicate that they would not attain widespread use. If other omissions occur, they are unintentional. Not all the media listed in Tables I and I1 will be discussed; the original publications give further details concerning these media.
B. CRYSTAL VIOLET(GENTIAN VIOLET) Churchman (1912) was the first to study extensively the selective bactericidal action of gentian violet, although earlier observations had been made by Drigalski and Conradi (1902). Churchman’s work was followed closely by the investigations of Krumwiede and Pratt ( 1914), who found that some streptococci were more resistant to gentian violet than were other bacteria. Haxthausen (1927a,b) added 0.001% crystal violet to glucose broth to produce a selective medium. Edwards ( 1931) modified this liquid medium to contain 0.0005% crystal violet, and devised an agar medium containing crystal violet plus 0.1% esculin (Edwards, 1933) for better colony differentiation. This latter medium was the predecessor of other esculin-containing media (A-24, A-25, A-27). Bryan (1932), apparently unaware of the publications of Haxthausen and the earlier work of Edwards, proposed the addition of 0.000677.1, gentian violet to improve the selectivity of blood agar. Brilliant green was equally satisfactory (Bryan, 1932; see also Litsky et al., 1952). Dick and Hucker (1940) utilized 0.00025% crystal violet to impart selectivity for “S. salivarius” in a broth medium used to detect oral contamination of drinking glasses, while Cooper and Linton (1947) utilized a similar medium (0.0033% crystal violet) for the detection of gonococci. These media served as the basis for development of many different media now used for isolation and enumeration of fecal streptococci. Although there is only one primary inhibitory agent ( a dye) in these media, the experiences of several workers utilizing such media with
ISOLATION AND ENUMERATION OF ENTEROCOCCI
261
streptococci will be mentioned to illustrate a point common to all selective agents. The workers cited used crystal violet at concentrations of 0.00025% or greater. McKenzie (1941) observed that crystal violet in a concentration of 0.00017, markedly inhibited growth of some streptococci; a level of 0.00005% was finally chosen for his medium. The selective action of crystal violet on streptococci also was noted by Packer (1943), who reported that a concentration of 0.0005% was inhibitory to many streptococci. A level of 0.0002% allowed the growth of all of the streptococci examined except a few strains of Streptococcus luctis. R. E. 0. Williams and Hirch (1950), using a tellurite-crystal violet medium (A-37), noted that a reduction of either ingredient led to some increase in the streptococcal count; an increase in either inhibitor decreased the count. Decreased levels of either inhibitor, however, resulted in a lowering of the selectivity of the medium. Thus, one might assume that the types of streptococci that would grow on a particular medium would vary somewhat with different concentrations of inhibitor (see also Splittstoesser et al., 1961). In addition, using the same medium, some cells of a specific species of streptococcus might be inhibited while others might grow, or the period of incubation might not be sufficiently long for maximum counts to be obtained. R. E. 0. Williams and Hirch (1950) found that the period of incubation considerably affected the results obtained; some strains of enterococci failed to survive for incubation periods that were sufficiently long to permit colony formation by other strains. Media containing dyes alone are not listed in Tables I and I1 because all these media lack the selectivity demanded by present-day workers. C. SODIUMAZIDE
After the studies of Loew (1891) of the inhibitory properties of azide to a variety of microorganisms, Schattenfroh (1896) examined the effects of various levels (0.05-1.0%) of sodium and ammonium azide on bacteria, yeasts, and molds. He noted that the test organisms differed considerably in their susceptibility to these compounds. Over 40 years elapsed, however, until azide was used to advantage in devising a selective medium for use in studies on fecal streptococci. Impetus was given these studies by Hartmann (1937), who examined the relative inhibition of Escherichia coli and streptococci by a variety of compounds. A large number of chemical agents had previously been studied by Kramer and Koch ( 1931 1, Garrod (1933), and Diernhofer (1936); see these publications for other citations. Most of the compounds tested did not lead to development of a practical medium, but some compounds of potential future use were discovered. Azide was the only compound that inhibited E . coli more than the streptococci at a relatively wide range of concentration (Hart-
262
HARTMAN, REINBOLD, AND SARASWAT
mann, 1937). Concentrations of 0.02-0.04% were recommended. Literature regarding the effect of varying concentrations of sodium azide on microorganisms has recently been summarized by Forget and Fredette ( 1962). Mallmann (1940) modified a medium devised by Darby and Mallmann (1939) by adding 0.02% sodium azide (B-5). He was able to estimate selectively the numbers of streptococci in samples of sewage. This was probably the first instance of selective enumeration of enterococci from a grossly contaminated environment without concomitant excessive inhibition of the enterococci themselves. A widely used modification of Mallmann’s broth medium was made by Rothe (1948). This medium (B-6) contained 0.02% sodium azide; changes were made in two of the other constituents. Mallmann and Seligmann (1950) reported on the use of this medium but, through a typographical error, the quantities of sodium chloride and glucose were in error. This latter formulation was used with success, however, by Morris and Weaver (1954). A similar medium (Ritter and Treece, 1948) also contained 0.027, azide, but less glucose was used. The medium supported luxuriant growth of all of the strains of streptococci and staphylococci tested. Commercial azide blood agar bases (A-5) apparently follow generally the work (A-2) of Snyder and Lichstein (1940; Lichstein and Snyder, 1941), with the azide concentration set at 0.02% according to the studies of the other workers cited previously. McKenzie ( 1941) preferred a thallous acetate-crystal violet medium (A-32) to a 0.01% sodium azide medium for isolation of mastitis streptococci. One might suspect from this work that use of the single selective ingredient, azide, leaves much to be desired as far as selectivity for fecal streptococci is concerned when grossly contaminated samples are to be examined. This has been found to be the case by a number of workers, including Burkwall and Hartman ( 1964), who included Azide Blood agar in their study (see also Richards et al., 1945a,b; Ritter and Treece, 1948; Zaborowski et al., 1958). Thus, the use of azide alone in media for food microbiology is usually limited to preenrichment prior to confirmation in a more selective broth or agar medium. In addition, altered hemolytic patterns may be obtained on media containing azide (Snyder and Lichstein, 1940; Packer, 1943), and the large quantities of glucose used in many of the media discussed in this section would tend to reduce the hemolytic properties of many of the streptococci growing there. One notable exception to the use of azide alone as a selective ingredient is the medium devised by Slanetz and Bartley (1957). This medium (A-13), intended for use with the millipore filter in water bacteriology, originated by substantial alteration of a medium (A-1) of Chapman (1944) by Slanetz et al. (1955). The relatively high level of azide, 0.04%,
ISOLATION AND ENUMERATION OF ENTEROCOCCI
263
might account for the selectivity of the medium, although Saraswat et al. (1963) found that a stock culture each of Streptococcus bovis and Lactobacillus acidophilus grew on this medium. Raibaud et al. (1961) obtained low recoveries and overgrowth by lactobacilli when pig cecal samples were plated in medium A-13. Burkwall and Hartman (1964) also reported that recovery was low when frozen foods were plated on medium A-13; however, greatly increased recovery was obtained when Tween SO ( W . L. Williams et al., 1947) and sodium carbonate (Chesbro and Evans, 1959) were added to medium A-13. Since pH changes were minimal, the increased recovery was not thought to be due to lowered selectivity resulting from higher pH, as has been reported by Edwards (1938) and Packer (1943). Further studies by Lachica and Hartman (1965) showed that medium A-13 yielded highest counts when supplemented with 0.075% Tween 80, 0.2% KH2P04, and 0.2% NaHC03 (medium A-16). If the higher counts are obtained because of increased recovery of fecal streptococci, medium A-16 holds promise of being better for many purposes than any medium tested by Burkwall and Hartman (1964). Whether the use of Tween and carbonate in some manner reverses in part the inhibitory power of sodium azide remains to be determined. Carbonate may even be contributing to the selectivity of the medium, as described in the section that deals with pH. Kenner et aE. (1961) described new solid (A-17) and liquid (B-19) media for enumeration of enterococci. These media contained, among other ingredients, 0.04% sodium azide, 0.0015% bromcresol purple, and 0.01% 2,3,5-triphenyltetrazolium chloride (TTC). The use of TTC in culture media is discussed in a later section. Media A-17 and B-19 seem to permit growth of S. bovis and Streptococcus equinus (Kenner et al., 1961), so they would not be appropriate for use when only enterococci are desired, e.g., when dairy products are to be examined (Saraswat et al., 1963). The broth (B-19) is probably slightly more inhibitory than the agar (A-17) to S. bovis, S. equinus, and Streptococcus mitis (Hall et al., 1963). Hall et al. (1963) and Burkwall and Hartman ( 1964) were favorably impressed with results obtained when frozen foods were plated on medium A-17. The latter workers obtained results using medium A-17 that were equivalent to those obtained using medium A-29. These two media were superior to many other direct-plating media examined for enumeration of fecal streptococci from frozen foods. In a recent study of freeze-dried foods (Saleh et uZ., 1966), higher counts were usually obtained on medium A-15 than on medium A-17; however, medium A-15 was generally the less selective of the two. Mossel et nl. (1957; Mossel, 1964) also were of the opinion that medium A-17 lacked selectivity because almost quantitative recoveries of Staphylococcus aureus were ob-
264
HARTMAN, REINBOLD, AND SARASWAT
tained on the medium. Raibaud et al. (1961) reported that they obtained low recoveries of enterococci and encountered overgrowth by lactobacilli when cecal samples were plated on medium A-17. Regardless of these shortcomings, KF agar (A-17) and broth (B-19) media are among the better yet developed for enterococci (Hall, 1964). Saleh et al. (1966) noticed that colony size on medium A-17 was larger when scrambled eggs were plated than when other freeze-dried foods were examined. This observation might be utilized in future improvement of medium A-17.
D. AZIDE PLUSDYES Edwards (1938) examined the efficacy of 0.0001% crystal violet in combination with 0.01% sodium azide (B-3) for the diagnosis of mastitis. In contrast, Packer (1943) selected higher concentrations of crystal or gentian violet and azide (A-21, B-20). Various concentrations of crystal violet and azide have been used by other investigators (Tables I and 11) in media developed for different purposes. Other dyes have been used in combination with sodium azide (see Schaedler et al., 1965). Some workers have added pH indicators so that acid production can be detected (see B-4, B-11, B-16, B-22; Zaborowski et al., 1958). For some purposes a great deal of selectivity is not necessary. All these azide-dye media, although more selective than media containing azide or dye alone, still require confirmation under most circumstances. Litsky et al. (1953) developed an ethyl violet-azide medium (B-17) ( Litsky et al., 1952) that reportedly possessed increased selectivity, although Ritter et al. (1956) thought that the new medium was equivalent to their own azide-crystal violet medium (B-10). Litsky et al. (1953) recommended use of the broth (B-5) devised by Mallmann ( 1940) for presumptive identification, followed by transfer into medium B-15 for confirmation, This combination of media has been widely used and is apparently very satisfactory for isolation and enumeration of enterococci from water. Only Streptococcus faecalis and Streptococcus faecium and their varieties were reported to survive such an isolation procedure (Litsky et al., 1953), but Kenner et al. (1961; Hall et al., 1963) noted that Lactobacillus plantarum and Pediococcus cerevisiae gave positive results with ethyl violet-azide medium. Materials, containing these and related bacteria might therefore give erroneously high counts (see also Splittstoesser et al., 1961). Medium B-15 would not be satisfactory if counts of S. bouis, S . salivarius, or S. equinus were among those desired. A similar medium containing agar (A-14) was devised for use by the drop-plate method, and Ferraro et al. (1958) used an ethyl violet-azide plating medium for confirmation of enterococci. Ethyl violet-azide media may suffice when used by the drop-plate procedure or for confirmation
ISOLATION AND ENUMERATION OF ENTEROCOCCI
265
of enterococci, but Saraswat et al. (1963) and Burkwall and Hartman (1964) found this medium (A-14) to be low in yield when used for direct plating. Croft ( 1959) obtained unimpressive results in some attempts to use medium A-14 in a millipore filter procedure for water analysis. E. AZIDE PLUSELEVATED INCUBATION TEMPERATURES Use of an incubation temperature of 45°C. in conjunction with azide originated with Hajna and Perry ( 1943), who developed a medium (B22) for presumptive enumeration of streptococci in milk. The elevated incubation temperature, following the work of Sherman (1937b)) was used to limit the type of streptococcus that would grow (see also Allen et al., 1949). Medium B-22, known as SF medium, was modified by Hajna ( 1951) by inclusion of glycerol (medium B-21). The medium also has been modified (B-13, B-14) by reduction of the azide concentration to 0.025%, but Hajna (1951) warned that this reduced the specificity of the medium. As far as is known, incubation temperatures within the ranges discussed would not prevent proliferation of any fecal streptococci, with the possible exception of some strains of S. mitis. Nevertheless, Ferraro and Appleman (1957) demonstrated clearly that erroneously low enterococcus counts were frequently obtained when citrus concentrates were inoculated into medium B-22; preliminary enrichment in phenol red lactose broth resulted in greatly increased counts. Childs and Allen ( 1953) suggested a “subculture” method ( B-14) whereby inoculated tubes are first incubated at 37°C. for 24-48 hours, then positive tubes are subcultured to fresh tubes of the same medium to be incubated at 45°C. There seems to be no reason why the subculture could not be eliminated; the original tubes could be incubated at 37°C. for several hours to permit growth initiation, then removed to a 45°C. incubator for the remainder of the incubation period. This application of 45°C. incubation also has been investigated by Allen et al. ( 1953), Burman ( 1961), and Mead ( 1963, 1964). Interaction between azide concentration and temperature of incubation was mentioned by Splittstoesser et al. ( 1961). An incubation temperature of 45°C. did not reduce counts of enterococci obtained on broth B-6, but did when samples were inoculated into medium B-22. A temperature of 39.5”C., rather than 37”C., was used with Packer’s medium (A-21) by Mossel et al. (1957; medium A-22); increased selectivity, but decreased yields, were obtained. Mossel (1964) seemed to prefer using a 37°C. incubation temperature to obtain greater recovery, combined with confirmation in another medium.
266
HARTMAN, REINBOLD, AND SARASWAT
F. AZIDE PLUSESCULIN The only azide-esculin medium to be proposed (A-24) was described by Morelis and Colobert ( 1958) and was later modified (A-25) by Horie and Saheki (1960). As is discussed in the section on thallium plus other agents, there are certain limitations to using esculin hydrolysis as an indicator of enterococci. In addition, 0.225% sodium azide is a high concentration, which probably accounts for the low yields of enterococci obtained on these two media in comparison with some others (Burkwall and Hartman, 1964). Selectivity of these media, however, is excellent. G. PRECAUTIONS WHEN USINGAZIDE-CONTAINING MEDIA Several precautions are necessary when using azide-containing media. Various investigators have reported that the selectivity may be altered if the azide is sterilized with the basal medium. Mieth (1960), for example, reported that the inhibitory action of a given quantity of azide was diminished when media were autoclaved at 110°C. Guthof and Dammann (1958) also stated that azide was very heat sensitive. Other workers (i.e., Snyder and Lichstein, 1940) did not notice any differences in the selectivity of azide-containing media whether the azide was sterilized separately or with the basal medium. Various investigators have approached this problem in different ways, ranging from a very cautious separate sterilization of the azide by filtration, through separate sterilization by steaming for a short period, to inclusion of azide in the medium before autoclave-sterilization. So many media have been proposed using the latter approach to sterilization that it appears that the method is adequate if the azide concentration is adjusted appropriately to allow for some (as yet unknown) loss. If this is done, the medium should always be sterilized in the same manner. Likewise, prepared media should not be held too long; storage for 1 week at room temperature does not seem to have a deleterious effect (Forget and Fredette, 1962; Pike, 1945a), although Pike (1944) observed a decrease in the inhibitory properties of blood-containing azide media stored for as little as 2 days at room temperature. Therefore, prepared media, especially those containing blood (Pike, 1945a), should be stored in the refrigerator, but not longer than 2 weeks. Care should also be taken in storing dehydrated media (Hall et al., 1963). Loss of azide is accompanied by formation of hydrazoic acid, which is volatile (Gerencser and Weaver, 1959) and probably more toxic (Smuckler and Appleman, 1965) than azide. Transformation of azide to hydrazoic acid is especially rapid in acid media (Gerencser and Weaver, 1959); consequently control of pH of azide-containing media is very
ISOLATION AND ENUMERATION OF ENTEROCOCCI
267
important. Furthermore, in plates containing much agar the release of hydrazoic acid is less (or reabsorption is greater) than in plates containing small volumes of medium; therefore, azide-containing media may become increasingly inhibitive as the volume of agar per plate is increased ( Gyllenberg and Niemela, 1960). Gerencser and Weaver ( 1959) took advantage of the volatile nature of hydrazoic acid (medium A-26) by incubating cultures in a hydrazoic atmosphere. Azide in acid solution is placed in a separate container in a closed jar, together with petri plates of nonselective medium; quantities of hydrazoic acid absorbed by the medium are determined by the amount of azide used, the size of the container, and the type and possibly the surface area of the culture medium. The hydrazoic acid procedure was used by Gyllenberg and Niemela (1960) in conjunction with the millipore-filter medium of Slanetz and Bartley (1957). Differences in the volume of agar per plate did not influence counts of fecal streptococci or colony colorization when the hydrazoic technique was used; these two factors were altered by volume of agar when the conventional Slanetz and Bartley procedure was used. Although Gerencser and Weaver (1959) reported that hydrazoic acid liberated from azide-containing media may contaminate other media stored in close proximity, Forget and Fredette (1962) and Smuckler and Appleman (1965) did not detect toxicity in media stored beside azidecontaining media or an azide stock solution. Azide apparently exerts its primary function by inhibiting metalloporphyrin enzyme systems, such as catalases and cytochrome c oxidases (see references in Gunsalus and Stanier, 1961, and Nicholls, 1964). Electron transport is interrupted. As mentioned previously, azide penetrates some cells only as the undissociated acid, so the pH of the medium can have a great effect on the selective properties of tce medium.
H. THALLIUM SALTS In 1918, Browning and Gulbransen (1918) reported that certain grampositive cocci were more resistant to thallium acetate (TA) than were other bacteria tested. The selectivity of thallium salts was subsequently examined in detail by McKenzie (1941), Cooper and Linton (1947), Richards et al. (1945a,b), Rantasalo ( 1947), and Sharpe ( 1955); these references indicate the spectrum of organisms inhibited by different concentrations of thallium salts. Kinnear (1931) reported that a broth containing 0.06% thallium nitrate ( T N ) and 0.005% crystal violet was a good selective medium for streptococci and staphylococci, but the thallium salt interfered with hemolysis when incorporated into an agar medium, Interference of hemolysis by 0.1% TA also was noted by Cooper and Linton (1947). McKenzie (1941) developed a 0.1% TA-crystal violet broth
268
HARTMAN, REINBOLD,
AND SARASWAT
(B-28) for use in the diagnosis of streptococcal mastitis and noted that TA and TN salts were equally selective. Thallium sulfate (TS) also has been used successfully (Kehler Ellingsen and Hauge, 1953), The selectivity of TA is not affected by minor alterations in pH, although the selectivity of sodium azide is ( McKenzie, 1941). At a level of 0.270 TA, growth of a culture of Streptococcus agalactiae was inhibited, but other streptococci and a culture of S. aureus grew well. Richards et al. (1945a,b) were the first to note that the group D streptococci were not inhibited at all by 0.1% TA, and Rantasalo (1947) recommended the inclusion of 0.087, TA in a medium (A-28) for the isolation of streptococci from the throat. A level of 0.05% TA was used by several investigators (cited by Sharpe, 1955) and by Cooper and Ramadan ( 1955; medium B-26) to isolate streptococci from various sources. The TA medium (B-26) was not as satisfactory for the examination of human feces as was a tellurite medium (B-24), yet Mead ( 1963, 1964) proposed a TA medium (A-33) that was supposedly superior to others for recovery of “human” fecal pollution of water (the level of TA was erroneously listed as 1%in Meads 1963 publication). The efficacy of medium A-33 was questioned by Mundt (1964); it appears to us that positive colonies on medium A-33 are indicative of, but far from specific for, human enterococci. In addition, the medium and culture method is fairly complex, and selectivity is obtained only at sacrifice of yield. Finally, it seems probable that some enterococci from plant sources would produce positive colonies on medium A-33. R. E. 0. Williams and Hirch (1950) obtained growth of greening micrococci and a few other bacteria in addition to the desired streptococci when 0.05% TA was used in a medium to study the distribution of streptococci in the air. A concentration of 0.1% TA was stated to be superior to concentrations of 0.05 or 0.2% (Sharpe, 1955). At a level of 0.1% TA, members of genus Lactobacillus also grew well, as did the two strains of S. aureus examined and an unidentified micrococcus. Two of 23 strains of Lactobacillus and the micrococcus were inhibited by 0.2% TA; however, there was no observable inhibition of the four fecal streptococci examined. Gonococci will grow if the TA concentration is reduced to O.O2C/o (Cooper and Linton, 1947). TA also is a good selective agent for isolation of pleuropneumonia-like organisms ( references are cited in Kleineberger-Nobel, 1962). Thus, different species are inhibited by thallous salts at different levels of thallium-ion concentration. The data of McKenzie ( 1941), Cooper and Linton ( 1947), and Sharpe (1955) indicate that levels of TA somewhat higher than 0.1% might be used successfully if a more selective medium for group D streptococci
ISOLATION AND ENUMERATION OF ENTEROCOCCI
269
was desired, but this problem has not been investigated further. The mechanism of selectivity exerted by thallium salts is unknown (Cooper and Linton, 1947; Sharpe, 1955).
I. TETRAZOLIUM SALTSAND THALLOUS ACETATE Laxminarayana and Iya (1953) observed that two strains of S. faecalis var. liquefaciens and a strain of “typical” S. faecalis reduced 2,3,5-triphenyltetrazolium bromide more rapidly than an ‘‘atypical” strain of S. faecalis (possibly S . faecium?) or 10 other streptococci belonging to five other species. Barnes (1956a) studied the reducing properties of many strains of fecal streptococci and noted that 2,3,5-triphenyltetrazolium chloride (TTC) was reduced rapidly by S . faecalis and its varieties, but not by s. faecium, s. faecium var. durans, or s. bovis. Earlier, H. G. Neumann (in Goetz and Tsuneishi, 1951) observed that TTC could be incorporated at a level of 0.4% in media containing customarily used inhibitory and selective agents, including azide media for isolation of enterococci, Reinbold et al. ( 1953) reported that 0.0025% ditetrazolium chloride was noninhibitory to enterococci. Further comment on the selectivity of tetrazolium compounds will not be made here, except to mention that Weinberg ( 1953) described toxicity of TTC at concentrations above 0.001% to gram-positive bacteria, and Solberg and Proctor (1960) developed a technique whereby the plate was flooded after incubation to circumvent any possible toxic effects. The differential reducing properties of different species of streptococci were used to advantage in devising two thallous salt-containing media (A-29 and B-27) for selective enumeration and differentiation of S . faecalis and S . faecium (Barnes, 1956a,b, 1959). In Barnes’s original procedure, a basal medium is made, sterilized, and partially cooled, and sterile solutions of TA, T’C, and glucose are added. Plates are poured and allowed to solidify, and the sample is then streaked on the surface of the medium, Streptococcus faecium and its variety durans reduce tetrazolium poorly or not at all at pH 6.0 and produce white colonies (Barnes, 1956a). Some strains of S. bovis grow well on the medium and produce white or pale pink colonies, while other strains grow poorly and produce minute red colonies ( Medrek and Barnes, 1958). Streptococcus faecalis and its varieties grow well1 and reduce tetrazolium strongly (Barnes, 1956a). The medium supposedly is highly selective for group D streptococci (Barnes, 1959), although Sharpe ( 1955) used a similar medium for the selective isolation of lactobacilli. In a survey of bacon factories (Barnes, 1956c; Barnes et al., 1956), only group D and a few group N streptococci were isolated. Lactobacilli apparently caused no difficulty
270
HARTMAN, REINBOLD, AND SARASWAT
in this application when an incubation period of 24 hours was used. According to Barnes (1958), lactobacilli will form colonies when incubated for 48 hours, but Fanelli and Ayres (1959), Hartman (1960), Hartman et al. (1962), and Burmeister et al. (1966) utilized an incubation period of 48 hours and did not encounter excessive overgrowth by unwanted organisms (also see next paragraph). On the other hand, Saraswat et al. (1963) observed that medium A-29 was unsatisfactory for dairy bacteriology because a dense background of lactic bacteria often obscured the results. Analogous difficulties were encountered by Mieth (1960) and Raibaud et al. (1961) when fecal samples were streaked on medium A-29, and Burkwall and Hartman (1964) obtained reduced selectivity as the incubation period was increased when frozen foods were plated. Nevertheless, yields on medium A-29 were greater than on most media for isolation and enumeration of enterococci in frozen foods, and selectivity was fairly good (Burkwall and Hartman, 1964). Modifications of medium A-29 and of procedures for its use have been made by several investigators. Extended incubation periods have already been discussed. In addition, Burkwall and Hartman (1964) used the pour-plate, rather than the streak-plate, method of inoculation. The solidified pour-plate was overlayed with sterile agar; thus, all colonies were subsurface. Use of this overlay may have eliminated growth of some of the unwanted organisms because difficulties were rarely experienced in overgrowth by other bacteria (see also Fry, 1932). Under these conditions, however, group D enterococci could not be differentiated on the basis of tetrazolium reduction; almost all samples of frozen foods (Hartman, 1960; Hartman and Huntsburger, 196l), rumen fluid (Hartman et al., 1962), and ensiled corn (Burmeister et al., 1966) yielded only red subsurface colonies. Another modification of the medium was made by Vera (1961); TA was included in the basal medium, so only sterile TTC had to be added to the basal medium immediately prior to use. McKenzie ( 1941), Cooper and Ramadan ( 1955), and Sharpe ( 1955) also had added TA before sterilization of their media. In some preliminary comparisons of the various modifications of the medium, equivalent results were obtained ( Hartman and Lachica, unpublished data, 1965). Two further modifications of medium A-29 have been made to increase the selectivity. Franklin and Sharpe (1963) used an incubation temperature of 45°C. for 2 days (A-30) with apparent success to adapt the medium for use on dairy products. Lachica and Hartman (1965) examined the combination of 2% citrate ( Reinbold et al., 1953) with medium A-29 and discovered that toxicity of the medium to staphylococci but not to streptococci was increased (medium A-31). These two modifications have not been tested extensively in comparison with other media.
ISOLATION AND ENUMERATION OF ENTEROCOCCI
271
J. THALLIUM SALTSPLUSOTHERINGREDIENTS Stableforth et al. (1949) and Wilson and Slavin (1950) incorporated TS or TA at a concentration of 0.033% to improve the selectivity of the medium of Edwards (1933) in the diagnosis of mastitis. No recognition was given by these or subsequent authors to the inhibitory effect of TA on hemolysis that was reported by Kinnear (1931), and one must assume that thallous salts do not produce substantial changes in hemolytic reactions until TA concentrations of about 0.1% are attained (Cooper and Linton, 1947). Hauge and Kghler Ellingsen (1953; Kghler Ellingsen and Hauge, 1953; Sandvik and Hauge, 1954 ) incorporated staphylococcus p-toxin and named the resultant medium TKT agar ( Thalliumsulfat-KristallviolettToxinblutagar). The addition of p-toxin arose as a result of studies by Munch-Petersen and Christie (1947 and earlier) on the use of this substance in a blood-containing medium for the identification of s. agalactiae. This medium also was used by Seelemann and Obiger ( 1956), who were probably the first to note specifically that it supported the growth of enterococci. The medium contains 0.5% NaCl, in spite of the fact that McKenzie (1941) mentioned that TA reacts with NaCl to yield insoluble and nonselective thallium chloride. Levels of NaCl supposedly cannot exceed about 0.1% in thallium-containing media ( McKenzie, 1941); however, Barnes (1956c, p. 193) could not obtain evidence that concentrations of NaCl up to 0.5% reduced the inhibitory preperties of TA. Gyllenberg and Koine (1957) used 2% NaCl in a thallium-containing medium, modified after one developed by Garey et al. (1941). Nevertheless, samples containing large quantities of salt may alter the selectivity of TA-containing media. Mieth (1960) used medium A-27 to isolate enterococci in the examination of the fecal fiora of various animals and noted that the 0-toxin was not necessary when the medium was used for this purpose. Better recovery of S. bovis was observed with medium A-27 than with an enrichment procedure using medium B-12, but S. faecium was better detected using the enrichment. Medium A-27 was preferred to medium A-29, because A-27 had no background colonies to interfere with isolation (Mieth, 1960). Judging from the variety of strains of enterococci described by Mieth (1960, 1961, 1962a,b), all group D streptococci grow well on the medium. Yields on a modification of medium A-27 were only one fifth of those obtained on medium A-15 (Burkwall and Hartman, 1964). Although only one of 700 strains of enterococci examined by Mieth ( 1960, 1961, 1962a) was esculin negative, he selected only esculin-positive colonies for further examination. Rochaix (1924) and Swan (1954) concluded earlier that all enterococci fermented esculin (see also Kohler
272
HARTMAN, REINBOLD, AND SARASWAT
Ellingsen and Hauge, 1953, and Seelemann and Obiger, 1956). Esculin had been used for many years in differential culture media (F. C. Harrison and Vanderleck, 1909a,b). Burkwall and Hartman (1964) noted, however, that not all colonies that grew on medium A-27 were positive for hydrolysis of esculin within the recommended incubation period. Similar probdems had been encountered by R. E. 0. Williams and Hirch ( 1950, p. 517). Many of the esculin-negative colonies were enterococci and many were not ( Burkwall and Hartman, 1964). When esculinnegative colonies are excluded from the count, the estimate of the population of fecal streptococci might be low. On the other hand, if esculin-negative colonies are included in the count, some bacteria other than fecal streptococci may be included, Variability in the ability of an organism to ferment esculin was noted years ago (F. C. Harrison and Vanderleck, 1915); some organisms are weakened and give atypical colonies, yet their activity is regained upon repeated culture. Such alteration of apparent fermentative properties by unfavorable environment was noted as early as 1905 ( MacConkey, 1905). Esculin also has been used in media containing sodium azide (media A-24 and A-25) and sodium taurocholate (media A-35 and A-36). At least the first two, and probably all four, of these media have the same shortcoming insofar as colony recognition is concerned. The use of sorbitol fermentation and tyrosine decarboxylase production (medium A-33) as criteria for "specific" biotypes of fecal streptococci would present similar difficulties.
K. CITRATE The use of citrate (2%) as a selective ingredient in media (A-3) for enterococci originated with the studies of Reinbold et al. (1953). Campbell and Gunsalus (1944) and Abd-El-Malek and Gibson (1948) had previously studied citrate utilization by S. faecalis, while MacLeod and Snell (1947) had reported that 2% citrate inhibited the growth of Leuconostoc mesenteroides and all five species of Lactobacillus examined, but had little effect on the growth of S . faecalis (see also Campbell and Gunsalus, 1944). Medium A-3 was modified by Saraswat et al. (1963) by raising the azide concentration to 0.04% to increase the selectivity (medium A-18) for use in dairy bacteriology. Saraswat et al. ( 1963, and unpublished ) found that the increased azide concentration did not lower yields under practical conditions. Burkwall and Hartman (1964) did not encounter organisms other than enterococci when the lower azide concentration (0.01%; Reinbold et al., 1953) was used for plating frozen foods, although yields were rather low, SO it may be that Citrate-Azide agar has certain desirable properties related to selectivity that most
ISOLATION AND ENUMERATION O F ENTEROCOCCI
273
other media do not possess. This is especially true with samples containing lactobacilli or other organisms that grow on many other commonly used media. The use of citrate in combination with thallous acetate (Lachica and Hartman, 1965) is described in an earlier section. Citrate is inhibitory at concentrations of 1% to such organisms as S. lactis and Streptococcus cremoris, this inhibition being reversed by Ca++ and Mg++ (McDonald, 1957). Low levels of citrate also inhibit certain strains of S , aureus (Rammell, 1962), and the inhibition is reversed completely by Ca+ + and partially by Mg+ +; Mn+ + seems to be an additional element of importance in the reversal of citrate inhibition of Lactobacillus spp. and L. mesenteroides ( MacLeod and Snell, 1947). Citrate metabolism has been discussed recently by Deibel (1964) and and Srere (1965).
L. SODIUMCHLORIDE The addition of 6.5% sodium chloride (Sherman, 1937a) to medium B-22 was used by Ostrolenk and Hunter (1946) to reduce the number of false-positive reactions occurring at 45°C. (medium B-23). The sodium chloride, however, did not appreciably reduce the occurrence of falsenegative reactions. Splittstoesser et al. (1961) used as a confirmatory medium broth containing 6.5% NaCl and incubated at 45°C. (medium B-33). Previously, sodium chloride alone, in concentrations of 6 to 15%, had been shown by Hill and White (1929) to impart selectivity to media for culturing streptococci from clinical specimens. Neter ( 1939) also had used 6.5% NaCl in his studies, while Snyder (1940) devised a 4% NaC1-blood agar ( A-43). Mastromatteo and Baldini (1963) recently described a 5% NaC1-blood agar (see also Kramer and Koch, 1931) for isolation of group A streptococci from the throat and showed that the NaC1-blood agar medium was more selective than an azide-crystal violet medium (Wahl and Meyer, 1957; Pettenela, 1957) for group A streptococci. The NaGl was more selective in agar than in broth (Mastromatteo and Baldini, 1963)) which is of interest because Guthof and Dammann (1958) successfully employed a broth medium (B-W), pH 8.3, containing 3% NaCl and 0.03% sodium azide for presumptive isolation of enterococci from water. An incubation temperature of 45°C. and concentrations of 4.5% NaCl and 0.04% azide in agar were used for confirmation (A-20). It is difficult to reconcile the use of media containing high concentrations of salt with the observations of Mastromatteo and Baldini (1963) and Litsky et al. ( 1953). The latter workers reported that 1.5%salt (in broth) was slightly inhibitory to enterococci. In addition, Mayeux and Colmer (1961) de-
274
HARTMAN, REINBOLD,
AND SARASWAT
scribed a medium containing 10% sucrose and only 0.005% sodium azide that was selective for isolation of Leuconostoc spp. from materials that probably also contained large numbers of plant forms of enterococci. These reports would indicate that at least some strains of enterococci are more susceptible than is commonly believed to environments with high osmotic pressures. Therefore, media containing high levels of sodium chloride may give erroneously low recoveries, although these same media might be adequate for confirmation of salt- and temperature-tolerant streptococci ( Splittstoesser et al., 1961).
M. TELLURITE Tellurite was first used as a selective agent in culture media as early as 1912, when Conradi and Troch (1912) described an improved medium
for isolation of diphtheria bacilli, These authors noted that streptococci and several other groups of microorganisms grew in the presence of 0.02y) potassium tellurite, but the growth of many bacteria commonly found in the throat was inhibited, Other aspects of early studies with tellurite have been reviewed by Gilbert and Humphreys (1926) and Whitley and Damon (1949). Observations similar to those made by Conradi and Troch (1912) were made by Smith ( 1914, 1918); however, Cooper and Ramadan (1955) discovered that a broth (B-25) containing 0.02% POtassium tellurite was quite suitable for isolating streptococci from feces of various animals. An agar medium containing 0.2% tellurite was also proposed (Cooper and Ramadan, 1955). Perry and Petran (1939) had proposed the use of tellurite agar slants specifically for preliminary isolation of hemolytic streptococci from the throat and nose. Fleming (1929) studied the effects of penicillin on different bacteria and observed that this antibiotic exerted a selective action on different species of streptococci. The complementary action of penicillin and potassium tellurite was later used (Fleming, 1932) in devising media for selective isolation of various groups of microorganisms, including streptococci. In a later study (Fleming and Young, 1940), levels of 0.02% to about 0.0002% tellurite were suitable for inhibition of E. coli in fecal material; susceptibility of Protcus spp. was variable ( see also Lichstein and Snyder, 1941). In the same year (Bornstein, 1940) the resistance of enterococci to penicillin and tellurite was confirmed; enterococci and S. (actis were resistant to penicillin, and Streptococcus oiridans strains were susceptible. All enterococci tested were resistant to 0.1% tellurite, while tolerance of other streptococci to tellurite (0.01% ) varied. A tellurite-taurocholate-esculin-sodium chloride agar of high pH (medium A-36) was described by Schafer ( 1953) but apparently was
ISOLATION AND ENUMERATION OF ENTEROCOCCI
275
never tested further, although recommended by a German commission on methods of examination of water ( Methodenkommission, 1960). A medium for the isolation of S. salivurius was devised by K. D. Rose and Georgi (1941), using 0.03% potassium tellurite and 0.00027, crystal violet. These concentrations are similar to those used earlier by Garrod (1933), but Pike (1945b) reduced the tellurite concentration to 0.01~0 (medium B-24). Chapman ( 1944,1946,1947) proposed a medium (A-38) containing only 0.002% potassium tellurite, but with 0.00008% crystal violet and 0.0075% trypan blue. Sucrose (5% ) was added as a substrate SO S. salivarius would produce polysaccharide and thus be differentiated from S . mitis and most hemolytic streptococci. Enterococci reportedly also produced distinctive colonies on this medium (A-38), known as MitisSalivarius agar. Sucrose ( 5 % ) was similarly used by R. E. 0. Williams and Hirch ( 1950) to examine streptococci from air in a medium (A-37) that contained less tellurite and crystal violet than Chapman’s medium. In addition, trypan blue was omitted. This medium would be expected to be less selective than Mitis-Salivarius agar, but even at these concentrations of- inhibitors growth of some streptococci (none of them S. salivarius) was severely retarded. The mechanism of growth inhibition by tellurite is not fully understood. Of the enterococci, S. faecalis is resistant to 0.05% tellurite; most others are susceptible to 0.05% tellurite (Deibel, 1964; Hartman et al., 1966), although tellurite-resistant strains can be selected from a susceptible population ( McDowell and Hartman, unpublished data, 1964). Fleming and Young (1940) had observed earlier that tellurite-resistant strains of E . coli could be obtained by adapting cultures to increasing concentrations of the inhibitory agent. Tucker et al. (1962) reported that S. faecalis reduces tellurite to pure tellurium metal, and Walper et al. (1962) developed a method for the quantitative determination of tellurite in microbiological media. Tellurite reduction by protoplasts and cell-free preparations has been studied (Thomas et al., 1963), and progress has been made in investigations on the site and nature of tellurite reduction (Tucker et al., 1964; Thomas et al., 1965). Several additional observations are worthy of note in regard to the use of tellurite in culture media. Smith (1918) reported that, “On standing, the tellurate solution gradually loses its antiseptic action, whereas telluric-acid solution, if sterilized by heat, keeps indefinitely.” R. E. 0. Williams and Hirch (1950) reported that storage of tellurite plates for up to 7 days in a refrigerator before incubation had no detectable effect on the streptococcus counts. Fleming and Young (1940) found that, upon extensive incubation (48 hours ), resistant strains could “absorb or render
276
HARTMAN,
REINBOLD, AND SARASWAT
inert at least 90 per cent of the tellurite over a distance of 16 mm” from the resistant colony. Tellurite causes hemolysis in blood agar in concentrations as low as 0.002% (Edwards, 1933). Obviously many fecal streptococci are inhibited by relatively low concentrations of tellurite or do not reduce tellurite to produce black colonies. These bacteria would not be included in counts made on tellurite-containing media.
N. PENICILLINAND OTHERANTIBIOTICS J. C. White and Sherman (1944) were the first to combine penicillin with azide for isolation of enterococci (medium A-9). Methylene blue was added in a modification of the medium (A-10) by Winter and Sandholzer (1946a,b), so debris would be stained deep blue whereas colonies were white with a blue center. This permitted better differentiation of colonies from debris. In addition, the penicillin level was doubled, which resulted in better selectivity for fecal streptococci. A penicillincontaining enterococcus confirmation medium ( A-19) was also proposed by Winter and Sandholzer (1946b) for water analysis; enterococci would have to survive a rigorous presumptive test ( B-15a) before encountering the penicillin. M. F. White et al. (1947) omitted penicilhn from the confirmation medium. Yields on these media would probably be low compared with yields attainable on certain other media discussed in previous sections. Other antibiotics have been included in media used to isolate streptococci, and a few of these media have been examined for their application to the isolation of fecal streptococci. Burkwall and Hartman (1964) studied two media (A-40 and A-41), both containing neomycin. These media were not sufficiently selective for enumeration of enterococci in frozen foods; however, changes in formulation of the media might increase the efficacy of these media for such uses. 0. SODIUMTAUROCHOLATE AND BILE SALTS Sodium taurocholate was first used as a selective agent for the isolation of coliform bacteria ( MacConkey, 1905). Application of this selective agent to media for isolation of streptococci was reviewed by Koch (1935), who devised an improved medium (A-35) that apparently possessed good selectivity. Weissenbach (1918; see also Bagger, 1926) preferred ox bile (medium B-30) to the sodium taurocholate preparations available at that time. Mieth (1960) used a taurocholate enrichment broth (B-12) of quite different composition in that sodium azide and sodium chloride were inoluded as additional selective agents. These media, except B-12, probably lack the selectivity necessary for primary isolation of enterococci from foods and other sources. Szita (1957) pro-
ISOLATION AND ENUMERATION OF ENTEROCOCCI
277
posed a taurocholate-crystal violet-tellurite medium that seemed to function well as a confirmatory agar. Rycroft ( 1956) developed a taurocholate-mannitol broth (B-31) with incubation at 44°C. for use in confirmation of fecal streptococci. Two unusual features daimed of preparations grown in this medium were extensive chaining of cells and prolific production of group D antigen for serological tests. This medium may be superior to that developed by Medrek and Barnes (1962) for group D antigen production by mannitol-fermenting enterococci; however, no one has examined this possibiIity. P. PHENETHYL ALCOHOL A phenethyl alcohol medium (A-42; Lilley and Brewer, 1953; Anonymous, 1956) was found by Burkwall and Hartman (1964) to lack the selectivity necessary for enumeration of fecal streptococci in frozen foods. Nevertheless, phenethyl alcohol is another potentially useful ingredient for increasing the selectivity of existing media. Phenethyl alcohol apparently inhibits growth of gram-negative bacteria (Lilley and Brewer, 1953; Berrah and Konetzka, 1962), except gramnegative anaerobes (Dowel1 et al., 1964). Inhibition probably occurs through blockage of DNA synthesis (Berrah and Konetzka, 1962; Treick and Konetzka, 1964), although the synthesis of messenger RNA and the process of enzyme induction evidently also are affected by phenethyl alcohol (Rosenkranz et al., 1964, 1965).
Q. PH Chesbro and Evans (1959) devised a medium of pH 10.0 for tolerance tests of enterococci. A modification of this medium (pH 9.6) gave low yields when used for direct plating ( Hartman, unpublished data). Yet high pH may stilI be suitable for prdiminary enrichment or for selectivity after preliminary enrichment. Some investigators have devised media with rather high pH values (media A-19, A-20, A-24, A-25, A-35, A-36, B-14, B-15a). Tolerance of enterococci to high pH values is well known (see Chapman and Rawls, 1936))and several workers (Mueller and Whitman, 1931; Stainsby and Nicholls, 1932) inoculated fecal material or pathological specimens into sodium bicarbonate solutions of 0.2 to 1.0% which were incubated for a period prior to plating. This method has also been applied to milk samples (Groesbeck, 1935). The high pH destroys many gram-negative contaminants. Carbonate and bicarbonate have also been used to stimulate growth of fecal streptococci (Burbank, 1929; C. W. Langston, cited in Deibel, 1964; media A-15, A-16, A-17, B-19). The mechanism of stimulation has
278
IIARTMAN,
HEINBOLD, AND SARASWAT
been investigated (see Martin and Niven, 1960; Wright, 1960; Deibel, 1964). Of interest in this regard is the finding of Mickelson (1964) that some strains of S. pyogeries required C02, supplied as NaHCO:,; one COZrequiring strain could be adapted to grow in bicarbonate-free medium. An interesting discovery was reported recently by Sims ( 1964). Streptococcus bovis would grow in media acidified to pH 5.0 and incubated at 37°C. in an atmosphere of 5% C 0 2 in air. Other streptococci cannot grow under these conditions, The potential use of this medium has not been explored. Contaminating lactobacilli could probably be inhibited by 2 F citrate or some other additional ingredient. K. SELENITE
A selenite (about 0,024F )-aniline blue (about 0.071% )-blood platc w ~ devised s (medium A-34; Guthof, 1952) to differentiate streptococci by modification of a medium originally intended for diagnosis of diphtheria ( Herrmann, 1939). Although not proposed for primary enrichment or plating (which is performed with other media), the selenite-
indicator-plate ( medium A-34) may prove valuable for subsequent differentiation of enterococci (Guthof and Winkler, 1955; Guthof and Dammann, 1958). It could be used to isolate $trains of S. bovis, S. salivarius, or S. mitis, the growth of which might be inhibited on other more commonly used media. S. TETRATHIONATE
Many of the selective media now used for isolation of fecal streptococci were first applied to examination of gram-negative rods of intestinal origin. Thus, it is not surprising that tetrathionate broth, which was used by Cooper et al. (1942) to isolate Salmonella paratyphi from feces, would be applied (medium B-29) to isolate enterococci (Cooper and Ramadan, 1955). Several workers (see Prescott and Baker, 1904) have noted that in these media coliforms grow profusely at first, then are followed, after incubation for 2 or 3 days, by increases in the streptococcal populations. Mallmann and Gelpi (1930) took advantage of this succession of culture types in use of a standard lactose enrichment broth for detecting streptococci. Confirmation was made by microscopic examination. Cooper and Ramadan ( 1955) discovered that tetrathionate broth, though ohten successful with human feces, failed significantly in conccntrating streptococci from sheep and bovine feces. An incubation period of 24 hours was used, however. Perhaps modifications of the constituents of the medium and procedure of isolation (such as use of a longer incubation period ) will enable tetrathionate-based media to be used successfully in food microbiology. The striking differences in
ISOLATION AND ENUMERATION OF ENTEROCOCCI
279
recovery of streptococci from human, bovine, and sheep sources with tetrathionate versus tellurite and thallium acetate media emphasize the fact that different media do have different selectivities. This fact might be used to good advantage when more is known about the distribution of various fecal streptococci in nature.
Ill. Comparative Studies on Media and Methods So far, we have discussed various media with little regard to the type of sample to be examined (water versus a food versus intestinal contents, etc.), technique to be used for the examination (most probable numbers (MPN) versus pour plate versus surface or smear plate versus membrane filter), or objective of the examination (isolation vs. enumeration). Obviously, no one culture method will be the most suitable for all applications. In like manner, no one medium will be most appropriate for all culture methods or all types of natural materials. Comparative studies on the efficacy of a medium for a specific purpose, therefore, must be interpreted with caution, especially when translating the efficiency of the medium to a different type of material or to a material that has been treated differently. A. GENERALDISCUSSION Many, if not a large majority, of the microorganisms in certain foods and other natural environments are in a different physiological state than similar strains cultivated in the laboratory. This fact had been noted earlier by many investigators (see F. C. Harrison and Vanderleck, 1915; Darby and Mallmann, 1939). Stock cultures may be valuable for initially determining the approximate selectivity of a medium, but frequently these preliminary tests have not been followed by examination of sufficient samples of natural products. With laboratory strains as the test material, continuation of a rapidly growing culture is the usual concern, whereas with bacteria from the natural environment the problem often seems to be not only growth but also growth initiation (see also Ferraro and Appleman, 1957, and Slanetz and Bartley, 1957). The problem of growth initiation has received little attention in research because it is difficult to approach experimentally. Nevertheless, the over-all effect is that a medium usually will be more inhibitory to bacteria in natural products than to rapidly growing laboratory cultures. Because there are so many unknown factors, one studying the complete flora of a material might be well advised to use several media, each with a different selective ingredient or group of selective ingredients (see Cooper and Ramadan, 1955) .
280
HAHTMAN, REINBOLD, AND SARASWAT
Another factor that seems to be well known but usually is equally well ignored is that various media have different selectivities. Too often the method of defining a group of indicator organisms, such as “coliforms” or “enterococci,” has been to determine the flora that will grow on a medium used to detect the group, rather than to define the group first and then obtain media with appropriate selectivity. These factors become of paramount importance when natural materials, such as foods, are to be examined. As improved classifications of the fecal streptococci become available (Deibel et al., 1963; Deibel, 1964; Hartman et d., 1966), comparative studies will be facilitated. Meanwhile, it might be well to point out that organisms within a culture vary in their ability to grow under one set of conditions (see Whittenbury, 1963), and no single medium or set of conditions is likely to result in selective recovery of all of the fecal streptococci in a sample containing quantities of other, sometimes closely related bacteria. Preliminary enrichment may overcome some of these difficulties. The value of preliminary enrichment in sugar broth had been described as early as 1952 by Allen and co-workers (cited in Childs and Allen, 1953) and was emphasized again recently by R. E. Rose and Litsky (1965). Other workers have chosen to inoculate “presumptive” media (i.e., B-13 or B-16), transferring later to more selective “confirmatory” media (i.e., A-19 or B-15). Still other workers have preferred to use very highly selective media (i-e., A-3, A-8, A-18, A-24, A-25, A-27) with some sacrifice in yield. A list of all publications in which each of the various media and methods has been used for specific applications would mean little. Most workers have compared their new medium with only one or two other media. These studies, although they may be excellent, are so difficult to correlate with each other that adequate conclusions cannot be reached. On the other hand, there have been some rather comprehensive comparisons involving several or many types of media and methods simultaneously. In only two of these studies (Saraswat et al., 1963; Burkwall and Hartman, 1964) have different pour-plate media been compared extensively. Some key comparative studies have been selected to illustrate the present status of media and methods for the determination of enterococci. IN WATER B. ENTEROCOCCI
The anallysis of enterococci in water was given impetus by the work of Litsky et al. ( 1953), who found that a most probable number (MPN) procedure involving selective enrichment in B-6 followed by confirmation in B-17 gave much greater yields than use of media B-15a or B-22. The B-6, B-17 combination has been widely used in water bacteriology
ISOLATION AND ENUMERATION OF ENTEROCOCCI
281
and seems better than many other media, such as a B-21, B-17 combination (Croft, 1959) and B-21 alone (Kenner et al., 1961). Slanetz and Bartley (1957) devised a membrane filter procedure (A-13) that gave higher yields from water than the B-6, B-17 combination. Kenner et al. (1961) also reported that A-13 was superior to the B-6, B-17 combination, although Croft (1959) found the two methods equivalent. New media (A-17, B-19) were developed by Kenner et al. (1961) for MPN, membrane filter, and direct plating techniques. The MPN procedure gave highest yields; direct plating in A-17 was probably intermediate and slightly higher in yield than the membrane filter method; all were superior to A-13. The membrane filter and direct plating methods were recommended where applicable because colonies could be confirmed without additional plating. Mallmann and Kereluk ( 1957) reported that considerably greater numbers of enterococci were recovered by the drop-plate method using medium A-14 than by an MPN method using B-6, B-17, but these observations have not been applied to other media. MPN versus direct plating methods are discusssed in the next section. Although many combinations of media and methods await extensive testing, the three most logical selections for use in water bacteriology at present are the B-6, B-17 combination for use with the MPN procedure, A-13 for the membrane filter procedure, and A-17 and B-19 for MPN, membrane filter, or direct plating. The method chosen would be dictated by the circumstances of analysis. Regardless of the procedure selected, isolates should be confirmed, at least in preliminary experiments. Confirmation, especially from filter membranes, shouId probably be preceded by enrichment on a nonselective medium (Croft, 1959). Such a practice also might be necessary for confirmation in medium B-17, because this medium inhibits many strains of S . bovis and S. equinus (Kenner et al., 1960). The value of preenrichment for MPN or membrane filter techniques has already been discussed.
C. ENTEROCOCCI IN NONDAIRY FOODS The first comprehensive comparative study of methods for enumerating enterococci in foods was done by Zaborowski et al. (1958). Medium B-15 was the best of four MPN presumptive media examined; medium B-6 gave higher counts but was not as selective. Medium B-17 was satisfactory for confirmation. Thus, the optimum procedure developed by Zaborowski et al. (1958) would be use of B-6 or B-15a with confirmation in B-17. Fanelli and Ayres (1959) also found the B-6, B-17 combination good for MPN determinations, and Splittstoesser et al. (1961) reported that medium B-6 gave higher counts than three other presumptive MPN media. Hall et al. (1963), however, preferred medium B-21 to the B-6,
282
HARTMAN, REINBOLD, AND SARASWAT
B-17 combination. The latter workers reported that B-21 was equivalent to B-19, a finding in contrast to previous reports on the inadequacy of medium B-21 for food analyses. In summary, the best of the MPN procedures so far developed is use of the B-6, B-17 combination, or possibly B-19. Hall et al. (1963) reported that B-19, a broth, was slightly more inhibitory than an agar medium of the same formulation (A-17) to some, but not all, species of enterococci, Fanelli and Ayres (1959) also obtained highest yields by using an agar medium (A-29) and direct platins procedures, rather than MPN techniques. Medium A-29 was found by Burkwall and Hartman (1964) to be the best of 15 direct plating media examined. Medium A-17 also was satisfactory, as was a new medium ( A-15, improved formula, A-16). These media, although they are high in yield, lack selectivity and need at least some confirmation. This has been emphasized by Mossel ( 1964) and Saleh et al. (1966). Some agar media apparently possess sufficient selectivity that confirmation is unnecessary. The best of these, as determined by Burkwall and Hartman ( 1964), was medium A-3. ( A modification of this medium has been proposed recently, medium A-18.) Lower yields might be obtained on this medium than on the media discussed in the preceding paragraph. On the other hand, one might wish to use a very high-yielding medium, such as A-42, but there may be many false-positive results (Burkwall and Hartman, 1964), so confirmation would probably be essential. D. ENTEROCOCCI IN DAIRY PRODUCTS
Of ten media examined, only medium A-18 met the rigid requirements established by Saraswat et al. (1963) for enumeration of enterococci in dairy products. Media A-30 and A-31 also may be suitable, but their efficacy in relation to medium A-18 has not yet been well established.
E. ENTEROCOCCI IN INTESTINAL CONTENTS There is such divergence of opinion with regard to the best medium for isolation and enurneration of fecal streptococci from intestinal ingesta that we only refer readers to the publications of Kenner et al. (1960), Mieth ( 1960), and Raibaud et al. ( 1961) as examples of what some investigators think of other investigators’ media. F. FUTURE NEEDS
A comprehensive comparison of media and methods representative of all of those presently available is urgently needed. These media and methods should be utilized simultaneously, using the same samples or
ISOLATION AND ENUMERATION OF ENTEROCOCCI
283
subsamples. Subsamples would probably be necessary because the study should include interlaboratory or, preferably, intercontinental collaboration. Isolation and quantification of fecal streptococci in natural materials of all types should be examined. This would be a gigantic undertaking because confirmation of positive colonies and presumptive tests would be necessary. Likewise, negative presumptive tubes (colonies) should also be examined ( a matter that few investigators have considered). Before such a large screening program is undertaken, there would have to be agreement on what bacterial species or strains are desired and what are not, and then procedures would have to be developed to evaluate the eficacy of the media. Only when such experiments are completed can optimum procedures be selected for specific applications with a reasonable degree of confidence.
REFERENCES Abd-El-Malek, Y., and Gibson, T. (1948). J. Dairy Res. 15, 233-248. Allen, L. A., Brooks, E., and Williams, I. L. (1949). J. H y g . 47, 303-319. Allen, L. A., Pierce, M. A. F., and Smith, H. M. (1953). J. Hyg. 51, 458-467. Anonymous. (1953). “Difco Manual,” 9th ed. Difco Lab., Detroit, Michigan. Anonymous. ( 1956). “BBL Products,” 4th ed. Baltimore Biol. Lab., Baltimore, Maryland. Bagger, S. V. (1926). J. Pathol. Bacteriol. 29, 225-238. Barnes, E. M. (1956a). J. Gen. Microbid. 14, 57-68. Barnes, E. M. { 1956b). J. Gen. Microbiol. 14, v. Barnes, E. M. ( 1 9 5 6 ~ ) J. . AppZ. Bacteriol. 19, 193-203. Barnes, E. M. (1958). Personal communication. Barnes, E, M. (1959). J. Sci. Food Agr. 10, 656-662. Barnes, E. M., Ingram, M., and Ingram, C. C. (1956). J. Appl. Bacteriol. 19, 204211. BBL (Baltimore Biological Laboratory). ( 1963 ). Personal communication. Berrah, G., and Konetzka, W. A. (1962). J. Bacteriol. 83, 738-744. Bornstein, S. (1940). J. Bacteriol. 39, 373-387. Braun, W. ( 1965). “Bacterial Genetics,” 2nd ed., 380 pp. Saunders, Philadelphia, Pennsylvania. Browning, C. € I . , and Gulbransen, R. (1918). In “Applied Bacteriology” (C. H. Browning, ed.), pp. 147-149. Oxford Univ. Press, London and New York. Bryan, C. S. (1932). Am. J. Public Health 22, 749-751. Burbank, R. (1929). Bull. N.Y. Acad. Med. [ 2 ] 5, 176-187. Burkwall, M. K., and Hartman, P. A. (1964). Appl. Microbiol. 12, 18-23. Burman, N. P. ( 1961). J. Appl. Bacteriol. 24, 368-376. Burmeister, H. R., Hartmarl, P. A., and Saul, R. A. (1966). Appl. Microbiol. 14, 31-34. Burnett, G. W., and Scherp, H. W. (1962). “Oral Microbiology and Infectious Disease,” 1003 pp. Williams & Wilkins, Baltimore, Maryland. Campbell, J. J. R., and Gunsalus, I. C. (1944). J. Bacteriol. 48, 71-76. Chapman, G. H. (1944). 1. Bacteriol. 48, 113-114.
284
HARTMAN, REINBOLD, AND SARASWAT
Chapman, G. H. (1946). Am. 1. Digest. Diseases 13, 105-107. Chapman, G. H. (1947). Trans. N.Y. Acad. Sci. [2] 10, 45-55. Chapman, G. H., and Rawls, W. B. (1936). J. Bacteriol. 31, 323-331. Chesbro, W. R., and Evans, J. B. (1959). 1. Bacteriol. 78, 858-862. Childs, E., and Allen, L. A. (1953). J. Hyg. 51, 468-477. Churchman, J. W. (1912). J. Ezptl. Med. 16, 221-247. Colobert, L., and Morelis, P. (1958). Ann. Inst. Pasteur 94, 120-122. Conradi, H., and Troch, P. (1912). Muench. Med. Wochschr. 59, 1652-1653. Cooper, K. E., and Linton, A. H. (1947). Monthly Bull. Min. Health Lab. S e w . 6, 204-208. Cooper, K. E., and Ramadan, F. M. (1955). J. Gen. Microbiol. 12, 180-190. Cooper, K. E., Wood, N., Elliot, E., Caswell, M., and Small, W. (1942). J. Pathol. Bacteriol. 54, 345-353. Croft, C. C. (1959). Am. 1. Public Health 49, 1379-1387. Crowe, H. W. (1913). Proc. Roy. SOC. Med. 6, 117-125. Darby, C. W., and Mallmann, W. L. (1939). J. Am. Wuter Works Assoc. 31, 689706. Deibel, R. H. (1964). Bacterial. Reu. 28, 330-366. Deibel, R. H., Lake, D. E., and Niven, C. F., Jr. (1963). 1. Bacteriol. 86, 12751282. Dick, L. A., and Hucker, G. J. (1940). J. Milk Technol. 3, 307-313. Diernhofer, K. (1936). Mikhwirtsch. Forsch. 18, 83-86. Dowell, V. R., Jr., Hill, E. O., and Altemeier, W. A. (1964). 1. Bacterwl. 88, 18111813. Drigalski, V., and Conradi, H. (1902). Z. Hyg. Infektionskrankh. 39, 283-300. Edwards, S. J. (1931). Proc. Roy. SOC. Med. 24, 1369-1370. Edwards, S. J. (1933). 1. Comp. Pathol. Therap. 46, 211-217. Edwards, S. J. (1938). J. Comp. Pathol. Therap. 51, 250-263. Fanelli, M. J., and Ayres, J. C. (1959). Food Technol. 13, 294-300. Ferraro, F. M., and Appleman, M. D. (1957). Appl. Microbiol. 5, 300-303. Ferraro, F. M., Appleman, M. D., and Severens, J. M. (1958). Bacteriol. Proc. p. 24. Fitzgerald, R. J., and Keyes, P. H. (1960). J. Am. Dental Assoc. 61, 23-33. Fleming, A. (1929). Brit. I. Exptl. Pathol. 10, 226-236. Fleming, A. (1932). I. Pathol. Bacteriol. 35, 831-842. Fleming, A., and Young, M. Y. (1940). J. Pathol. Bacteriol. 51, 29-35. Forget, A., and Fredette, V. (1962). 1. Bacteriol. 83, 1217-1223. Franklin, J. G., and Sharpe, M. E. (1963). I. Dairy Res. 30, 87-99. Frazier, W. C. ( 1958). “Food Microbiology,” 472 pp. McGraw-Hill, New York. Fry, R. M. (1932). Brit. I. Exptl. Pathol. 13, 456-457. Fujiwara, K., Sekiya, K., and Bamba, K. (1956). Japan. J. Bacteriol. 11, 411-415. Garey, J. C., Foster, E. M., and Frazier, W. C. (1941). J. Dairy Sci. 24, 1015-1025. Garrod, L. P. (1933). S t . Burthotomew’s Hosp. Rept. 66, 203-252. Gerencser, V. F., and Weaver, R. H. (1959). Appl. Microbiol. 7, 113-115. Gilbert, R., and Humphreys, E. M. (1926). J. Bacteriol. 11, 141-151. Goetz, A,, and Tsuneishi, N. (1951). 1. Am. Water Works Assoc. 43, 943-984. Greer, J. E., and Britt, E. M. (1959). Bacteriol. Proc. p. 63. Groesbeck, W. M. (1935). Am. I. Public Health 25, 345-346. Gunsalus, I. C., and Stanier, R. Y., eds. (1960). “The Bacteria,” Vol. 1, 513 pp. Academic Press, New York.
ISOLATION AND ENUMERATION OF ENTEROCOCCI
285
Gunsalus, I. C . , and Stanier, R. Y., eds. (1961). “The Bacteria,” Vol. 2, 572 pp. Academic Press, New York. Gunsalus, I. C., and Stanier, R. Y., eds. (1962a). “The Bacteria,’’ Vol. 3, 718 pp. Academic Press, New York. Gunsalus, I. C., and Stanier, R. Y., eds. (1962b). “The Bacteria,” Vol. 4, 460 pp. Academic Press, New York. Gunsalus, I. C., and Stanier, R. Y., eds. (1964). “The Bacteria,” Vol. 5, 517 pp. Academic Press, New York. Guthof, 0. (1952). Zentr. Bakteriol., Parasitenk., Abt. I . Orig. 158, 87-95. Guthof, O., and Dammann, G. (1958). Arch. Hyg. Bakteriol. 142, 559-568. Guthof, O., and Winkler, F. (1955). Zentr. Bakteriol., Parasitenk., Abt. I . Orig. 162, 236-255. Gyllenberg, H., and Niemela, S. (1960). Ann. Med. ExptZ. Bid. Fenniae (Helsinki) 38, 303-308. Gyllenberg, H., and Roine, P. (1957). Acta Pathol. Microbiol. Scand. 41, 144-150. Hajna, A. A. (1951). Public Health Lab. 9, 80-81. Hajna, A. A., and Perry, C. A. (1943). Am. J. Public Health 33, 550-556. Hall, H. E. (1964). In “Examination of Foods for Enteropathogenic and Indicator Bacteria” (K. H. Lewis and R. Angelotti, eds.), pp. 20-25. Public Health Service, U.S. Department of Health, Education and Welfare, Washinpton, D.C. Hall, H. E., Brown, D. F., and Angelotti, R. (1963). I . Food Sci. 28, 566-571. Hannay, C. L., and Norton, I. L. (1947). Proc. SOC. Appl. Bacteriol. pp. 39-45. Harrison, A. P., Jr., and Hansen, P. A. (1950). J. Bacteriol. 59, 197-210. Harrison, F. C., and Vanderleck, J. ( 190%). Zentr. Bakteriol., Parasitenk., Abt. 11 22, 547-551. Harrison, F. C., and Vanderleck, J. ( 1909b). Zentr. Bakteriol., Parasitenk, Abt. 1I 22, 551-552. Harrison, F. C., and Vanderleck, J. (1915). Trans. Roy. SOC. Can., Sect. IV 131 9, 207-217 and 4 plates. Hartman, P. A. (1960). Appl. MicrohioZ. 8, 114-116. Hartman, P. A., and Burkwall, M. K. (1964). Appl. Microbiol. 12, 18-23. Hartman, P. A., and Huntsberger, D. V. ( 1961). Appl. Microhiol. 9, 32-38. Hartman, P. A,, Johnson, R. H., Brown, L. R., Jacobson, N. L., Allen, R. S., Shellenberger, P. R., and Van Horn, H. H., Jr. (1962). Iowa State J. Sci. 36, 217-231. Hartman, P. A., Reinbold, G. W., and Saraswat, D. S. (1965). J. Milk Food Technol. 28, 344-350. Hartman, P. A,, Reinbold, G. W., and Saraswat, D. S. (1966). Intern. Bull. Systematic Bacteriol. 16, 197-221. Hartmann, G. ( 1937). Milchwirtsch. Forsch. 18, 116-122. Hauge, S., and Kflhler Ellingsen, J. (1953). Nord. Veterinamned. 5, 539-547. Haxthausen, H. (1927a). Lancet 11, 370-373. Haxthausen, H. (1927b). Ann. DermatoZ. Syphilog. 8, 201-212. Herrmann, W. (1939). Z . H y g . Infektionskrankh. 121, 540-556. Hill, J. H., and White, E. C. (1929). J. Bacteriol. 18, 43-57. Horie, S., and Saheki, K. (1960). BUZZ. Japan. SOC.Sci. Fisheries 26, 623-626. Kavanagh, F., ed. (1963). “Analytical Microbiology,” 707 pp. Academic Press, New York. Kenner, B. A., Clark, H. F., and Kabler, P. W. (1960). Am. 1. Public Health 50, 1553-1559. Kenner, B. A., Clark, H. F., and Kabler, P. W. (1961). Appl. Microbiol. 9, 15-20.
286
HARTMAN, REINBOLD, AND SARASWAT
Kereluk, K. ( 1960). Personal communication. Keyes, P. H. (1962). Intern. Dental J. 12, 443-464. Kinnear, J. (1931). Brit. J. Exptl. Pathol. 12, 384-389. Kjellander, J. (1960). Acta Pathol. Microbiol. Scand. 48, Suppl. 136, 1-124. Kleinebcrger-Nobel, E. ( 1962). “Pleuropneumonia-Like Organisms ( PPLO ) MYCOplasmataceae.” Academic Press, New York. Koch, F. E. (1935). Zentr. Bakteriol., Parasitenk., Aht. I . Orig. 134, 348-367. KGhler Ellingsen, J., and Hauge, S. ( 1953). Nord. Veterinurmed. 5, 653-662. Krlmer, E., and Koch, F. E. (1931). Zentr. Bakteriol., Parasitenk., Abt. I . Orig. 120, 452-464. Krantz, G. E., and Dunne, H. W. (1965). Am. J. V e t . Res. 26. 951-959. Krumwiede, C., and Pratt, J. S. (1914). J. Exptl. Med. 19, 20-27. Lachica, V. F., and Hartman, P. A. (1965). Bacteriol. Proc. pp. 2-3. Langston, C. W., and Bouma, C. (1960). Appl. Microbiot. 8, 212-222. Laxminarayana, H., and Iya, K. K. (1953). Indian J . Dairy Sci. 6 , 75-91. Lichstein, H. C., and Snyder, M. L. ( 1941). J. Bacteriol. 42, 653-664. Lilley, B. D., and Brewer, J. H. (1953). J. Am. Pharm. Assoc., Sci. Ed. 42, 6-8. Litsky, W., Mallmaim, W. L., and FSeld, C. W. (1952). Stain Technol. 27, 229-232. Litsky, W., Mallmann, W. L., and Fifield, C. W. (1953). Am. J. Public Health 43, 873-879. Loew, 0. (1891). Ber. Deut. Chem. Ges. 24, 2947-2953. McCarty, M. (1958). In “Bacterial and Mycotic Infections of Man” (R. J. Dubos, ed. ), 3rd ed., pp. 248-276. Lippincott, Philadelphia. McDonald, I. J. (1957). Can. J. Microbiol. 3, 411-417. McKenzie, D. A. ( 1941 ) . Vet. Record 53, 473-480. MacConkey, A. (1905). J. Hyg. 5, 333-379. MacLeod, R. A., and Snell, E. E. (1947). J. Biol. Chem. 170, 351-365. Mallmann, W. L., (1940). Sewage Works J. 12, 875-878. Mallmann, W. L., and Gclpi, A. G., Jr. (1930). Michigan State Coll., Eng. Expt. Stu. Bull. 27, 1-16. Mallmann, W. L., and Kereluk, K. (1957). Bacterial. Proc. p. 142. Mallmann, W. L., and Seligmann, E. B., Jr. (1950). Am. J. Public Health 40, 286289. Martin, W. R., and Niven, C. F., Jr. (1960). J. Bacteriol. 79, 295-298. Mastromatteo, L., and Baldini, I. (1963). J. Bacteriol. 86, 1131-1133. Mastromatteo, L., and Pisu, I. (1959). Boll. Inst. Sieroterap. Milan. 38, 347-358. Mayeux, J. V., and Colmer, A. R. (1961). J. Bacteriol. 81, 1009-1011. Mead, G. C. (1963). Nature 197, 1323-1324. Mead, G . C. (1964). Nature 204, 1224-1225. Medrek, T. F., and Barnes, E. M. (1958). J. Appl. Bacteriol. 21, 79. Medrek, T. F., and Barnes, E. M. (1962). J. Gen. Microbiol. 28, 701-7C9. Methodenkommission. ( 1960). Milchwissenschaft 15, 340-345. Mickelson, M. N. (1964). J. Bacteriol. 88, 158-164. Mieth, H. ( 1960). Zentr. Bakteriol., Parasitenk, Aht. I. Orig. 179, 456-482. Mieth, H. ( 1961 ). Zentr. Bakteriol., Parasitenk., Abt. I . Orig. 183, 68-89. Mieth, H. (1962a). Zentr. Bakteriol., Parasitenk., Abt. Z. Orig. 185, 47-52. Mieth, H. (1962b). Zentr. Bakteriol., Parasitenk., Abt. 1 . Orig. 185, 166-174. Morelis, P., and Colobert, L. (1958). Ann. Inst. Pasteur 95, 667-680. Morris, W., and Weaver, R. H. (1954). Appl. Microbiol. 2, 282-285. Mossel, D. A. A. (1964). J . Sci. Food Agr. 15, 349-362.
ISOLATION AND ENUMERATION OF ENTEROCOCCI
287
Mossel, D. A. A., van Diepen, H. M. J., and de Bruin, A. S. (1957). J. Appl. BUCten‘ol. 20, 265-272. Mueller, J. H., and Whitman, L. (1931). I. Bacteriol. 21, 219-223. Munch-Petersen, E., and Christie, R. (1947). J. Pathol. Bacteriol. 59, 367-371. Mundt, J. 0. (1964). Nature 204, 201-202. Neter, E. (1939). PTOC.SOC. Exptl. Biol. Med. 42, 668-672. Nicholls, P. (1964). Biochem. J. 90, 331-343. Niven, C. F., Jr. (1963). I n “Microbiological Quality of Foods” ( L . W. Slanetz et ul.: eds.), pp. 119-131. Academic Press, New York. Ostrolenk, M., and Hunter, A. C. (1946). J. Bacteriol. 51, 735-741. Packer, R. A. (1943). J. Bucteriol. 46, 343-349. Perry, C. A., and Petran, E. (1939). Am. J . Clin. Pathol. 9, Tech. Suppl. 3, 70-71. Pettenella, G. ( 1957). Boll. Inst. Sieroterup. Milan. 36, 329-337. Pike, R. M. (1944). Proc. SOC. Exptl. Biol. Med. 57, 186-187. Pike, R. M. (1945a). Am. J. Hyg. 41, 211-220. Pike, R. M. (194513).J. Buctem‘ol. 50, 297-300. Prescott, S. C., and Baker, S. K. (1904). J. Infect. Diseuses 1, 193-210. Raibaud, P., Caulet, M., Galpin, J. V., and Mocquot, G. (1961). J. Appl. Bacteriol. 24, 285-306. Rainbow, C., and Rose, A. H. ( 1963). “Biochemistry of Industrial Microorganisms,” 708 pp. Academic Press, New York. Raj, H., Wiebe, W. J., and Liston, J. (1961). Appl. Microbiol. 9, 295-303. Rammell, C. G. (1962). J. Bucteriol. 84, 1123-1124. Rantasalo, I. (1947). Ann. Med. Internae Fenniae 36, 341-348. Reinbold, G. W., Swern, M., and Hussong, R. V. (1953). J. Dairy Sci. 36, 1-6. Richards, T., Soulides, D. A., and Soulides, E. (1945a). Proc. SOC.Appl. Bacteriol. pp. 41-43. Richards, T., Soulides, D. A,, and Soulides, E. (1945b). Proc. SOC. Appl. Bacteriol. pp. 44-46. Ritter, C., Shull, I. F., and Quinley, R. L. (1956). Am. J. Public Health 46, 612-618. Ritter, C., and Treece, E. L. (1948). Am. J. Public Health 38, 1532-1538. Rochaix, A. (1924). Compt. Rend. Soc. Biol. 90, 771-772. Rose, A. H. ( 1961 ). “Industrial Microbiology,” 286 pp. Butterworth, London and Washington, D. C. Rose, K. D., and Georgi, C. E. (1941). Proc. Soc. Exptl. Biol. Med. 47, 344-347. Rose, R. E., and Litsky, W. (1965). Appl. Microbiol. 13, 106-108. Rosebury, T., ( 1965). “Microorganisms Indigenous to Man,” 435 pp. McGraw-Hill, New York. Rosenkranz, H. S., Carr, H. S., and Rose, H. M. (1964). Biochem. Biophys. Res. Commun. 17, 196-199. Rosenkranz, H. S., Carr, H. S., and Rose, H. M. (1965). J. Bucteriol. 89, 1354-1369 and 1370-1373. Rothe, W. C. (1948). Cited in “Difco Manual” (see Anonymous, 1953). Rycroft, J. A. (1956). Monthly Bull. Min. Health Lab. Seru. 15, 197-200. Saleh, B. A., Silverman, G. J., and Goldblith, S. A. (1986). Food Technol. 20, 671674. Sandvik, O., and Hauge, S. (1954). Nord. Veterinarmed. 6, 255-268. Saraswat, D. S., Clark, W. S., Jr., and Reinbold, G. W. (1963). J . Milk Food Technol. 26, 114-117. Schaedler, R. W., Dubos, R., and Costello, R. (1965). J. Exptl. Med. 122, 59-66.
288
HARTMAN, REINBOLD, AND SARASWAT
Schafer, W. (1953). Zentr. Buktm‘ol., Parusitenk., ,4bt. I . Orig. 160, 54-62. Schattenfroh, A. (1896). Arch. Hyg. Bakteriol. 27, 231-233. Seelemann, M., and Obiger, G. (1956). Mlkhwissenschaft 11, 98-103 and 134-140. Sharpe, M. E. (1955). J. Appl. Bacteriol. 18, 274-283. Shattock, P. M. F. ( 1962). In “Chemical and Biological Hazards in Food” (J. C. Ayres et al., eds.), pp. 303-319. Iowa State Univ. Press, Ames, Iowa. Sherman, J. M. (1937a). Bacteriol. Reu. 1, 3-97. Sherman, J. M. (193713). 1. Bacteriol. 33, 26-27. Sherman, J. M., Niven, C. F., Jr., and Smiley, K. L. (1943). 1. Bactetiol. 45, 249-263. Sims, W. (1964). J. Appl. Bacteriol. 27, 432-433. Slanetz, L. W., and Bartley, C. H. (1957). J. Bacteriol. 74, 591-595. Slanetz, L. W., Bent, D. F., and Bartley, C. H. (1955). Public Hedth R q t . ( U . S . ) 70, 67-72. Smith, J. F. (1914). I. Pathol. Bacteriol. 19, 122-124. Smith, J. F. (1918). In “Applied Bacteriology” (C. H. Browning, ed.), pp. 141-146. Oxford Univ. Press, London and New York. Smuckler, S. A., and Appleman, M. D. (1965). Appl. Microbwl. 13, 289. Snyder, M. L. (1940). J. Infect. Diseuses 66, 1-16. Snyder, M. L., and Lichstein, H. C. (1940). 1. Infect. Diseases 67, 113-115. Solberg, M., and Proctor, B. E. (1960). Food Technol. 14, 343-346. Splittstoesser, D. F., Wright, R., and Hucker, G. J. (1961). Appl. Microbiol. 9, 303-308. Srere, P. A. (1965). Nature 205, 766-770. Stableforth, A. W., Hulse, E. C., Wilson, C. D., Chodkowski, A., and Stuart, P. ( 1949). Vet. Record 61, 357-362. Stainsby, W. J., and Nicholls, E. E. (1932). J. Lab. Clin. Med. 17, 530-536. Swan, A. (1954). J. Clin. Pathol. 7, 160-163. Szita, J. (1957). Acta Microbiol. Acad. Sci, Hung. 4 , 289-293. Thiercelin, M. E. (1899). Compt. Rend. SOC. Bwl. 51, 269-271. Thomas, J. W., Appleman, M. D., and Tucker, F. L. (1963). Bactetiol. Proc. p. 124. Thomas, J.. Tucker, F. L., and Appleman, M. D. (1965). Bacteriol. Proc. p. 25. Treick, R. W., and Konetzka, W. A. (1954). J. Bacteriol. 88, 1580-1584. Tucker, F. L., Thomas, J. W., Appleman, M. D., and Bils, R. F. (1964). Bacteriol Proc. p. 113. Tucker, F. L., Walper, J. F., Appleman, M. D., and Donohue, J. (1962). J. Bacteriol. 83. 1313-1314. Vera, H. D. ( 1961). Baltimore Biological Laboratory, personal communication. Vera, H. D. (1963). Personal communication. Wahl, R., and Meyer, P. (1957). Ann. Inst. Pasteur 92, 43-55. Walper, J. F., Tucker, F. L., and Appleman, M. D. (1962). Anal. Biochm. 3, 298-301. Wang, W.-L. L., and Dunlop, S. G. (1951). Public Health Rept. ( U . S . ) 66, 1212-1218. Weinberg, E. D. (1953). 1. Bacteriol. 66, 240-242. Weissenbach, R.-J. (1918). Compt. Rend. Sac. B i d . 81, 559-561. White, J. C., and Sherman, J. M. (1944). J. Bacteriol. 48, 262. White, M. F., Kasper, J. A., and Cope, E. J. (1947). 1. Bactmiol. 54, 545. Whitley, 0. R., and Damon, S. R. (1949). Public Health Rept. ( U . S . ) 64, 201-212. Whittenbury, R. (1963). 1. Gen. Microbiol. 32, 375-384. Williams, R. E. O., and Hirch, A. (1950). J. Hyg. 48, 504-524.
ISOLATION AND ENUMERATION OF ENTEROCOCCI
289
Williams, W. L., Broquist, H. P., and Snell, E. E. (1947). J. B i d . Chern. 170, 619630. Wilson, C . D., and Slavin, G . (1950). J . Comp. PathoE. 60, 230-234. Winter, C. E., and Sandholzer, L. A. (1946a). J. Bacterial. 51, 588. Winter, C. E., and Sandholzer, L. A. (1946b). U. S. Fish Wildlife Sew., Fishery LeafEet 201, 1-9. (rev. 1957). Wright, D. E. (1960). J . Gen. Microbio2. 22, 713-725. Zabirowski, H., Huber, D. A., and Rayman, M. M. (1958). AppZ. Microbiol. 6, 97-104.
This Page Intentionally Left Blank
Crystal-Forming Bacteria as Insect Pathogens MARTINH. ROGOFF International Minerals 6. Chemical Corporation, Bioferm Division, Wasco, California
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Historical ....................
C. The Phospholipase Enzymes . . . . . . . . . . . . . . . . . . . 111. Host Susceptibility and the Toxic Factors Produced by Bacillis thuringiensis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Some Industrial Considerations . . . . . . V. Future Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . .......... ..........
291 291 293 294 296 303 305 306 308 312 312
I. Introduction
A. HISTORICAL Insect pathogens have received the attention of bacteriologists and insect pathologists for almost 100 years. Pasteur himself carried out a classic investigation of diseases of the silkworm (Pasteur, 1870). Later, Cheshire and Cheyne (1885) studied foulbrood disease of the honeybee, and in 1902 Ishawata in Japan isolated a spore-forming aerobic bacterium from diseased silkworms. These early studies of insect pathogens were basically directed toward control of epidemic diseases in economically important insects. Ishawata’s spore-former, which he called “Sotto-Bacillen” (“suddencollapse bacillus”), was not subjected to detailed study until 1915, when two other Japanese, Aoki and Chigasaki (1915), reopened the case and looked again at the disease the bacillus produced in silkworms. At about the same time in Thuringia, Germany, Berliner independently isolated a similar organism from diseased larvae of the flour moth. This isolate he called Bacillus thuringiensis (Berliner, 1915). Later, Mattes ( 1927) reisolated the bacillus and described the disease it caused in some detail. These early workers made several important observations whose significance was not recognized in their time. First, Aoki and Chigasaki (1915) observed that only old, well-sporulated cultures of Ishawata’s baciIlus could cause disease of silkworms by feeding; vegetative cultures were inactive. These investigators also noted the extreme rapidity with which 291
292
MARTIN H. ROGOFF
the disease symptoms became apparent, larvae often becoming paralyzed within 1 hour after ingestion of sporulated cultures ( Sotto-Bacillen!), and they suggested that the symptoms represent expression of the action of a toxin. The most characteristic feature of these bacilli was noted by the German investigators. Both Berliner and Mattes observed that the sporulated cells of these cultures contained not only the expected spore, but also a second body, an irregular rhomboid. As the spore developed a spherical granule appeared in the other half of the cell, which became rhomboidal as the spore matured. The inclusion was called a “Restkorper” by Berliner and was felt by Mattes to be a part of the nuclear apparatus of the bacterial cell. These observations passed quietly onto the library shelves, and their relationship to use of these bacteria to control insect populations remained dormant with them for some 20 years. Then a Canadian worker, Hannay, while studying sporulation in Bacillus thuringiensis, resurrected Berliner‘s “Restkorper,” observing that when spores were formed they were invariably accompanied by bodies which appeared to be diamondshaped crystals ( Hannay, 1953). When he observed mature sporangia in his cultures each contained a spore at one end of the cell and a crystal at the other. He suggested that these parasporal bodies or inclusions might be involved in the insect diseases caused by these bacteria. It then remained for another Canadian worker, T. A. Angus, to begin the elucidation of the role of the crystal in the observed pathogenicity of the crystalliferous bacilli for insects. After a series of studies (Angus, 1954, 1956a) he confirmed the work of Aoki and Chigasaki as well as Hannay’s suggestion of relationship between pathogenicity and the crystal. He was able to conclude that the toxic principle associated with the crystalline inclusions of the bacillus (he worked with a strain we would now call a Sotto-type) was not a typical exotoxin but required solubilization for activity and that this could be done by treatment of the crystals with dilute alkali or insect gut juice. He stated (Angus, 1956a) that “paralysis is not caused by the growth of the microorganism in the host tissue,” and herein lies the tale. On ingestion by a susceptible larva of a lepidopterous insect the crystal inclusion, which is protein in nature (Hannay and Fitz-James, 1955), can kill an insect via a characteristic pathology. The usual course of a bacterial infection, involving ingestion of living vegetative cells (or viable spores), reproduction of bacteria in the host tissues, production of invasive factors by the pathogen, and toxin production within the host, does not need to be followed in order to produce a toxemia. Ingestion of toxin formed external to the host can be sufficient to cause death. This ability
CRYSTAL-FORMING BACTERIA AS INSECT PATHOGENS
293
of the crystal alone to kill suddenly put the bacilli on an equal footing with chemical insecticides and opened the door to their use for field control of susceptible insect populations. Lest the preceding sketch lead to a misconception concerning a microbiological “Dark Age” in insect control with microbial pathogens, let me add that some workers were quite active in the period between Mattes and Hannay in evaluating B. thuringiensis as an insect control agent. Husz (1931) carried out field trials with the Mattes strain of B . thuringiensis for control of the European corn borer with good results. Extensions of this work to other insects were made, again with encouraging results. Jacobs (1950) in Great Britain brought out another application of the B. thuringiensis material when he used a commercial preparation (Sporeine) to protect flour against infestation by the flour moth. In this country, Steinhaus used the Mattes strain as a water-based spray to obtain control of alfalfa caterpillar ( Steinhaus, 1951). This study pointed out the advantage of toxin versus infection for microbial insect control. Prior work had attempted control of this insect with polyhedrosis virus. The incubation period of the virus in the host allowed a long period of continued feeding by the insect to take place between spraying and death of the larvae. When the bacillus preparation was used cessation of feeding by the larvae immediately followed ingestion of the sprayed leaves; little loss of foliage was observed. These early studies paved the way for increased field trials and expansion of a new field of microbiological activity. OF CRYSTAL-FORMING BACTERIA B. TAXONOMIC CONSIDERATION
The rapid increase in the number of investigators actively studying crystalliferous bacilli has, of course, resulted in descriptions of these organisms appearing in the literature in some quantity. Proposals as to taxonomic status followed (Angus, 1956b; Delaporte and Bkguin, 1955; Steinhaus and Jerrel, 1954; Toumanoff, 1952), as did some confusion. Since the multiplicity of isolates required some broad taxonomic consideration of the Bacillus thuringiensis group, several investigators initiated studies along taxonomic lines. This particular group of bacilli is quite similar to Bacillus cereus, which is also in actuality a group of closely related organisms and shares with it a common spore antigen. It also shows some cross reactions with B. cereus bacteriophages (Norris, 1963) and was found to have the characteristic B . cereus exosporium (Norris and Burges, 1965). Norris even feels that for each type of B. cereus a corresponding crystal-former exists. However, the ability to produce crystals and cause disease in Lepidoptera, and the stability of the crystal-forming property on the part of these
294
MARTIN H. ROGOFF
bacilli, mandates their consideration as separate species. Three lines of approach have been taken toward classifying these bacteria. Heimpel and Angus (195s) followed the lines laid out in Bergey, departing from B. cereus on the basis of parasporal body formation. These investigators defined two species, one of three varieties and one of two, and a species with a single representative, B. finitimus. The French workers deBarjac and Bonnefoi (1962) proposed a subdivision into six subgroups to which they later added two more (Bonnefoi and deBarjac, 1963), largely on the basis of a study of flagellar antigens of the vegetative cells. Norris (1964) added another serological subgroup and reported a very elegant study of esterase enzyme patterns produced by these bacteria, using a starch gel electrophoresis technique. Norris found striking similarities between the esterase patterns and antigenic subgroupings, supporting the results obtained by the earlier workers. A summary of current taxonomic status in accord with the findings of the aforementioned investigators is given in Table I. The reader is referred to Norris’ paper (1964) for detaiIs of methods. Norris’ feelings about nomenclature of this group were recently presented to an international body convened for this purpose, the “International Symposium for the Identification and Assay of Viruses and Bacillus thuringiensis Used for Insect Control.” He did not believe it possible to be definitive concerning the relationship to B . cereus and suggested that the bacteria be referred to simply as B. thuringiensis, used as a general description applied to any crystal-forming insect pathogen. To this appellation could be added a serotype or loosely a “type” designation added for descriptive purposes. Thus serotype 1 would correspond to the “Berliner-type” of B . thuringiensis and so on. These phrases would, of course, have no nomenclatural significance (Norris and Burges, 1965); this is where the matter now stands.
II. Toxic Materials Produced by the Crystal-Forming Bacilli It may be best in treating this subject to separate the materials formed by the various B. thuringiensis types which elicit a toxic response in susceptible insects into two groups. The first of these includes the true toxins only, these being ( 1 ) the crystalline protein, ( 2 ) a heat-stable, water-soluble exotoxin active against certain Diptera, ( 3 ) a heat-stable, water-soluble dialyzable exotoxin toxic to Lepidoptera on injection but not ingestion, and ( 4 ) a heat-labile, water-soluble exotoxin toxic to only one known host, the larch sawfly (Smirnoff, 1964). The second group of materials would include (1) viable spores, germination of which in the gut may or may not result in tissue invasion and disease symptoms. On germination the spores would, however, give
CRYSTAL-FORMING BACTERIA AS INSECT PATHOGENS
295
rise to vegetative cells, which might then produce (2 ) exoenzymes such as the phospholipases ( lecithinases ) or hyaluronidase. Production of the phospholipases could result in direct symptoms of toxicity as a result of their action on phospholipid-containing tissues. The effects of hyaluroniTABLE I COMPARATIVE CHARACTERISTICS OF CRYSTALLIFEROUS BACTERIA^ Biochemical reactions
__
~-
+ + + + + + + + + + + - - +
+ + + + -
+
+ + + - - + + + - + * + - + - - + + -
+ - - + + + + + - + + - - + + + + + - + a
1
1
Berliner
11 111
2 3
Finitimus Alesti
IV(a)
4A
Sotto
IV(a) Iv(b)
4A 4B
v
5 6
Dendrolimus Kenya Galleriae Entomocidiis
6 7 8 9
Entomocidus Galleriae Morrison Tolwor t h
VI
VI VII VIII IX After Norris and Burges (1965).
B. thuringiensis var. thuringiensis B. finitimus B. thuringiensis var. alesti B . thuringiellsis var. sotto
B. entomocidus var. subtoxicus B. entomocidus var. entomocidus
dase on animal tissues are morphologically similar to the breakdown of insect gut wall in the presence of B . thuringiensis preparations. It is theoretically possible that all of these entities might be present in a given culture of B . thuringiensis in amounts sufficient for their activities to be quantitated. It is, however, an unlikely situation. Toxin production may be influenced by medium, strain characteristics of the culture in question, and stability of the toxic entities themselves. These considerations will be dealt with later. At this juncture we may proceed to
296
MARTIN H. ROGOFF
consider the toxic entities individually in the light of current knowledge of each. A. THE CRYSTALLINE PROTEIN 1 . Physicochemical Nature of the Protein Hannay and Fitz-James ( 1955) carried out electron micrography studies on crystals of B. thuringiensis and found them to be tetragonal and quite regular in shape; very few misformed crystals were encountered. They also observed a periodicity of the crystal surface consisting of successive striae, or parallel rows, of material extending around the corners of the crystal. When they examined effects of alkali in the crystals they found that the crystal protein dispersed by alkali was made up of small globular units. The unit size of these particles was smaller than the width of the parallel rows they had observed on the surface. Thus, it seemed that the crystal might be built not of discrete molecules of the size of the alkali-dispersed units but of larger units susceptible to mild hydrolysis. Later, Norris and Watson (1960) examined crystals of the Alesti-type bacillus, resolved the surface periodicity in two dimensions, and suggested that the ridging previously observed reflected a tetragonal packing of molecules in the crystal. Both groups of authors obtained evidence which made the original contention by Hannay (1953), that the crystals were encased in a shell or membrane, unlikely. It should be pointed out here not only that crystal shape is a function of strain but that crystal size and dimension may even be affected by cultural conditions. Differences between crystal proteins produced by different strains of crystal formers also exist. It would seem best that detailed observations on crystals of one strain be regarded as applying only to the strain under study. Generalizations regarding crystal structure will require further experimental evidence. The protein itself is quite inert but has no unique characteristics, other than its particular crystal form, which are not shared by other protein molecules. Hannay and Fitz-James (1955) reported 17% nitrogen for Berliner-type crystal protein, and Angus found about 1870for Sotto-type (1956b). These proteins were hydrolyzed to yield 17 to 19 amino acid residues, none of which was unusual or present in unusual concentration. A low content of sulfur-containing amino acids seems characteristic. The protein is heat sensitive and when dissolved in dilute alkali absorbs in the ultraviolet region with a maximum at 280mp, normal for absorption by the aromatic amino acids. Inertness of the crystals is reflected in their solubility characteristics in aqueous systems. These are such that the toxicity of dissolved crystal protein to insects is always suspect and loss of activity due to denatur-
CRYSTAL-FORMING BACTERIA AS INSECT PATHOGENS
297
ation can be observed. Unfortunately, quantitation in the biological test systems employed leaves something to be desired, and quantitative aspects of subunit toxicity have not yet been reported with any authority. Apparently the protein is soluble only in relatively strong aqueous alkali. Solution of the protein at neutrality in any solvent system has not been reported to date. Solubility characteristics as reported by various investigators are as follows: ( a ) soluble in 0.01-0.05 N sodium hydroxide (Sottotype; Angus, 1956b); ( b ) insoluble in alkali at pH 11.5 but soluble at pH 10.5 in the presence of thioglycollate ( Alesti-type; Young and FitzJames, 1959); ( c ) soluble at pH 11.8 (Berliner-type; Hannay and FitzJames, 1955); ( d ) soluble at pH 10.0 (Sotto-type; Angus, 1956b); ( e ) soluble at pH 11.0-12.2 (Alesti-type; Fitz-James et d.,1958). These data on differences in the pH levels required for solubilization of the proteins produced by different strains may reflect differences in the proteins themselves as a function of strain. This has been suspected but not yet demonstrated. We have also found, in accord with Young and Fitz-James, that certain compounds permit solubilization of the protein at lower pH values, and we have observed solution of Berliner-type crystals at pH 9.5 in the presence of urea and potassium borohydride. Once in solution the Berliner-type protein was reported to have an isoelectric point between pH 4.9 and 5.2 (Hannay and Fitz-James, 1955). Reduction of the pH of the protein solution to pH 4.0 resulted in precipitation, which could also be brought about by addition of trichloroacetic acid. Precipitation of dissolved crystal protein has also been accomplished by the addition of 2% ammonium sulfate to a solution (Angus, 1956b) and by dialysis of an alkaline solution. The resultant precipitates from the above treatments are, however, all amorphous. Recrystallization of the dissolved crystal protein has yet to be accomplished. To date little work has been completed on characterization of the toxic unit of the crystal protein; the mass of indirect evidence points to some subunit of the crystal protein being responsible for the toxicity to lepidopterous larvae observed. Even the question of homogeneity of the protein in the crystal of a given B. thuringiensis type still has not been definitively answered. Some physicochemical and immunological work on characterization has been carried out. Angus ( 1956b) studied electrophoretic characteristics of dissolved crystal protein of a Sotto-type culture. Electrophoretic mobilities were determined in a 0.1 N Tris buffer system at pH 9.0. He observed only one single fraction, with a slowmoving minor shoulder. We have looked at Berliner-type crystal protein, partially solubilized (pH 9.5) with a paper ionophoresis system in 0.6% acetic acid. Such preparations show five differentiable ninhydrin-positive zones. Immunochemical studies carried out by Monro (1961) on a highly
298
MARTIN H. ROGOFF
purified material derived from a Berliner-type strain indicated at least two, and perhaps more, distinct components in the crystal protein. Krywienczyk and Angus (1960) studied crystal protein from Berliner-, Sotto-, and Entomocidus-types and reported that each protein contained several components, one of which was common to all three preparations. On the basis of these studies it seems probable that the crystal contains more than one component, albeit a single entity may be responsible for toxicity. This makes the last-mentioned work most interesting, since it is a direct indication of minor differences in the proteins of crystals produced by different strains of the bacillus. One does not readily rid himself, however, of the gnawing feeling that methods of solubilization of the crystal are reflected in the components observed. This area definitely requires a good deal of very careful work. 2. Crystals and Cell Physiology Again we enter an area where observations to date have not delved deeply into the subcellular level, into the metabolic systems responsible for synthesis of the crystal protein and deposition of the crystal. What has been observed can be readily summarized. When a crystalliferous bacillus is cultivated in a medium containing a limiting nutrient, the entire culture usually sporulates shortly after exhaustion of the limiting component. Glucose serves admirably. If the culture is examined microscopically at about the point of nutrient limitation it will be seen that simultaneous with the appearance of spores in the cells the crystalline parasporal bodies appear. On the basis of their electron-microscopic observations, Norris and Watson (1960) forwarded the hypothesis that the crystals are laid down in close proximity to nuclear elements within the cell. Following sporulation the sporangia undergo autolysis and both mature spores and crystals are released into the growth menstruum. With certain strains under appropriate conditions high protease activity in the lysates may be observed and dissolution of crystals by the proteases can be observed. Cells of crystal-forming bacilli apparently can rely on protein turnover and amino acid pools to synthesize the crystal protein independent of external nutrients. One can readily demonstrate endotrophic sporulation with many of these cultures. If a well-grown culture in the granular stage is harvested, washed free of medium, and resuspended in distilled water (specific compounds required for sporulation may have to be added), the washed cells will then form both spores and crystals in the endotrophic system (Monro, 1961; Singer et d.,1966). Toxicity of such crystals for insects does not appear to differ from that of crystals formed in the presence of external nutrients. The normal synchrony of crystal and
CRYSTAL-FORMING BACTERIA AS INSECT PATHOGENS
299
spore formation in the cell can be upset by rigorous environmental conditions. Smirnoff ( 1963a) demonstrated that on prolonged cultivation at a low temperature crystal formation would take place without concurrent spore formation. He also demonstrated that by inclusion of urea in culture media ( a t concentrations proper to elicit the response in a given strain) crystal formation could be inhibited without, apparently, affecting sporulation (Smirnoff,1963b). Use of mutagenic agents on the bacilli can also result in loss of crystal-forming ability ( Fitz-James and Young, 1959). Monro’s immunological studies ( 1961) demonstrated that crystal protein antigens were absent from vegetative cells of his strain and in fact were not demonstrable at all until optically visible crystal structure was apparent in sporulating cells. None of the above precludes definitive relationships between the sporulation process and crystal formation in the cells. The visible evidence is difficult to deny. Apparently the crystal protein-synthesizing systems are repressed until the onset of sporulation. It seems logical to assume that sporulation is related to crystal formation at least insofar as the regulation systems are involved. There is no reason to suspect that a normal cell constituent such as the crystal protein would not be under the control of all the internal regulatory mechanisms normally operative in the cell. Crystal-forming ability is a very stable characteristic in these bacteria. Cultures under prolonged laboratory culture show no tendency to lose their crystal-forming ability. Some of these, such as the Sotto-type and the Mattes strain, have been cultured outside of their respective hosts for over 50 years. The nature of the crystals formed is also a constant strain characteristic as evidenced by differences in the insect susceptibility spectra for the toxins of given strains of the bacilli, as well as the fact that there are variations in toxin potency between closely related strains. This latter characteristic according to Heimpel and Angus (1958) is sufficiently stable to have warranted their use of relative toxicity of crystal from different strains for silkworm larvae as a taxonomic criterion for establishing species and variety status. We are probably dealing here with a situation which is evidenced in the case of other protein or polypeptide materials produced by bacilli which have physiological activity ( bacitracin ) , Such materials are not single molecular entities but are rather families of closely related compounds which differ in specific activity in accord with molecular structure. This will probably prove true for the crystal proteins. Crystal shape is fairly constant, but crystal size is not and can be affected by growth conditions. It is probably safe to assume that availability of any individual amino acid may directly affect crystal protein synthesis. The amount of crystal protein produced by the cell is, interest-
300
MARTIN H. ROGOFF
ingly enough, relatively huge considering that none of the material is transported outside of the cell. Fitz-James et al. (1958) analyzed an Alesti-type culture and found that 32% of the dry weight of the sporulated cell mass could be accounted for as crystal protein. 3. Crystal Protein and Toxic Action
What does one observe when a silkworm larva is fed crystals of Bacillus thuringiensis? Grossly, within a few minutes, cessation of feeding is seen. This is followed by a general paralysis of the larva within 1 to 7 hours; the insect is usually dead within 24 hours. Closer investigation would reveal paralysis of the midgut shortly after ingestion of crystals, probably within 20 to 30 minutes in the case of the silkworm. This was demonstrated experimentally by Heimpel and Angus ( 1959), who followed by X-ray observation the movement of barium sulfate mixed with food and toxin through the larval gut of a number of susceptible Lepidoptera. In the silkworm, which is very susceptible to crystal toxin, a rapid increase in pH of the blood (hernolymph) can be observed some 10 minutes after crystal ingestion, even prior to observable gut paralysis. Following gut paralysis the contents of the midgut begin to leak into the hemocele; the alkalinity of the hemolymph continues to rise until an increase of 1.0 to 1.5 pH units has been reached. This amount of increase in the pH level is sufficient to bring on the total paralysis which eventually results in the death of the insect. Death may be the result of starvation, although this has been questioned. The paralysis effect can be artificially induced by injection of sufficient alkaline buffer into the hemocele to raise its pH to 8.0 (Norris, 1963). Disorganization of the larval midgut epithelium can be seen in stained sections made within 45 minutes of crystal ingestion (Heimpel, 1963). This very interesting pathological picture raises two questions concerning the action of the protein crystals. First, what is the actual toxic entity derived from the crystal, and, second, what is its site (or mode) of action? A beginning toward answering these questions was made by Angus’ now classic study which originally demonstrated the role of crystals in the infective process (Angus, 1954). He took a washed aqueous suspension of Sotto-type spores, crystals, and debris and divided it into two parts. One part was diluted with water. The second part was treated with alkali until the crystals dissolved, at which point the spores and debris were centrifuged out. The pellet was resuspended in water. A portion of the supernatant was dialyzed against water and another portion heated to 70°C. for 30 minutes. The five materials thus obtained were then tested against silkworm larvae by feeding and direct injection into the hemocele. Angus’ observations of the test are given in Table 11.
CRYSTAL-FORMING BACTERIA AS INSECX PATHOGENS
301
TABLE I1 EFFECTOF FEEDING AND INJECTINGLARVAE OF Bomb~pmori WITH FRACTIONS OF AN ALKALI-TREATED CULTURE OF Bacillus sottoa Culture Original culture (spores and crystals) Alkali-treated culture Spores alone Supernatant Supernatant dialyzed Supernatant heated at 70°C. for 30 min. a
Fed
Iniected
Paralysis followed by septicemia No effect
Septicemia, no paralysis
Paralysis, no septicemia Paralysis, no septicemia No effect
No effect No effect No effect
Septicemia, no paralysis
After Angus (1954).
The results of this experiment indicated that the paralysis which foL lowed ingestion of crystals by susceptible larvae was due to a heat-labile, nondialyzable toxic substance, toxic on ingestion but not on injection into the hemocele. Crystal-free spores, on the other hand, could cause the septicemia following injection into the hemocele, but were not able to invade the blood when ingested in the absence of crystal. Since 1954 a number of studies have attempted to answer the questions concerning the nature of the toxin and its action. I will attempt to summarize these into a representative current viewpoint. First as to active form of the toxin. It had been proposed (Heimpel and Angus, 1958) that the toxin had to be in solution for expression of its action. This correlates well with the spectrum of susceptible hosts which, in the main, consists of insects with a high midgut pH. In general, larvae of those Lepidoptera with a gut pH of 9.0 to 10.5 are highly susceptible to the crystals; solubiIity of the crystal protein in the presence of reducing substances at these pH values is quite probable. Having noted the presence of enzymes in silkworm foregut which broke the crystal protein down to amino acids, Heimpel and Angus (1959) suggested that the crystal might be a protoxin. The actual toxic entity would then be a smaller, enzymatically formed subunit of the protein. Angus (1964) has recently obtained evidence to support the hypothesis of solubilizing action of gut juice on the crystal. First he demonstrated that clarified silkworm gut juice did not contain any toxic principle (certain other investigators had run into trouble with another insect, imported cabbageworm, whose gut juice contained a toxic substance). He then incubated a clean preparation of Sotto-type crystals in the gut juice. The filtered reaction mixture was then fed or injected into silkworm larvae. Those larvae which received solubilized crystals by oral ingestion rapidly exhibited the expected paralytic symptoms; larvae which had received an injection into the hemocele
302
MARTIN H. HOGOFF
of 10 times the amount of toxin used to elicit paralysis by oral ingestion showed no toxic symptoms at all. The experiments indicated not only that incubation with gut juice yielded a soluble toxin active by the normal oral route, but that toxin action was specific to the gut since the general paralysis must then be due to something other than the toxin itself. The experiments allow no conclusion concerning gut juice enzyme action on crystals. I t was felt that leakage of alkaline midgut contents into the hemocele might account for the paralysis, and the proposed mode of action of the toxin on midgut epithelium was supported by the experiments. Histological evidence obtained from studies of several susceptible insects indicated that after toxin ingestion cells of the midgut become free from one another and also from the basement membrane of the gut. This can be noted only 45 minutes after toxin ingestion by silkworm larvae. Heimpel (1963) suggested that the cell-cementing substances might be the site of toxin action. Data obtained by Fast and Angus (1965), however, show the effects of the toxin to be a bit more complex. These investigators studied the effect of Sotto-type toxin on permeability of the gut wall of silkworm larvae by means of radioactive tracers. They reported that transfer of UC14-glucose into the hemocele was inhibited by toxin, while transfer of sodium-C14-carbonate was accelerated. The data were felt to support the proposal that the general paralysis as a symptom of toxemia was due to changes in hemolymph pH induced by altered gut wall permeability. The authors also pointed out, on the basis of differences in the level of radioactivity derived from carbonate compared to that derived from glucose in the hemolymph 90 minutes after intoxication, that free diffusion through the gut wall was still not obtained (as proposed by Heimpel and Angus, 1960), although visible damage is evident. They prefer, in the light of their data, an alternative hypothesis also forwarded by Heimpel and Angus (1960). This hypothesis stated that the toxin in some way inhibits the active transport mechanisms in the gut cells, which would explain the increased carbonate and decreased glucose levels observed and would also be consistent with the histological evidence of cell breakdown in the late stages of toxemia. The dependence of spore effectiveness on crystal action was also indicated by Angus’ (1954) early study. Spores from which all crystal had been removed by alkali leaching produced no pathology in the insect when taken by the oral route. On direct injection into the hemocele septicemia rapidly set in. Therefore, without concomitant crystal toxin action the spore or its derived vegetative cell cannot pass through the gut wall; it retains its integrity.
CRYSTAL-FORMING BACTERIA AS INSECX PATHOGENS
303
B. THE WATER-SOLUBLE EXOTOXINS The crystalline protein toxin, owing to its historical role, visibility, assayability, and the fact that it is the basis of commercial exploitation of Bacillus thuringiensis, has received more attention than the other metabolic products of the crystal-forming bacilli. The attempts on the part of various investigators to study mode of crystal action, host spectrum of B. thuringiensis, and characteristics of commercial microbial insecticides have led to the discovery of several as yet unidentified materials in the culture fluids of crystal-forming bacilli which show toxicity for certain insects. These are commonly referred to as the group of watersoluble exotoxins. Our present knowledge of these compounds is even less definitive than our knowledge of crystal protein in regard to both structure and function. Indeed in certain cases even host spectra are not well understood. In short, the waters in this area are still muddy and the problem of presenting current knowledge is not simple. In this area, then, it would seem best to be brief and objective, simply leading the reader into the subject and allowing his curiosity to prompt him to seek greater detail. The relationships between soluble exotoxins and the characteristic pathology observed when a susceptible host larva ingests a B. thuringiensis preparation containing crystals are not known. In fact, the chances are good that none exists. The bulk of the reports of susceptible host spectra of the soluble toxins shows them to be mainly effective against other groups than the Lepidoptera, the main group of insects susceptible to the protein crystal. It is possible to sift through the reports of effects of soluble exotoxins and studies on their nature and recognize three types of material which may be produced by specific strains of crystal-forming bacilli under appropriate conditions. These are: ( 1 ) a heat-stable exotoxin, toxic when ingested by larvae of certain flies (Muscidue),which prevents completion of development to the adult in the pupal stage (Briggs, 1960); ( 2 ) a heat-stable exotoxin, toxic on injection into the hemocele which produces a mortality in certain Lepidoptera ( McConnell and Richards, 1959); ( 3 ) a heat-stable exotoxin, toxic on ingestion to several species of Larch Sawfly larvae (Smirnoff, 1964). The first of these exotoxins, distinguished by its heat stability and characteristic pathology, has thus far received the most attention and been most clearly defined. The material has often been referred to as “fly toxin” or “fly factor,” although this author prefers the appelation “heat-stable exotoxin.” It is produced in the growing bacterial cultures prior to the onset of sporulation and remains in the supernatant liquid of sporulated cultures (Cantwell et al., 1964; Mechalas and Beyer, 1963). The toxin
304
MARTIN H. ROGOFF
kills larvae and/or pupae of some Diptera and Lepidoptera and acts primarily at some point in the pupation process, manifesting its action in arrested pupal development. Even when adult flies emerge from pupae derived from larvae dosed with the exotoxin they are malformed in any of several ways, A characteristic malformation of the terminal end of the abdomen is often observed. One cannot read the literature pertinent to this toxin without getting the feeling that we may be dealing with more than one molecular entity, albeit the modes of action may be quite similar. Without delving into certain inconsistencies between the work of various investigators concerning toxicities for various insects and routes of administration of toxin for efficacy, let the following discussion suffice to clarify this feeling. Two groups of investigators, Cantwell and his co-workers and deBarjac and his group in France, are currently investigating and attempting to purify and identify the heat-stable exotoxin. The French group has isolated a nucleotide which on hydrolysis gave adenine, ribose, and phosphate residues and showed the same toxic properties toward insects as the thermostable exotoxin. This material, as one would expect, has a at 257 mp and characteristic ultraviolet absorption spectrum with a,,A a hmin at 228-229 mp ( deBarjac and Dedonder, 1965). The material separated by Cantwell (Cantwell et al., 1964) is reported to have a ,,A at 270 mp, certainly not characteristic for a nucleotide. Their compound also produces the proper pathology in insects. The conclusion must thus be drawn at present that more than one substance capable of producing the observed pathology is produced by crystal-forming bacilli and can be separated by appropriate means. Continued interest in this exotoxin stems from the fact that it may well prove to be an excellent means of commercial fly control. It can be fed to a number of domestic animals, pass through the digestive tract, and be deposited in the feces. Toxin which has passed through the gut remains in an active form in sufficient concentration to prevent emergence of adult flies which normally would develop from eggs deposited in the fecal mass. Clarification of the structure and function of the exotoxin would be most valuable at this juncture. The next toxin to be considered is that reported by McConnell and Richards (1959). This was derived from a Berliner-type culture and assayed by injection into wax moth larvae. A number of other larvae were also found susceptible (roaches, flies, mosquitos) but all on injection; when given orally the toxin was ineffective. Studies by other investigators demonstrated susceptibility of most of these test insects to the exotoxin on ingestion at proper dosage levels (Briggs, 1960; Burgerjon and deBarjac, 1960; Hall and Arakawa, 1959). Thus, precise details con-
CRYSTAL-FORMING BACTERIA AS INSECT PATHOGENS
305
cerning mode of administration of the toxin may well have indicated a difference in toxins which might not actually exist. Current opinion is almost unanimous that the "McConnell-Richards factor" and the heatstable exotoxin are identical. Lastly, we may consider the toxin reported by Smirnoff (1964) to be definitely different from the heat-stable factor as indicated by its heat lability. This toxin was obtained from a sporulated commercial preparation of a Berliner-type bacillus. It was found by Smirnoff to be highly toxic to 18 species of larch sawfly larvae. No further studies on this toxin have been reported at this writing. ENZYMES C. THEPHOSPHOLIPASE Phospholipase enzymes, in particular lecithinases, which are produced by various crystal-forming and non-crystal-forming spore-forming bacilli, have been thought to play a role in the ability of these bacteria to infect and kill insects. There have been several reports that test insects such as the wax moth (Lepidoptera) or larch sawfly (Hymenoptera) die after lecithinase is injected into or ingested by larvae (Bonnefoi and Bbguin, 1959; Heimpel, 1955; Kushner and Heimpel, 1957; Toumanoff, 1953). The lecithinases catalyze reactions which cleave the lecithins at various places on the molecule dependent on the specific site of action of the lecithinase involved. There are four lecithinases: A, C, and D plus a lysolecithinase. Since histological examination shows damage to midgut cells of bacillus-infested larvae and since the damaged tissues are known to contain phospholipids, some investigators felt that lecithinase or other phospholipase activity might well be involved in the observed pathology. Conceivably, lecithinase-catalyzed reactions could lead to disruption of the gut wall by destruction of phospholipid cementing material and thus permit invasion of the hemocele by spores or vegetative cells of the pathogen. Typical bacteremias would result. It is really doubtful that this is the case. There are several considerations which lead the writer to relegate phospholipase activity to a very minor role in the pathology produced by crystal-forming bacillus preparations. First, no one has succeeded in demonstrating that any relationship exists between phospholipase production by a given strain of B. thuringiensis and its effectiveness as a pathogen for lepidopterous larvae. Second, phospholipases active in the pathology would have to be produced by vegetative cells of the pathogen. Activity of the crystal protein is already seen to be sufficient to cause all observed symptoms, so one must suppose that in the case of the normally susceptible host insect, midgut epithelium disruption is due to the action of the crystal protein, requiring and probably receiving no assistance from vegetative cell
306
MARTIN H. ROGOFF
products; the damage caused by the crystal protein is massive. Third, the pH of the midgut of most of the susceptible larvae is much higher than the pH values (6.8 to 7.4) which have been reported as optimum for the lecithinases (Bonnefoi and Bkguin, 1959). The larch sawfly (and other insects of its type) with a gut pH more nearly in the neutral range ( p H 7.0 to 7.8) may well prove to be more susceptible to lecithinase action. Lastly, even in those lepidopterous larvae in which spore germination is known to occur and active multiplication of vegetative cells to be initiated, the gut contents have been shown to remain in the gut and not to leak into the hemocele (Heimpel, 1963). Thus, even here no lecithinase activity of any consequence is seen. In short, we should be aware of the potential activity of these enzymes in infestation without attributing to them a greater role than they actually play. In Table 111 there is a summary list of the various toxic agents mentioned in the preceding sections. TABLE I11 TOXIC AGENTSPR0DUCE.D BY CHYSTALLIFEHOUS BACILLIWHICH TO THE OBSERVED PATHOLOGY IN INSECTS MAY CONTRIBUTE Component Crystal protein Spore Heat-stable soluble exotoxin McConnell-Richards factor (may be identical with above exotoxin ) Heat-liable soluble exotoxin Exoenzymes ( phospholipases, hyaluronidase ) Vegetative bacterial cells
111.
Action or Pathological Symptom Midgnt paralysis in susceptible Lepidoptera Septicemia following penetration to hemocele Interference with pupal development; death Mortality in various insect larvae
Mortality in larch sawfly larvae May be partially responsible for destruction of gut wall epithelium May produce toxins and enzymes above, plus other metabolic products on growth in host gut and hemocele
Host Susceptibility and the Toxic Factors Produced by Bacillus thuringiensis
As in any biological system, there are no sharp breaks in the responses of various host insects to the ingestion of a preparation of crystal-forming bacilli. The entire range of reaction from exquisite sensitivity to complete resistance is encountered. Lepidoptera can be broadly broken down into two major groups, those insects susceptible to crystal action alone and those showing little or no susceptibility. Susceptibility is characterized by the gut paralysis previously described, Crystal-susceptible insects can be further divided into two subgroups according to whether or not general paralysis follows gut paralysis.
CRYSTAL-FORMING BACTERIA AS INSECT PATHOGENS
307
The general paralysis is a symptom of toxemia in a restricted group of insects whose larvae are characterized by a comparatively high gut pH. After crystal ingestion changes in the permeability of the gut wall can be observed (Fast and Angus, 1965); the high gut pH then falls owing to leakage of gut contents into the hemocele. The pH of the hemolymph concurrently rises as a result of the same phenomenon. The general paralysis takes place as the hemocele pH rises; the same effect, as mentioned before, can be demonstrated by simple injection of buffer into the hemocele raising the p H to 8.0 (Norris, 1963). Thus, it is seen that the general paralysis is only a secondary effect of toxin action, in reality an indicator of degree of gut wall damage. Those Lepidoptera displaying the general paralysis symptom are called type I insects by Heimpel (1963; Heimpel and Angus, 1958), who classifies insects into four groups on the basis of their responses to the crystal protein. Representatives of this extreme sensitivity group are the silkworm, the Chinese oak silkworm, and the tobacco and tomato hornworms. The type I1 insects, which represent the majority of the Lepidoptera according to Heimpel ( 1963), include all of the crystal-susceptible insects other than type I. When these larvae are fed B. thuringiensis preparations there is no leakage of gut contents, no rise in p H of the hemolymph, and no general paralysis. After ingestion of the crystal, the onset of gut paralysis is observed in a few hours, feeding ceases, and a slow decrease in pH of the gut occurs. A similar decrease in pH of the gut is normally seen when healthy larvae are allowed to enter a state of starvation. In type I1 insects, apparently, death is the result of starvation, toxin action being insufficient physiologically to cause the secondary symptoms observed in the type I host; it must be recognized, however, that even in the absence of the secondary symptoms toxin action is the prime factor in the death of the insect. Norris (1963) broadens this concept a little by considering that when the gut pH falls, it falls to a low enough level to allow initiation of germination of ingested spores and rapid multiplication of vegetative cells. He feels that growth of bacteria in the larval gut results in breakdown of the gut wall and subsequent penetration of the bacteria into the hemocele. In the course of this type of infection, general septicemia develops and the insect, when it succumbs, is packed with spores and crystals. He does, however, raise the question of just how much of a role crystals vs. toxins of the growing cell (lecithinases) play in the attack on the larval gut wall. The proportional role of each may well prove to be a function of the particular host species. Heimpel and Angus’ (1958) third group of insects is probably represented by only one lepidopterous host, Anagasta kuhniella. In this case,
308
MARTIN H. ROGOFF
neither spores nor crystals alone killed all the insects tested (spores alone gave 44% mortality of test larvae, crystals gave 12% mortality). However, feeding a whole culture with a spore-to-crystal ratio of 1:l caused 80-90oJo mortality in the test population. This appeared to be a synergistic effect hitherto unreported. Heimpel now feels that this unique reaction may be due to the action of the aforementioned McConnellRichards toxin complex which is reported as a soluble component found in the culture fluid. This material, if a true toxic entity dried with spores and crystals, might well be responsible for the response of Anagmta. Other investigations ( Burgerjon and Yamvrias, 1959; Krywienczyk and Angus, 1960) tend to support Heimpel’s hypothesis. The fourth group of insects are the resistant or nonsusceptible Lepidoptera. This group would include such species as Agrotis, Peridroma, Euxoa, and Mamestra. This fourth classification is an addition to the scheme by the French workers in the field (Burgerjon and deBarjac, 1960). A summary of this spectrum of response to B. thuringiensis preparations is given in Table IV. It is somewhat difficult to summarize the heterogeneous information presented here. One could say that we are dealing with a complex biological activity of some unique organic compounds, plus the activity of a living entity, the B. thuringiensis cell itself. The crystalline protein toxin is the prime factor in the effects, although its relative efficacy remains a function of the host insect. However, since all components of the system can play a role in the host’s death, it is desirable that the B. thuringiensis preparations used as toxicants contain crystal, spore, and all soluble toxins, characterized ur not.
IV. Some Industrial Considerations This essay is primarily intended to explore what is currently known about the characteristics of Bacillus thuringiensis that make it a good insect pathogen, but since it is being produced commercially and is currently being used to control insects in the field, certain aspects of commercial interest should be considered. The materials presented to an insect in the field are considerably different from the pure crystal or soluble toxin preparations used in laboratory studies. In the final formulation stage a microbial insecticide based on B. thuringiensis is a very complex material indeed. In the fermentation sufficient nutrient is provided to allow development of the fermentor population through the life cycle to complete sporulation. At sporulation, of course, crystal formation is concomitant. Media currently used in commercial processes for B. thwingiensis preparations are largely
CRYSTAL-FORMING BAClTXIA AS INSECT PATHOGENS
TABLE IV ANGUS' (1958) CLASSIFICATION OF HOSTS BASED SUSCEPTIBILITY TO PREPARATIONS OF CRYSTAL-FORMING BACILLI
SUMMARY ON
309
OF HEIMPELAND
Inseot type
Primary toxic agent
I
Crystal
Silkworm, Chinese oak silkworm Tobacco homworm Tomato hornworm
11
Crystal
Bulk of susceptible
Representative hosts
Lepidoptera
Symptoms 1. Rapid midgut paralysis with lowering of pH of midgut contents. Rapid midgut disorganization 2. Leakage of midgut contents into hemocele with rise in hemocele pH 3. General paralysis in 1-7 hours 1. Midgut paralysis 2. Slow drop in midgut pH (probably due to starvation) 3. No histopathological changes in midgut epithelium; no general paralysis, no rise in hemolymph
PH 4. Germination of
111
Soluble toxins
Anagasta kuhniella
IV
-
Leptinotarsa decemlineata Euxoa segetum Lycophotia sancia
spores and multiplication of vegetative bacilli in the larval gut with later hemolymph invasion 1. No gut or general paralysis 2. Death in 2-4 days Not susceptible
based on rich, insoluble, complex natural materials such as cottonseed, soybean, and fish meals (Megna, 1963; Drake and Smythe, 1963); one particular method uses a semisolid bran medium, which after development of the culture is simply dried and ground to final product (Mechalas, 1963). In contrast, laboratory media are often very simple defined formulations (Singer et al., 1966). By the time the fermentor population has sporulated it has converted a portion of the supplied nutrients into vegetative cells, spores, crystal protein, soluble toxins, exoenzymes, and other metabolic excretion products. Synchronous growth in the fermentation is not absolutely necessary, but nearly simultaneous sporulation is desirable from the viewpoint of obtaining a uniform product. The commercial products are prepared as dusts, wettable powders, or sprayable liquid formulations. Therefore, depending on methods of recovery for specific products, they may contain the insoluble materials including spores, crystals, cell debris, and
310
MARTIN H. ROGOFF
residual medium ingredients. In addition to the insoluble materials, any soluble materials from the culture fluids remaining in the final product are included. To the harvested fermentation product are then added the diluents, vehicles, stickers, and chemical protectants as the requirements of the individual product may dictate to ensure field performance. With all of these in the final product it is readily seen that the prospective insect target ingests a very complex material. Regardless of its complexity the final preparation must be attractive to its intended host larvae. I t must always be borne in mind that the microbial insecticides are not contact poisons and must be consumed by the larvae. At present operations have been carried out on a commercial scale in France, Germany, Czechoslovakia, and the United States. These products have appeared under a variety of trade names: Thuricide, Larvatrol, Agritol, Sporeine, and Bakthane. At present control of the insects listed in Table V is either a commercial reality or at the development stage of TABLE V INSECT PESTSCuRnENTLY CONTROLLED WITH Bacillus thuringiensis PREPAIUTIONS Insect pest
Species designation
Cabbage looper
Trichoplusia ni
Imported cabbageworm
Pieris rapae
Tobacco hornworm Tomato hornworm
Protoparce sexta Protoparce quinquemaculata Colias eurytheme Porthetriu dispar Paleacrita vemata Alsophila pometria Malacasoma sp. Platyptilim pusillodactyla Ceramidia sp., Platynota sp., Opsiphanes sp.
Plants affected Broccoli, flower, potato, Broccoli,
cabbage, caulicelery, lettuce, melon cabbage, cadi-
flower
Alfalfa caterpillar Gypsy moth Spring cankerworm Fall cankerworm Tent caterpillar( s ) Plume moth Defoliating and peelscarring caterpillars
Tobacco Tomato Alfalfa Forest trees Forest and shade trees Forest and shade trees Forest and shade trees Artichoke Banana
extensive field testing. Results of laboratory testing indicate over 120 susceptible insects to date. These include insects which are pests of agricultural crops, forests, orchards, vineyards, and stored foods. They also represent the entire range of susceptibility. Considerations of interest to commercial developers of B . thiiringiensis preparations concern assay, effective dose rates for field use, development of resistance on the part of the host, and safety. Of these, the last is of extreme importance, but it is generally accepted today that prepa-
CRYSTAL-FORMING BACTERIA AS INSECT PATHOGENS
311
rations of B. thuringiensis are very safe for vertebrates. In addition, its specscity among the insects themselves is quite striking. Other than those Lepidoptera which are target insects for the preparations only some Diptera and sawflies appear susceptible. Bees and earthworms are unaffected, and no predatory and parasitic insects have yet been found which are susceptible and would be killed in fields in which microbial insecticides were being used. In fact, the bulk of even the lepidopterous fauna will probably not suffer from commercial use of these insecticides. The question of resistance has not yet been raised in a practical way, no cases of resistance to crystal toxin having been reported at this juncture. More probably it is precisely in the instances of those insects which have developed resistance to certain chemical insecticides that B . thuringiensis preparations will play a major role in control. It is probably safe to say that assay or rather standardization of not only commercial but laboratory preparations is not as yet satisfactory. Bioassay is the rule for quantitation of toxin; in the United States viable spore count is used as a label specification in commercial products. Spore count is basically inadequate, since it is not necessarily an indicator of toxicity to insects of the crystal protein, the agent which determines the efficiency of short term insect kill of the preparation (Burges, 1964). Differential staining to allow numerical counting of spores and crystals is an aid to gross determination of the presence of crystal protein but, again, not an actual measure of toxicity. For toxicity measurements bioassay against susceptible larvae is used, and estimates of lethal doses based on larval mortality are used, An example of such a technique is that of Splittstoerser and McEwen (1961) in which the larvae feed on an agar mixture containing the preparation under test. Burgerjon (1962) has suggested a rapid assay based on measurements of leaf area coated with test preparation ingested by a susceptible test insect. In any case, differences in susceptibilities of various test insects to the toxins, as well as variation in the toxins produced by the bacteria themselves complicate the situation. Standardization is severely handicapped. Certain European countries favor bioassay with standardized insects and a selected bacterial preparation as a standard for comparison of test material. Discussion of standardization at an international symposium held in London in 1964 has initiated efforts to establish international standard preparations to be used by those requesting them for standardization applications. More definitive assay methods are required but are not yet available, although we may assume they are being actively sought. Last to be considered is dose rate, with its important ramification of cost of insect control by means of B. thuringiensis preparations. At present the field application is considerably higher than the absolute amount
312
MARTIN H. ROGOFF
of toxin required to kill the larvae present on the vegetation. The absolute dosage required for kill is roughly equivalent to that of a chemical insecticide such as D.D.T.; approximately 0.5 pg. of Sotto-type whole dried culture is sufficient to kill 1 gm. of a highly susceptible larva such as a silkworm. It is possible to administer lethal dose rates economically as evidenced by current economic control in the field. However, increases in both unit toxicity of the commercial preparations and efficiency of delivery to the prospective host are under intensive scrutiny.
V. Future Considerations We have, in the crystal-forming bacilli, a potential for an entire new concept in insect control, A wide spectrum of specific, safe, effective, and economical control agents are available to us almost as quickly as our research and development capabilities can uncover them. The crystalforming bacilli, which can be regarded as true toxicants in view of their potent crystal protein toxin, have an advantage over many other pathogens whose dependence on septicemia for kill makes the expression of their effects slow. Inhibition of feeding by a susceptible insect larva soon after ingestion of crystal protein results in considerable control of crop spoilage. Utilization of the concept of endemic or epidemic disease in insect populations cannot as yet give this effect. Future investigations in this area will range from the practical aspects of process improvement, better formulations, improved application methods, and compatibility with more conventional control methods, to the more fundamental studies of mode of action and nature of the crystalline protein toxin. Since the position of the crystal-forming bacilli in the field of biological insect control may be likened to that of penicillin among antibiotics, we would hope that pursuance of the fundamental studies, to include the search for new types of Bacillus thuringiensis and the investigation of the precise nature of the toxin, will soon make available a whole range of specific insecticides with all of their desirable characteristics intact.
REFERENCES Angus, T. A. (1954). Nature 173, 545-546. Angus, T. A. (1956a). Can. j . Microbwl. 2, 122-131. Angus, T. A. (1956b). Can. 3. Microbiol. 2, 416-426. Angus, T. A. (1964). 1. Insect Pathol. 6, 254-257. Aoki, K., and Chigasaki, Y. (1915). Mett. Med. Fuk. Kais. Uniu., Tokyo 13, 419-440. Berliner, E. (1915). 2. Angew. Entomol. 2, 29-56. Bonnefoi, A,, and Be@, S. (1959). Entomophuga 4, 193-199. Bonnefoi, A., and deBarjac, H. (1963). Entomophugu 8, 223-229. Briggs, J. D. (1960). J. Insect Pathol. 2, 418-432.
CRYSTAL-FORMING BACERIA AS INSEm PATHOGENS
313
Burgerjon, A. (1962). Ann. Epiphyties 13, 59-72. Burgerjon, A., and deBarjac, H. (1960). Compt. Rend. 251, 911-912. Burgerjon, A., and Yamvrias, C. (1959). Compt. Rend. 249, 2871-2872. Burges, H. D. (1964). World Crops 2, 8. Cantwell, G. E., Heimpel, A. M., and Thompson, M. H. (1964). J. Insect Pathol. 6, 466-480. Cheshire, F. R., and Cheyne, W. W. (1885). J. Roy. Microscop. SOC. [21 5, Pt. 2, 581-601. deBarjac, H., and Bonnefoi, A. (1962). Entomophaga 7, 5-31. deBajac, H., and Dedonder, R. (1965). Compt. Rend. 160, 7050-7053. Delaporte, B., and Bkguin, S. (1955). Ann. Inst. Posteur 89, 632-643. Drake, B. D., and Smythe, C. V. (1963). U. S. Patent 3,087,865. Fast, P. G., and Angus, T. A. (1965). I . Invert. Pathol. 7 , 29-32. Fitz-James, P. C., and Young, I. E. (1959). J. Bact&E. 78, 743-754. Fitz-James, P. C., Toumanoff, C., and Young, I. E. (1958). Can. J. Microbiol. 4, 385392. Hall, I. M., and Arakawa, K. Y. (1959). J. Insect Pathol. 1, 351-355. Hannay, C. L. (1953). Nature 172, 1004. Hannay, C. L., and Fitz-James, P. C. (1955). Can. J. Microbiol. 1, 694-710. Heimpel, A. M. (1955). Can. I. 2001.33, 311-326. Heimpel, A. M. (1963). Advan. Chem. Ser. 41, 64-74. Heimpel, A. M., and Angus, T. A. (1958). Can. I. Microbiol. 4, 531-541. Heimpel, A. M., and A n g u s , T. A. (1959). J. Insect Pathol. 1, 152-170. Heimpel, A. M., and Angus, T. A. (1960). Bacteriol. Rev. 24, 266-288. Husz, B. (1931). Sci. Rept. Intern. Corn Borer Invest. 4, 22-23. Jacobs, S. E. (1950). Proc. SOC. AppZ. Bacteriol. 13, 83. Krywienczyk, J., and Angus, T. A. (1960). J. Insect Pathol. 2, 411-417. Kushner, D. J., and Heimpel, A. M. (1957). Can. I . Microbwl. 3, 547-551. McConnell, E., and Richards, A. G. (1959). Can. I. Microbiol. 5, 161-168. Mattes, 0. (1927). Sitzber. Ges. Befoerder. Ges. Ndurw. Marburg 62, 381-417. Mechalas, B. J. (1963). U. S. Patent 3,086,922. Mechalas, B. J., and Beyer, 0. (1963). Develop. Ind. Microbiol. 4, 142-147. Megna, J. C. (1963). U. S. Patent 3,073,749. Monro, R. E. (1961). J. Biophys. Biochem. Cytol. 11, 321-331. Norris, J. R. (1963). Sci. Progr. (London) 51, 188-197. Norris, J. R. (1964). 1. Appl. Bacterwl. 27, 439-447. Norris, J. R., and Burges, H. D. (1965). Entomophagu 10, 41-47. Norris, J. R., and Watson, D. H. (1960). J. Gen. Microbiol. 22, 744-749. Pasteur, L. (1870). ‘‘Etudes sur la maladie des vers B soie,” Vol. I, 322 pp. and Vol. 11, 327 pp. Fanthier-Vellois, Paris. Singer, S., Goodman, N. S., and Rogoff, M. H. (1966). Ann. N . Y. Acad. Sci. (in press). Smimoff, W. A.( 1963a). J. Insect Pathol. 5, 242-250. Smimoff, W. A. (1963b). I . Insect Pathol. 5, 389-392. Smirnoff, W. A. (1964). Entomophagu, Mem. 2, 249-254. Splittstoerser, C. M., and McEwen, F. L. (1961). J. Insect Pathol. 3, 391-398. Steinhaus, E. A. (1951). Hilgardiu 20, 359-381. Steinhaus, E. A., and Jerrel, E. A. (1954). Hilgardiu 23, 1-23. Toumanoff, C. (1952). Ann. Inst. Pusteur 82, 512-516. Toumanoff, C. (1953). Ann. Inst. Pusteur 85, 90-98. Young, I. E., and Fitz-James, P. C. (1959). J. Biophys. Bwchem. Cytol. 6, 483-498.
This Page Intentionally Left Blank
Mycotoxins in Feeds and Foods EMANUEL BORKER,NINO F. INSALATA, COLETTE P. LEVI,AND JOHN S. WITZEMAN General Foods Technical Center, White Plains, New York I. Introduction ..................................... 11. Aflatoxin ........................................ A. Investigation of Hepatotoxic Feeds . . . . . . . . . . . . . . B. Toxin-Producing Organisms .................... C. Physical and Chemical Properties . . . . . . . . . . . . . . . D. Biological Effects ............................. E. Biochemical Effects ........................... F. Test Methods ................................ C. Agricultural Commodities Affected . . . . . . . . . . . . . . H. Governmental Actions ......................... 111. Other Mycotoxicoses .............................. A. Responsible Fungi ............................ B. Identified Toxins ............................. C. Pathological Implications for Man . . . . . . . . . . . . . . . IV. Control Mechanisms .............................. A. Prevention of Mold Growth .................... B. Removal of Contaminated Products .............. C. Inactivation of Toxin .......................... References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
315 316 316 318 319 322 329 330 335 335 336 336 338 342 343 343 345 345 346
1. Introduction Mycotoxins are metabolites of molds which can produce pathological or undesirable physiological responses in man and other warm-blooded animals. These metabolites are occasionally ingested as contaminants in foods subjected to mold attack and can be responsible for disease outbreaks. Some of these metabolites under proper conditions of use and dosage are valuable therapeutic agents, i.e., the antibiotics. The toxic and therapeutic effects of the metabolites produced by Claviceps purpurea have been known since Biblical times (Barger, 1931). Despite this early knowledge of ergotism, little scientific attention has been focused on other mycotoxicoses until recent years. Forgacs (1962) in a review article accurately described mycotoxicoses as the neglected diseases. Before 1960, the bulk of the literature on the subject appeared in veterinary journals. Except for ergotism and reports on certain fungal diseases in Russia, little information was available on mycotoxicoses in man. The recent discovery of an extremely potent carcinogen, aflatoxin, produced by Aspergillus flavus has dramatically changed the status of mycotoxin research. A symposium in March, 1964, at the Massachusetts Institute of Technology with the participation of WHO, FAO, UNICEF, 315
316
BORKER, INSALATA,
LEVI,
AND WITZEMAN
and the Pan American Health Organization brought together many investigators of mycotoxicoses. The proceedings of the symposium have been published ( Wogan, 1965). Since then many scientific societies, incIuding The American Chemical Society, The American Oil Chemists Society, The American Society of Microbiology, The American Public Health Association, The Association of Official Analytical Chemists, and The Institute of Food Technologists, have presented symposia on mycotoxins.
II. Aflatoxin A. INVESTIGATION OF HEPATOTOXIC FEEDS Concurrent outbreaks of disease in poultry and fish in several different parts of the world during early 1960 focused much attention on the significance of diet contamination in the induction of cancer in animals. The first epidemic was uncovered as a result of regulatory seizure of a large shipment of diseased Idaho hatchery-raised trout at a California border station. Many of the fish were found to have hepatomas. This incident led to national studies that demonstrated the prevalence of hepatic carcinoma in this commercially important species of fish (Ashley, 1962). Thirty years ago Haddow and Blake (1933) reported sporadic cases of liver tumors in trout. However, within the past 10 years the disease has assumed epidemiological importance in the United States and Europe (Ghittino and Ceretto, 1962). The high prevalence of trout hepatomas occurred with the wide-scale use of dry pelleted commercial feeds (Dollar and Katz, 1962). The vast majority of trout raised in the United States come from federal, state, or commercial fish hatcheries. Consequently, there is considerable opportunity for exposure to commercially available feeds. A survey of federal and state hatcheries illustrated the presence of the disease in the majority of trout-yielding areas in the country (Hueper and Payne, 1961). Some hatcheries demonstrated hepatomas in 50% to 75% of the fish population (Wood and Larson, 1961). Originally, the constituents of the dry pelleted trout feeds were dried fish meal, cottonseed meal, meat scraps, and dried milk. However, owing to increased demand for meat in livestock feeds, fish meal and cottonseed meal have become the predominant components of the trout feed. Before the use of dry pelleted feeds the fish received a wet diet composed primarily of sheep, swine, and cattIe viscera combined with ground beef and horse meat. These diets of unprocessed products were not known to produce hepatomas ( Shimkin and Kraybill, 1964). Recently, Halver ( 1962, 1965b; Halver et al., 1962) has shown that hepatomas in trout can be caused by aflatoxin. LaRoche et al. (1962) have reported on hepatoma-
MYCOTOXINS IN FEEDS AND FOODS
317
inducing agents in trout diets. Aflatoxin has been found in the cottonseed meal portion of the diet (Engebrecht et al., 1965). Another outbreak of a similar disease producing hepatomas which killed in excess of 100,000 turkey poults occurred in England in 1960. The malady, which became known as the “turkey X disease,” was characterized by acute hepatic necrosis associated with generalized bile duct proliferation. Coincidentally, outbreaks of a similar condition in ducklings and young pheasants were reported, Blount (1961) examined the incidence of the disease in turkey poults, and Asplin and Carnaghan (1961) examined its incidence in ducklings and chickens in a number of areas. They showed that the common factor in these outbreaks was Brazilian groundnut meal in the rations. Intensive investigations were carried out by a number of laboratories for the presence of known toxic agents in samples of the meal, but they were unable to demonstrate any specifre known poisonous agent. The toxic agent could not be attributed to contamination from pesticides or plant alkaloids. However, the toxic peanut meal fed at 20% levels in the diets of rats produced hepatic carcinoma in weanling rats within 30 weeks (Lancaster et al., 1961). Reports from Europe were relatively slow concerning outbreaks of this disease due to toxic groundnut meal, although large quantities of a similar meal were being imported into many European countries. The first case of the disease on the European continent was diagnosed by Carnaghan in chickens in Spain (Carnaghan and Allcroft, 1962). Two outbreaks of the disease in ducks were reported, one in Austria by Kohler and Swaboda (1962), and the other in Hungary by Derzsy et al. ( 1961). The low incidence of this disease in Europe may have been due to the fact that soya meal is generally fed to young animals while groundnut meal is generally used in the diets of mature animals (Allcroft and Carnaghan, 1962). In the same article the authors concluded that some cases of groundnut poisoning in animals had been incorrectly diagnosed before 1960. Paget (1954) observed exudative hepatitis and other lesions in laboratory animals such as guinea pigs when peanut meal was used in the diet. At the same time as the outbreak of turkey X disease in England, a similar disease of ducklings was noted in Kenya and reported to Central Veterinary Laboratory in Weybridge, England. The ducklings’ feed ration contained a groundnut meal grown and processed in East Africa. When the meal was withdrawn, animal deaths ceased. When it was discovered that the peanut meal was contaminated by mold, an important clue was provided in defining the etiology of the toxin. This became the first indication that the problem was not confined solely to Brazilian groundnut meal (Allcroft and Carnaghan, 1962). By growing some of the fungal species, Sargeant et al. ( 1961b) identified the toxin-producing
318
BORKER,
INSALATA, LEVI,
AND WITZEMAN
fungus as Aspergillus flavus. The British investigators therefore designated aflatoxin as the name for the toxic components. Four of these components have been separated and identified. B. TOXIN-PRODUCING ORGANISMS
Aspergillus flavus is a common saprophytic mold from many foodstuffs and soils (Raper and Fennell, 1965). It grows rapidly but requires somewhat more moisture than most other molds. At tropical temperatures (30°C.) it will grow at 80 to 85% relative humidity (Spensley, 1963). Austwick and Ayerst (1963) have shown that the optimum temperature is 33°C. In the initial investigations of the toxigenic properties of A. flavus the fungus was cultured on sterilized, nontoxic groundnut kernels (Codner et al., 1963). It has been shown that animal products, rice, and pork appear to support toxin production better than peanuts. Soybeans or soya protein show weak production of the toxin. Other protein sources such as wheat, oats, millet, egg solids, and skim milk powder appear to support good growth and yield of toxin (Shimkin and Kraybill, 1964). They reported that a medium proposed by Brian et al. (1961), utilizing glucose, ammonium nitrate, zinc sulfate, potassium dihydrogen phosphate, calcium chloride, and other salts, supported good fungal growth. In studies reported by Wogan et al. (1963) most toxin production was carried out in liquid media by growing mold spores from a stock culture on Czapek's agar for 5 to 9 days at 30°C. In this work two methods were used, either stationary growth or submerged growth in shake flasks. These methods may not initially give a high yield of toxin compared to similar methods using infected peanuts. Yeast extract or corn steep liquor added in concentrations of 1 gm. per liter of medium is superior for the yield of toxin. Since protein or protein hydrolysates will also cause marked increase in toxin production, Wogan (1963; Wogan et al., 1963) studied the influence of various amino acids on growth. Alanine, lysine, methionine, tyrosine, aspartic acids, histidine, arginine, glysine, isoleucine, hydroxyproline, ornithine, and phenylalanine have no effect. However, glutamic acid and proline stimulate production of one of the toxic metabolites, whereas leucine, threonine, and tyrosine have a less pronounced effect. Tryptophan stimulates production of another toxic metabolite. In agreement with these findings, Davis et al. (1966) found that composition of the medium and the environment surrounding the medium greatly influences the extent to which A. flavus synthesizes aflatoxin. They reported that the production of aflatoxin is highly aerobic. Comparisons of standing cultures with submerged cultures show that the highest yields of toxin are obtained under high aeration. When yeast extract is added to the medium and cultures are incubated in containers having
MYCOTOXINS IN FEEDS AND FOODS
319
large surface area-to-volume ratios, 3 days is sufficient for surface mycelial mats to produce aflatoxin. Aflatoxin is also produced by other members of the A. fEavus-oryzae group (Codner et ah, 1963) and by Pencillium puberulum (Hodges et al., 1964). De Vogel et al. (1965) described a rapid screening test for aflatoxin producers of the A. flavus-oryzae group using ultraviolet examination of the culture plate. Mateles and Adye (1965) describe conditions for aflatoxin production on a synthetic medium in submerged culture. Glucose, sucrose, or fructose were the preferred carbon source, cassaminic acid the preferred nitrogen source. Zinc was required at a minimum of 0.4 mg. per liter. In addition to the aflatoxins, A. flavus can produce other toxic substances on food materials. For example, Wilson and Wilson (1964) have reported recently on a substance causing tremors in mice, produced by the mold grown on cereal grains. Studies on the production, isolation, and properties of other toxins produced by A. flavus (such as @-propionic acid, aspergillic acid, etc.) have been reported by Bush et aZ. (1945, 1951), White and Hill (1943), Tilden et al. (1961), and Wynston and Tilden (1963). A report of fatalities to cats and rabbits from contaminated peas did not identify the toxin (Kulik, 1954). Wilson ( 1966b) has prepared a review on these other toxins.
C. PHYSICAL AND CHEMICAL PROPERTIES Chromatographic purification techniques, such as column chromatography and countercurrent distribution, have been used to separate crude extracts of aflatoxin-contaminated nuts into a number of fluorescent compounds-from 6 to 15, and 20 on synthetic media-depending upon the fungal strain used, the substrate, and the environmental conditions (Sargeant et al., 1963; Hartley et al., 1963). Of these fluorescent components, four closely related chemical compounds have been definitely correlated with toxic activity (de Iongh et al., 1962; Nesbitt et aZ., 1962). The generally accepted nomenclature follows the pattern of thin-layer chromatography separation and the particular fluorescence of each compound when viewed under long-wave-length UV light. Thus, the four compounds are known as aflatoxin B1 and Bz showing blue fluorescence, and aflatoxin GI and Gz showing green fluorescence, and the numbers following the capital letters are in decreasing order of mobility in thinlayer chromatographic separation. The values of absolute Rf's are, of course, not strictly reproducible, but in a separation obtained with methanol-chloroform as developing solvent, the reported Rf's of B1 and B2 are 0.56 and 0.53 and those for G1 and GP are 0.48 and 0.46 respectively.
320
BORKER,
INSALATA, LEVI,
AND WITZEMAN
Preliminary work on extraction and purification of the toxin progressed quite rapidly, with many workers participating, including Armbrecht et al. ( 1963) and van der Zijden et al. (1962). Data obtained for the melting point, optical rotation, molecular weight (determined by mass spectroscopy), and elementary analysis of the aflatoxins are summarized in Table I. These, together with UV, IR, and NMR studies of the isolated compounds as well as their reduction products and comparison with NMR
I
I1
I11
TV
V VI Aflatoxins Bz and GZ are the dihydro derivatives of the corresponding aflatoxin of the “1”series, where the double bond in the terminal furane ring is reduced ( Hartley et al., 1963). The toxicity of aflatoxins Bz and Gz is lower than that of aflatoxins B1 and G1indicating that the reduction of the double bond of the dihydro-furane ring decreases the toxic activity (Carnaghan et al., 1963; Hartley et al., 1963).
TABLE I CHEMICALAND PHYSICAL PROPERTIES OF AFLATOXIN~
Matoxin Bl
Molecular formula C17H120f3
Cl,Hl,O,
C17H1!207
G2
a
C17H1407
Molecular weight 312
314
328
330
Ultraviolet absorption EtOH
Infrared absorption Fluorescence CHCl, emission A,, at 365 mF cm-1 excitation mp
Melting point, "C.
Specific rotation a,23
my
&
268-269 (dl
-559
223 265 362
25,600 13,400 21,800
1760 1684 1632 1598 1562
425
286-289
-492
222 265 362
19,600 9,200 14,700
1760 1685 1625 1600
425
(a)
244-246
(a)
237-240 (d)
-556
-473
~
m
m
8
0
E 5
2 r
E
E 4
243 257 264 362
11,500 9,900 10,000 16,100
1760 1695 1630 1595
450
21 245 265 365
28,000 12,900 11,200 19,300
1760 1694 1627 1597
450
Data compiled from Asao et al. (1963, 1965), Chang et al. (1963), and Wogan (1966).
it
3 r 0 v1
322
BORKER, INSALATA, LEVI, AND WITZEMAN
spectra of synthetic coumarins, indicated all of the compounds to have a furano coumarin structure. It was concluded that aflatoxin B1 contains a carbonyl group in a five-membered ring cross conjugated with a, 0 unsaturated lactone function. Furthermore, NMR studies showed that aflatoxin B1 and GI are very similar, the only difference being that aflatoxin B1 has a five-membered ring with no oxygen in the ring, whereas aflatoxin GI has a six-membered ring with an oxygen in the ring. Formulas I and TI show the structures for aflatoxin B1 proposed by Asao et al. (1963), and formulas I11 and IV show alternative structures proposed by van der Merwe (1963). Later studies by Asao et al. (1965) confirmed structures I and 11. Aflatoxins Bg and G2 are the dihydro derivatives of the corresponding aflatoxin of the “1”series, where the double bond in the terminal furane ring is reduced (Hartley et al., 1963). The toxicity of aflatoxins BQ and G2 is lower than that of aflatoxins B1 and GI indicating that the reduction of the double bond of the dihydro-furane ring decreases the toxic activity (Carnaghan et al., 1963; Hartley et al., 1963). Alkaline hydrolysis opens the lactone ring; partial recyclization after acidification has been reported by de Iongh (1962). Catalytic reduction of affatoxin Bl produces affatoxin Bz by the uptake of 1mole of hydrogen (van Dorp et al., 1963),and it is completely reduced after the addition of 3 moles of hydrogen. The fragmentation of aflatoxin B1 by ozonolysis results in levulinic, succinic, malonic, and glutaric acids. The additive product formed by aflatoxin B1 with hydroxyl groups catalyzed by strong acids (Andrellos and Reid, 1964) is the basis of a confirmatory test for aflatoxin B1. The adducts formed with formic acidthionyl chloride, acetic acid-thionyl chloride, and trifluoroacetic acid show a changed and characteristic chromatographic pattern with little change in the fluorescence characteristics.
D. BIOLOGICAL EFFECTS Aflatoxin is an extremely potent hepatotoxic and hepatocarcinogenic agent. When compared to other known hepatocarcinogens such as p-dimethylaminoazobenzene, carbon tetrachloride, 0-propiolactone, other quinoline and lactone analogs, and even certain antibiotics (Brown et al., 1961; Eschenbrenner and Miller, 1946; Fitzhugh and Nelson, 1948; Kawamata et al., 1958, 1959), the potent toxicity and carcinogenicity of aflatoxin can be readily recognized, as shown in Table 11. Butler and Barnes (1964) conclude that aflatoxin is one of the most active carcinogenic substances known, based on the appearance of hepatic tumors in rats with dosage levels as low as Spg. per day. This dose is considerably lower than the dose which is required by other carcinogens such as p-dimethylaminoazobenzene (0.75 pg. daily) and dimethylnitrosamine ( 9 pg. daily). With
323
MYCOTOXINS IN FEEDS AND FOODS
TABLE I1 LACTONES AND RELATED SUBSTANCES WHICH HAVEBEENBIOASSAYED FOR CARCINOGENIC ACTIVITY IN THE RATTHROUGH REPEATED SUBCUTANEOUS INJECTIONS~ ~~~
Dose per injection (oil)
Tumors Time o f Treatment First per observation, time, wk. tumor, wk. survivors wk.
Compound injected Control ( arachis oil )
0.5 ml.
Control ( arachis oil)
0.5 ml.
61
0-propiolactone
1.0nig.
44
54
0/6
54
-
0/3
107
29
10/10
44
-
Patulin
0.2 mg.
61
58
4/4
69
Penicillic acid Penicillin G (sodium salt)
1.0 mg.
64
48
4/4
67
2.0 mg.
46
59
1/4
100
4-Hex-2enolactone
2.0 mg.
64
79
2/4
102
4-Hex-4enolactone
1.0 mg.
58
63
3/5
99
a-Angelica
2.0 mg.
61
-
0/5
100
lactone y-Butyrolactone
2.0 mg.
61
-
0/5
100
S-2-CarboxyethylL-cysteine
0.5 mg.
52
87
1/1
87
Aflatoxin B or G (fed)
0.006 mg.
28
-
5/6
26
a
Taken from Dickens and Jones ( 1961) and Lancaster et al. ( 1961).
most species liver lesions are accompanied by hemorrhage and growth retardation. The mature animals of most species are better able to detoxify or inactivate aflatoxin. According to Newberne et al. (1964; Newberne and Carlton, 1963) the order of susceptibility is as follows: ducklings, guinea pigs, rabbits, rats, and mice. Shimkin and Kraybill (1964) offer this order of toxicity in poultry: ducklings, turkeys, and chickens; while for larger farm animals, the order is swine, cattle, horses, and sheep. Specific observations on a variety of animals have been reported. 1 . Laboratory Animals
Rats It appears that rats are somewhat resistant to aflatoxin. They are able to survive short-term feeding experiments with a diet containing as much
324
BORKER, INSALATA, LEVI, AND WITZEMAN
as 50% toxic groundnut meal (Butler and Barnes, 1964). However, prolonged feedings of a 207. toxic meal diet reduces the growth rate of these animals as well as the food intake, and prolonged feeding beyond a few weeks’ time gives rise to further liver lesions and hepatomas (Lancaster et al., 1961). Schoental (1961) fed the toxic guinea pig diet which presumably contained aflatoxin and obtained liver damage in rats. Salmon (Salmon and Newberne, 1963; Salmon et al., 1963) produced hepatomas in 98 of 113 rats by feeding commercial peanut meal as the sole protein source, or by supplementing the peanut meal with beef or casein. Butler and Barnes (1964) have demonstrated the effects of aflatoxin on pregnant rats. Their work shows that pregnancy increases the susceptibility of the animal to aflatoxin, although resulting litter weights were normal and no teratogenic effects were observed. However, when high doses were given halfway through pregnancy the litter was stunted and some animals died. The mothers also exhibited pronounced liver changes.
Mice According to Allcroft et al. (1961; Allcroft and Carnaghan, 1962, 1963a,b), mice are resistant in some degree to short-term feedings. Platonow (1964) has shown that continued feedings of toxic groundnut meal to mice for at least 3 months has no deleterious effects. Levels of aflatoxins B1 and G1 in the diet were 4.5 p.p.m. and of aflatoxins B2 and G2, 0.6 p.p.m. No anorexia or loss in body weight occurred. In addition, no mortalities nor pathology of internal organs were observed.
Guinea Pigs Exudative hepatitis in guinea pigs fed contaminated groundnuts was found by Patterson et al. (1962). These animals were more susceptible than rats. Butler and Barnes (1964) determined that it was necessary to reduce the diet to 10% and 5% toxic meal for the animals to survive for a few months. On feeding 20% toxic meal, they found that all animals succumbed in 14 to 28 days. In addition to overt weight loss, postmortem examination showed ascites, pleural effusions, extensive edema of the abdominal wall, and pulmonary and adrenal hemorrhages. 2. Farm Animals
Cattle Calves from 1 to 6 months of age are highly susceptible to aflatoxin. They become more tolerant to aflatoxin with increased age. Long-term feeding studies have shown that 3- to 4-year-old heifers become clinically
MYCOTOXINS IN FEEDS AND FOODS
325
affected after continuous feedings on concentrated rations containing 20% of a strongly toxic groundnut meal, whereas 8- to 10-year-old cows under the same conditions show no adverse clinical effects. In older cows, no effects on conception or gestation have been noted. Calves exhibit cirrhosis and pallor of the liver associated with ascites and edema of the mesentery. Histologically the bovine liver shows one distinct lesion which does not occur in other species, namely, chronic centrilobular endophlebitis of the central hepatic vein ( veno-occlusive disease) ( Allcroft, 1965; Allcroft and Carnaghan, 1963a; Allcroft and Lewis, 1963b). It is interesting to note that Loosmore and Markson (1961) find the disease in cattle is indistinguishable from ragwort poisoning. Not only are the symptoms and gross morbid anatomy the same for both conditions, but the tetrad of hepatic lesions is the same, including the veno-occlusive lesion. Allcroft and Carnaghan (1963a, b ) demonstrated a toxin in milk from cows fed contaminated feeds. Although the pathology was the same as that of the aflatoxins, de Iongh (1965; de Iongh et al., 1964) showed that the milk toxin was another compound. Van der Linde et al. (1965) found that this toxin appeared in milk 12 to 24 hours after aflatoxin-containing diets were fed. Swine The most susceptible large farm animals appear to be pigs from 3 to 12 weeks of age. Of the mature swine, pregnant sows are the most commonly affected. Pigs are the only species to show generalized jaundice. The liver is discolored from white to bright yellow, and subcutaneous hemorrhages occur ( Harding et al., 1963). Strong circumstancial evidence implicates aflatoxin poisoning in a disease of pigs in Morocco in 1945 wherein these pigs were fed a diet containing groundnut meal. There was heavy mortality in young pigs, and those that survived the acute phase were found to have 100% incidence of hepatic carcinoma on postmortem examination ( Ninard and Hintermann, 1946).
Sheep Sheep are quite resistant to aflatoxin. When 3-month-old sheep are fed rations containing 20% strongly toxic peanut meal for 3 years they fail to demonstrate any obvious clinical effects other than a slight growth retardation. No liver damage was observed after 2 years on this ration (Allcroft and Carnaghan, 1963a)b; Allcroft and Lewis, 1963b). No field outbreaks have been confirmed. Allcroft and her colleagues (1966) investigated the metabolism of aflatoxin in sheep. Toxic compounds with chromatographic behavior identical to those of the cow “milk toxin” were found in the liver, kidneys, and urine of the sacrificed sheep. Butler and Clifford (1966) have shown
326
BORKER,
INSALATA, LEVI,
AND WITZEMAN
the presence of this component in the livers of rats given aflatoxin €31 orally or intraperitoneally. Allcroft suggests that compounds referred to as the “milk toxin” be designated aflatoxin M, since it appears in the organs and fluids of animals ingesting aflatoxin. 3. Poultry
Avian livers do not develop fibrosis to the extent seen in mammals. After 6 days of toxic peanut meal at the level of 10% in the diet, degenerative changes appear in the liver parenchymal cells. There is extensive proliferation of the bile duct epithelium. The kidneys usually show multiple and diffuse hemorrhages (Shimkin and Kraybill, 1964). Similar effects of ragwort poisoning in fowl have also been reported by J. G. Campbell (1956). Turkeys Blount (1961) has shown that postmortem findings in poults consist of profuse enteritis, congestion and swelling of the kidneys, and, on occasion, hemorrhagic involvement of the pancreas. Histopathologically, the renal changes indicate a glomerular nephritis. This is the only species exhibiting definite renal lesions. Siller and Ostler ( 1961) have described bile duct proliferation. Acute cases in turkeys showed diffuse degeneration of parenchymal cells with severe hepatic hemorrhages ( Carnaghan and Allcroft, 1962). Chickens Chickens are comparatively resistant to aflatoxin, and, although mortality is low, chicks killed at regular intervals when fed 15% toxic groundnut meal exhibit striking hepatic changes (Allcroft and Carnaghan, 1963a). In a feeding study by Asplin and Carnaghan (1961), the small number of chickens which died in the first 3 weeks of the feeding experiment had pale livers which were firm in texture; the kidneys were pale and enlarged. In birds sacrificed during the following 3 months, the liver and kidney changes were similar but more marked. In those killed in later periods, these organs were normal in texture but discrete white pinpoint lesions were consistently found in the liver. Early histological findings showed retrogressive and degenerative parenchymal changes in the liver. This persisted until the birds were between 3 and 4 months of age, when lymphoid hyperplasia became evident in those areas where degenerative cells were still active. After four months the multiple areas of lymphoid hyperplasia did not appear to increase in size or number. In addition, diffuse areas of degeneration of the acinar cells occurred in the pancreas. Carnaghan et al. (1965) confirmed the high tolerance of Rhode Island
MYCOTOXINS IN FEEDS AND FOODS
327
Red chicks to the effects of aflatoxin. When the chicks were fed continuously a ration with 15% of a highly toxic meal there was only one death and the main effect was retardation of growth rate. At 4 weeks, there was a significant difference between mean body weights of test chicks and controls. After 8 weeks controls weighed almost 45% more than the test chicks. The relative liver weights were much greater in the test group, although the liver weights for the two groups were not significantly different throughout the experiment. There was increased fat deposition in the livers of the test group initially. After 3 weeks the lipid content of the liver fell sharply, associated with a period of regeneration. After 7-8 weeks’ feeding, there was little difference in liver fat content between the two groups.
Ducklings Ducklings are very susceptible to the toxin, and histopathological hepatic lesions develop rapidly. Day-old ducklings have succumbed within 24 hours to doses as low as 2 pg. per kilogram. Within 3 to 4 days of feeding toxic meal, proliferation of bile duct epithelial cells is clearly visible; this is marked by 7 days. The pathology is characterized by diffuse renal hemorrhages and petechiae present in the pancreas. In older ducklings, the liver becomes more cirrhotic and eventually progresses to the stage of nodular hyperplasia. Ducklings over 3 weeks of age, particularly white-breast breeds, frequently develop severe subcutaneous hemorrhage in the legs and feet ( Allcroft and Carnaghan, 1963a; Asplin and Carnaghan, 1961; Wogan, 1965). The distinct acute onset of this disease in ducklings is a characteristic that contributes to the value of this species as a diagnostic aid in the bioassay for aflatoxin. 4. Other Animals
Dogs Holding (1964) has reported that postmortem examination of a dog that died with acute hemorrhagic gastroenteritis, fever, and jaundice revealed a friable, bright yellow liver and hemorrhages in many other organs. Aflatoxin was demonstrated in the food, which contained groundnut meal. Histological findings were compatible with aflatoxicosis. Bailey and Groth (1959) showed that an epizootic of hepatitis in dogs was due to the dual infection of maize with A. fEavus and P. rubrum. Rainbow Trout Halver (1965b) reports that after feeding of crude aflatoxin extracts to rainbow trout histopathological examination revealed liver lesions similar
328
BORKER,
INSALATA, LEVI,
AND WITZEMAN
to those observed in ducklings, consisting of massive hemorrhagic necrosis of the liver parenchyma with little or no bile duct proliferation. Based on these experiments the LD,o in a 10-day test with a 50 gm. rainbow trout is 1 to 3 mg. of crude aflatoxin per kilogram of body weight, which would represent 0.5 to 1 mg. of aflatoxins B1 and GI per kilogram of body weight. Considerable liver damage could be observed when 0.2 mg. of crystalline aflatoxin per kilogram of body weight was administered. Long-term feeding studies using crystalline aflatoxins revealed a high incidence of primary liver cell carcinoma when trout were fed only parts per billion of the compounds for 6 months to 1 year.
Monkeys Lesions of the liver similar to those generated in ducklings have been produced in monkeys by Tulpule et al. ( 1964) and others (Anonymous, 1964~).Pure aflatoxin (60% B1 and 40% GI) in doses of up to 250 pg. per kilogram of body weight, which were toxic to ducklings, guinea pigs, and rats, produced no ill effects in monkeys after 20 days. Higher doses were therefore given. One group of monkeys received 0.5 mg. per kilogram daily for 18 days, followed by 1 mg. per kilogram daily thereafter. Another group received 1 mg. per kilogram daily throughout the investigation. The latter dosage was highly toxic, though the effects were not seen until 2 weeks had elapsed. At this time changes in the liver were noted on biopsy, consisting of round cells collecting in the portal tracts and vacuolation of the parenchymal cells. During the third and fourth weeks after administration, the monkeys developed anorexia and apathy; the urine became highly colored and the animals died in coma. Examination of the liver and biochemical tests indicated impaired liver function. The serum albumin level and the albumin-globulin ratio were reduced. The liver lesions resembled those seen in biliary cirrhosis. 5. Man
Except for studies with human cell cultures (Legator and Withrow, 1964; Legator et al., 1965) there is no information on the effects of aflatoxin in man, There is much speculation about possible relationships between the importance of moldy corn in African native diets (Quass, 1959-1960), the high incidence of liver cancer in Africans ( Berman, 1951; Higginson and Oettle, 1960; Oettle, 1956), and aflatoxicosis and other mycotoxicoses. Despite the lack of any damage to man directly traceable to aflatoxin, the malignant liver tumors found in other species have created considerable concern to many governmental and regulatory agencies (Anonymous, 1964b).
MYCOTOXINS I N FEEDS AND FOODS
329
E. BIOCHEMICAL EFFECTS Many studies are being pursued currently in an attempt to understand the mode of action and biosynthesis of the aflatoxins. Physiological changes produced by aflatoxin on different warm-blooded animals were described by Philps (1964). They include an increase in serum alkaline phosphatase, sometimes followed by a decrease, in monkeys, swine, and calves; and a decrease of vitamin A in swine and calves. Decreased vitamin A levels in animals fed corn inoculated with nontoxic species of A. fEaous have also been reported. Carnaghan et al. (1965) studied the effect of feeding toxic groundnut to chickens. Their experiments indicate only minor differences in DNA values and a smaller RNA content in the liver of animals fed with the toxic meal; the RNA/DNA ratio found in the liver of chicks fed toxic meal was lower, which would suggest hepatic functional changes. They note that since there exists a relation between the RNA/DNA ratio and the rate of cytoplasmic protein synthesis, this would indicate that protein synthesis is impaired in the animals that were fed the toxic ration. They also note reduced vitamin A. No significant change was found for serum and liver alkaline phosphatase; these values were erratic. Smith (1963) showed that in vitro incorporation of C14-labeled leucine into protein of rat and duckling liver slices is inhibited by low concentrations of purified aflatoxin B1. Aflatoxins Bz, G1 and G:!produce identical effects. Shank and Wogan (1964) studied the in vivo incorporation of leucine into rat liver proteins. After a single sublethal oral dose of aflatoxin B1 the toxin immediately suppressed and then increased amino acid incorporation (4.76 mg. toxin/kg. body weight). They found that, in animals fed aflatoxins, the incorporation of C14 from labeled glucose and leucine was reduced in liver lipid and increased in liver protein, Their studies also showed decreased liver glycogen and increased liver lipids in ducklings and rats. A mixture of aflatoxins B1 and G1 fed to monkeys produced increased liver fat and decreased protein in liver and serum. The effect of the toxin on the synthesis of specific proteins such as liver tryptophan pyrrolase induction produced by hydrocortisone and by tryptophan in rats was studied by Wogan and Friedman (1965). LeBreton et al. (1964) reported studies on the effect of aflatoxin B1 on the gestation of rats on the development of hypertrophy and on the synthesis of RNA and DNA. It is concluded that there is an inhibition of DNA replication and increased synthesis and consequently increased mitosis in regeneration tissues. Wogan (1965) and Shank and Wogan (1965) usd C14-labeled compounds to study the metabolic fate of aflatoxin B1. The compound,
330
BORKER, INSALATA,
LEVI,
AND WITZEMAN
labeled either in the methoxy group or in the ring, was administered to rats intraperitoneally. In a study of distribution and excretion, the liver was the only tissue that retained a significant amount ( 6 to 9 % ) . The ring-labeled compound was mainly excreted in feces and urine, with the liver retaining approximately 8y0. These results indicate that the ring structures are metabolically stable and that a major quantity is excreted via the bile. This finding agrees with studies of Falk et al. (1965), who reported increasing fluorescence in the bile of rats fed aflatoxin B1 intravenously, reaching a maximum in 20 to 30 minutes, then decreasing to normal levels in 120 minutes. About 60% of the total fluorescence was found in the bile, the rest in urine. Seven fluorescent spots were detected by paper chromatography, one probably unchanged aflatoxin, the others probably conjugated metabolites of aflatoxin. The metabolic function of the liver remained unchanged. The metabolites are not yet identified. Adye and Mateles (1964) believe that the structure of aflatoxin suggests an aromatic biosynthetic pathway and studied the incorporation of various labeled precursors into resting cells respiring on synthetic media. They reported that the incorporation of phenylalanine and tyrosine is in accord with the hypothesis that the coumarin nucleus is derived from aromatic amino acids. The methyl group of methionine is probably incorporated into the 0-methyl group. The incorporation of acetate is clear, although there is lack of evidence as to its position. Moody (1964) suggests that mevalonate should be considered a possible precursor of aflatoxin. Holtka (1964) found sterigmatocystin, a metabolite of A. versicolor (Bullock et al., 1962), in A. fEavus cultures along with aflatoxin and suggested that it may be a precursor of aflatoxin, through cleavage and rearrangement. Wogan ( 1966) has reviewed much of the work on the biochemical effects of aflatoxin. The aflatoxins also produce changes in tissues of plant origin. Schoental and White (1965) note that the germination of the cress seeds (Lepidium sativum) is inhibited by a total aflatoxin concentration of 25 pg./ml. Concentrations of 10 pg./ml. apparently interfere with chlorophyll synthesis, a fact suggested by the complete absence of color. Black and Altschul ( 1965) report that gibberellic acid-induced increases in lipase and a-amylase activity of the germinating cottonseed are inhibited by aflatoxin. F. TESTMETHODS 1 . Chemical Assays In the area of analytical methodology, the main effort has been directed toward the development of a rapid and accurate procedure effective in
MYCOTOXINS IN FEEDS AND FOODS
331
the survey of a large number of samples, and capable of detecting a few micrograms of toxin per kilogram of sample. Four steps are common to all the chemical methods proposed: Sampling: An appropriate sampling procedure is obviously important. This is particularly true in the case of mycotoxins, occurring as they do by contamination of natural products, wherein the distribution of the compound in the tissues is heterogeneous and as a result marked differences can be found between individual nuts. G. J. van Esch (Anonymous, 1964a) reports levels of 8 to 29 mg. per kilogram in single nuts. Higher values were reported by Cucullu et al. (1966). Therefore a careful sampling procedure must be selected and enough sample (usually 2 kg. is recommended for peanuts) must be ground and mixed by an accepted method for the product being analyzed. Extraction of the Toxin: Aflatoxins are soluble in methyl alcohol, chloroform, and acetone. All methods developed use solvent extraction along two different procedures: ( 1) a 4- to 18-hour continuous treatment of the defatted sample, using a Soxhlet-type extractor, and ( 2 ) a rapid batch extraction with solvent mixtures of different composition, using a high-speed type of mixer. The latter procedure is preferred by American laboratories, while the former has been mainly used by European workers. Purification of the Crude Extract: Two purification techniques are mainly used: liquid-liquid partition and column chromatography. Chromatographic Separation and Semiquantitative Estimation: The four identified aflatoxins can be separated by chromatographic techniques involving paper or thin-layer chromatography; the latter method is preferred. The most common system uses silica gel as absorbent developed with 1.57% methanol in chloroform. The aflatoxins are excited by long wave length UV light (365 mp), and the intensity of the resulting fluorescence (425 to 450 mp) is proportional to the amount of aflatoxin. This provides a visual means of estimation on the developed chromatogram. This evaluation can be made either by direct visual comparison of fluorescence intensity with that of pure aflatoxin standards of known concentration or by serial dilution to extinction of visually detectable fluorescence.
a. European Method Development The early method of assay, developed by Sargeant et al. (1961a,b), was based on the techniques used in the isolation of the crude toxin. The extracts obtained were biologically evaluated using young ducklings and the samples examined were classified as “strong,” “moderate,” “slight,” and “negative” according to the extent of the liver lesions observed. The Tropical Products Institute of London (TPI) described a method in
332
BORKER, INSALATA,
LEVI,
AND WITZEMAN
which the defatted groundnut sample was continuously extracted with methanol (Anonymous, 1962a), this extract was then partitioned between aqueous methanol and chloroform using paper chromatography as a final step. Coomes and Sanders (1963) used a shortened period of methanol extraction. After clean-up by liquid-liquid partition and column chromatography, the concentrated chloroform extract was chromatographed on paper using a modification of a solvent system proposed by Hhodes et al. ( 1961). Paper chromatographic techniques were replaced by thin-layer chromatography (Coomes et al., 1964; Broadbent et al., 1963; Genest and Smith, 1963). Coomes and Feuell (1965) summarize the method now recommended by the TPI. The standard procedure for the sampling of oilseeds of the International Association of Seed Crushers is recommended for the preparation of the sample. The choice of the method of assay to be used depends upon the facilities available, the number of samples, and the manipulations involved. The method outlined consists of defatting, extraction, and thin-layer chromatographic separation. For each operation a rapid and a standard procedure are described. The extraction is made in a Soxhlet extractor for 4 hours, using methanol. The extract is then partitioned with chloroform, and convenient amounts are separated on silica gel chromatoplates. Different methods of extraction, other than the continuous extraction with methanol, have been proposed, such as shaking with chloroform after preliminary wetting of the defatted sample (Lee, 1965) or a methanol extraction followed by a chloroform extraction (de Iongh et al., 1964). A procedure for the determination of aflatoxin B1 in samples of high toxicity ( 1000 pg./kg.) has been developed by Nabney and Nesbitt (1964, 1965) based on the intensity of the UV absorption at 363 mp after separation by thin-layer chromatography. These authors mention the possibility of measuring fluorescence intensity in methanolic solution and the difficulty presented by the different relative fluorescent intensities of aflatoxin B, and BS. This problem emphasizes the necessity for prior separation of the four known aflatoxins if measurements of absolute fluorescence intensity are to be used for the assessment of aflatoxin content and toxicity of a given sample. TPI workers developed a procedure for the photorecording of fluorescent spots on chromatoplates and quantitative evaluation of aflatoxin that depends on the UV component of a photographic flash image to demonstrate the fluorescence (Anonymous, 1964b). The transmission of each recorded spot is measured with a densitometer and compared with that of a calibration curve similarly prepared.
MYCOTOXINS IN FEEDS AND FOODS
333
b. American Method Development The analytical procedures developed in the United States are directed toward achieving a rapid and sensitive method of detection of aflatoxin when applied to large numbers of samples. They differ from the European methods in the extraction and purification steps, in which the Soxhlet type of extractor has been replaced by a high-speed mixer. Various choices of extraction solvent and subsequent purification of the crude extract have been studied. Thus, Heusinkveld et aZ. (1965) proposed an acetone-hexane-water ternary solvent that is freed from fat by phase separation and from extraneous fluorescers using a Florisil column eluted with tetrahydrofuran, followed by elution of the toxin with acetone. Robertson et al. ( 1965) use an acetone-water-hexane extraction solvent mixture, followed by purification by liquid-liqujd partition between chloroform-water to which sodium chloride has been added. The rapid method proposed by Nesheim et al. (1964) involves the extraction of the toxin with a hexane-methanol-water mixture. An aliquot of the aqueous methanol extract containing the toxin is then purified on a Celite column from which the aflatoxins are eluted with a mixture of chloroform and hexane. All methods mentioned use thin-layer chromatography to separate the four aflatoxins in the purified extract. About 1to 5 pg. per kilogram of aflatoxin B1 can be detected. A program of statistical evaluation of these methods, selected because they were currently being employed and because they required relatively simple manipulation, had been undertaken in a collaborative effort by government, industrial, and independent laboratories ( Nesheim, 1964; Trager et al., 1964; Campbell, 1966; Campbell and Funkhouser, 1966; and Funkhouser and Campbell, 1966. The method preferred for peanut products was basically that of Nesheim et d.( 1964). Pons and Goldblatt (1965) have developed a rapid procedure for the extraction of aflatoxins in cottonseed products, extracting the toxin with 70% acetone; interfering gossypoI is removed from this extract by precipitation as a lead salt and the supernatant liquid is purified by extraction with chloroform for thin-layer chromatography. Engebrecht et al. (1965) proposed a method for the determination of aflatoxin B1 in cottonseed meals in which the defatted meal is extracted with acetone. Pigments and lipids are removed by cold filtration. Chloroform-acetone is used as a developing solvent in the thin-layer chromatography operation. Stoloff et al. (1966) proposed the extraction of cottonseed products with acetone-water. Interfering substances are removed by precipitation with lead acetate and acetic acid followed by liquid-liquid partition chromatography using powdered cellulose. Shui-Chin Chen and Friedman (1966) developed an assay method for aflatoxin in seed meals,
334
BORKER,
INSALATA, LEVI,
AND WITZEMAN
claiming a sensitivity limit of <0.02 pg./kg. The extraction is made with aqueous methanol. This extract is purified with lead acetate followed by partition chromatography on celite. The possibility of evaluating aflatoxin content by measuring fluorescence intensity has already been mentioned. Another approach using instruments was proposed by Gajan et al. (1964), who found that aflatoxin B1 and G1 give characteristic oscillopolarographic traces. The pure aflatoxins can be qualitatively detected at levels ranging from 0.3 to more than 50 pg. It was noted that the purification methods for crude extracts are not adequate for the application of the method. The use of a Photovolt Densitometer has been suggested to measure fluorescence intensity of the aflatoxins on the thin-layer chromatograms (Sinnhuber, 1965); the reported sensitivity is 2.5 X 10C4mcg. B1. The extracts obtained by any of the methods mentioned can still contain fluorescent compounds that may obscure the final estimation or identification of the toxins. Different development solvent mixtures for the thin-layer separation have been proposed, other than the methanol chloroform generally used. Adye and Mateles (1964) report that better resolution is obtained using 2:l formamide-water as stationary phase and benzene as mobile phase; A. D. Campbell (1966) proposed a benzeneethanol-water developing solvent mixture with which very good separation of the four aflatoxins is obtained. A clean-up step prior to the separation by thin-layer chromatography has been suggested. This would involve either washing the extract absorbed on a silica gel column with ethyl ether (Pons and Goldblatt, 1965) followed by elution with chloroform-methanol, or developing the chromatoplate with ethyl ether before the actual separation of the aflatoxins (Coomes et al., 1965). Three confirmatory tests for aflatoxin B1 have been devised by Andrellos and Reid (1964). The preparation of three addition products that produce characteristic blue fluorescent spots on thin-layer chromatography is described. Comparison of the Rf's of the adducts with those of pure aflatoxin and adducts of pure aflatoxin permits identification of the toxin in doubtful cases. 2. Bioassay During the early investigations of hepatotoxic diets and the biological effects of aflatoxin, bioassay procedures were established using very young ducklings as the test animals. The methods used currently for the duckling test are based on the work of Armbrecht and Fitzhugh (1964), Sargeant et al. (1961a), and Wogan ( 1965). The extract for this bioassay is obtained in the same manner used for obtaining a crude extract for the chemical assays. Recently A. D.
MYCOTOXINS IN FEEDS AND FOODS
335
Campbell et al. (1964) devised a rapid procedure for the extraction of peanut products that permits the extraction of samples as large as 2 kg. and can be adapted to prepare the amount of extract needed for the bioassay under study. In this procedure the sample is extracted with hexane-methanol-water by rapid mixing in a Waring Blendor-type mixer, the slurry is filtered in a basket centrifuge, and the separated aqueousmethanol extract is further freed from fat by extraction with hexane. The toxin is then transferred from the aqueous methanol into chloroform. After a number of purifications, the extract is dissolved in propylene glycol for submission to the biological system. More sensitive and less cumbersome techniques for bioassay have been developed and are under study. Spensley (1963) noted that chick embryos are highly sensitive to aflatoxin, with fractions of a microgram being fatal. A bioassay using chick embryos has been developed by Verrett et al. (1964). Pure aflatoxin B1 and extracts of contaminated peanuts and aflatoxin-free peanuts dissolved in propylene glycol were injected, before incubation, into the yolk or air cell of fertilized white Leghorn eggs. The mortality at the time of hatching was found to be related to the toxicity of the extract injected. Cell culture methods have been described by Daniel (1965) and Legator et al. (1965). Allcroft and Carnaghan (1966) find cell culture assay to give more precise and rapid results than the duckling test when a biological test is needed for confirmation of results obtained by thinlayer chromatography. Fishbach and Campbell (1965) noted that, owing to the toxic effect of aflatoxins on warm-blooded animals, these compounds should be regarded as potentially toxic to man and therefore adequate precautions should be taken in the laboratory handling of contaminated material and pure toxin. Since the compounds are heat stable, chemical means of decontamination were studied. They recommended the use of 5% NaOC1, an oxidizing agent, to dispose of contaminated materials and laboratory equipment.
G. AGRICULTURAL COMMODITLES AFFECTED In the development of the analytical methods and their application, aflatoxin has been found as a natural contaminant in many agricultural materials. A list of materials in which detection of aflatoxin has been reported is shown in Table 111.
H. GOVEHNMENTAL ACTIONS Because of the carcinogenic potential most governments are taking action to keep products suspected to be contaminated with aflatoxin out of the human food supply as well as animal feeds. Many nations use as a
336
BORKER,
INSALATA, LEVI,
AND WITZEMAN
TABLE I11 FOOD MATERIALS REPORTED AS SUSCEPTIBLE TO AFLATOXINCONTAMINATION Cassava Cocoa Coconut Corn Cottonseed meal Fish meal Peanut and peanut meal
Peas Potatoes Rice Sake Soy beans Tempe Wheat
maximum tolerance the 50 p.p.b. lower detectable limit of the initial duckling bioassay. In the United States, the limit under the Food and Drug Laws for a carcinogenic material is none. However, enforcement is obviously based on the detection limit of the method used. Thus, as a practical matter, the tolerance is about 10 p.p.b. A most stringent stated limit by an American agency is a 1 p.p.b. maximum of aflatoxin required by the Fish and Wildlife Service in finished feeds for fish. However, no analytical method has been specified by which feed manufacturers should measure the attainment of this limit (Halver, 1965a). In addition to the regulatory aspects, substantial budgets have been allocated for studies leading to the control of the toxin. As an example, the U. S. Department of Agriculture proposed an expenditure of $1,458,000 for a mycotoxin research program for Fiscal 1966 (Anonymous, 1965). The program has four objectives (Irving, 1965) : ( 1) to find practical methods to prevent mold growth on agricultural commodities, ( 2 ) to develop rapid methods for the detection of aflatoxin and solve any special problems in the analysis of each commodity, (3) to discover practicable processes for removing or destroying toxins in agricultural products that are contaminated, and ( 4 ) to elucidate the metabolic fate of aflatoxin in domestic animals.
111. Other Mycotoxicoses A. RESPONSIBLE FUNGI The discovery of the aflatoxins and their identification as hepatotoxic and carcinogenic fungal metabolites occurring in food commodities as a result of contamination brought a new interest in other toxic mold metabolites. There is already a substantial body of information on these poisons. In many outbreaks, it is evident that toxins produced by filamentous fungi are responsible for the diseases. The causative compounds have not been isolated, however, nor their structures established. Wilson (1966a) and Friedman (1964) have reviewed some of the literature on the fungal toxins. A list of fungi reported to cause disease in man or other animals is given in Table IV.
MYCOTOXINS IN FEEDS AND FOODS
TABLE IV FUNGI IMPLICATED IN MYCOTOXICOSES 1. Aspergillus flavus luchuensis flavus var. tremorgen niger clavatus ochraceus claviforme oryzae f uniigatus parmiticus giganteus tamarii terreus glaucus 11. Penicilliuni baarnense melenii citreoviride novae zeelandiae citrinium patulum claviforme puberulum commune purpurogenum cyclopium rubrum expansum terlikowski thoinii griseof ulvum toxicarium islandicum wticae j e nse ni parisiticus leucopus I l l . Fusarium sporotrichioides sporotrichella var. poae Eraminearum IV. Claviceps purpurea paspali V . Gymnoascus
SPP. VI. Trichoderma lignorum viride V I l . Pithomyces chartarum V I 1 1. S tachybotrys atra
I X . Dendrodochium toxicum X . Sclerotinnia sclerotiorum X I . Gibberella zeae
337
338
BOHKER,
INSALATA, LEVI,
AND WITZEMAN
TABLE IV (Continued) X U . Cladosporium epiphyllum fa@
_.___.__
X l l l . Seratostomella
fmbriata ~
XIV. Gliocladium firnbriatum
B. IDENTIFIED TOXINS Fungal metabolites shown to have adverse physiological effects are listed in Table V. No attempt is made to summarize properties or the available analytical methodology, since the knowledge of their chemistry, biosynthesis, and physiological effects and of inhibitory processes and control procedures is of a partial and scattered nature. If their potential presence in foods consumed by men and animals is recognized as a serious problem, practical analytical methods to detect trace amounts of these compounds in food commodities must be developed. The variety of chemical structures of the compounds described and the different physiological effects involved ( antibiotic activity, inhibition of seed germination, insecticidal activity, etc. ) renders difficult any attempt to correlate toxic activity and chemical structure. It is worth noting, however, that several of these toxins (highly hepatotoxic and carcinogenic) are unsaturated six-membered ring lactone compounds, as are the aflatoxins. Dickens and Jones (1961) studied the carcinogenic activity of a number of lactones and noted their possible importance to human cancer if consumed over a long period of time. In their work they compared the potency of a series of lactones and suggested the possible carcinogenic and noncarcinogenic structures shown (structures A through G ) . They noted “that the four-membered rings of the propiolactones ( A ) , penicil-
B
A
E
D
C
F
G
d
MYCOTOXINS IN FEEDS AND FOODS
, dU
o
+): v 0
C C
5
c
m
d
339
340
I
r"
Y
d d
/"
X g o
/ /
80
8
g@
O
Data from Miller (1961). van der Merwe (1965), and Wilson (1966a).
BOHKER, INSALATA, LEVI, AND WITZEMAN
p
0
a
MYCOTOXINS IN FEEDS AND FOODS
341
342
BORKER,
INSALATA, LEVI,
AND WITZEMAN
lin ( B ) , and various hexenolactones (C, D, and E ) can apparently be carcinogenic but apparently not the saturated five-membered lactone ( F ) nor the P-unsaturated lactoncs (G)." They also noted that hydrolysis of lactones as well as saturation of the double bond reduces the activity. C. PATHOLOGICAL IMPLICATIONS FOR MAN
With many mycotoxic agents, the over-all pathologic response can be divided into two categories: (1) an acute hemorrhagic syndrome, usually involving the kidney, liver, lungs, and intestine, sometimes accompanied by extensive bile duct proliferation, and ( 2 ) a chronic phase in which malignant hepatoma and other metastosarcomas are observed. In many cases overt symptoms such as anorexia, malaise, photophobia, reduced growth rate, weight loss, and dermatological reactions may mask the more serious involvement of various internal organs. There are many exceptions to this general pattern. For instance, ergot poisoning manifests itself in man in two distinct forms, gangrenous and convulsive. In the gangrenous form there is an intensive burning sensation in the extremities which may be followed by a progressive restriction in the blood supply to the limbs and feet which can result in a gangrenous necrosis. In the convulsive form a variety of symptoms are experienced that indicate neurological involvement, including hallucinations and convulsive seizures which may lead to death (Barger, 1931; Gabbai et al., 1951). Other than ergotism, most of the mycotoxicoses in man have been studied by Soviet investigators (Bilay, 1960; Army Medical Service, 1953; Anonymous, 1962b) Alimentary toxic aleukia ( ATA), which appears endemic to the rural people in many parts of the U.S.S.R., has been divided into three pathological stages. The first stage is characterized by stomatitis, gastrointestinal disturbance, nausea, emesis, and diarrhea. Subsequently, the symptoms disappear and the patient develops a general leukopenia, relative lymphocytosis, and corresponding reductions in erythrocytes, platelets, and hemoglobin level. The third stage begins acutely and is characterized by a rash on the trunk, pharyngitis, ulcerated larynx, and hemorrhagic diatheses involving the nasal and oral passages as well as the gastrointestinal tract. These symptoms are so severe that they may lead to strangulation and death ( Gajdusek, 1953a,b; Rubinshteyn, 1960; Mayer, 1953). Cereal fungi of FusaTium and Cladosporium genera were the dominant fungi implicated. All the toxic strains sporulated and produced toxin below O'C., which accounted for the serious epidemic due to human consumption of overwintered cereals in the mid-1940's (Joffe, 1962, 1965). A Fusariurn strain was found responsible for Kaschim-Back disease, which involves a manifestation of chondro-osteodystrophy in children (Perkel, 1960).
MYCOTOXINS IN FEEDS AND FOODS
343
Stachybotryotoxicosis is another fungal disease found to affect man and animals in the Soviet Union (Drobotko, 1945; Gajdusek, 1953a,b). The human form develops in various stages, ranging from a mild dermatitis and catarrhal inflammation of the mouth to a more severe form characterized by liver damage, anemia, leukopenia, and hemorrhages in various tissues and organs. Forgacs (1965, 1966) and Forgacs et al. (1963) have described the pathology for animals. The isolation of the toxin from Stachybotrys alba (alterans) was reported by Fialkov and Serebryanyy ( 1949). Japanese workers concerned with the implications for man are actively studying toxic molds associated with rice ( Kinosita and Shikata, 1965). Many studies have been made on the effects of Penicillium islandicum toxins (Miyake et al., 1955, 1959, 1960; Miyake and Saito, 1965; Uraguchi et at., 1961, 1964). In laboratory animals acute liver damage has been found in long-term feeding studies with contaminated rice. One of the toxins is a chlorine-containing peptide. Forgacs and his colleagues have extensively studied mycotoxicoses in animals. His reviews (1962, 1965, 1966; Forgacs and Carll, 1962) are valuable contributions to the literature. Other contributions from the veterinary field are too extensive to be included in this review. As an indication of the extent of the problem, Table VI lists animals reported susceptible to mycotoxicoses.
IV. Control Mechanisms
TO secure a food and feed supply free of mycotoxins, more attention to control techniques will be needed. These measures can be divided into three categories: ( 1 ) prevention of initial mold growth and subsequent mycotoxin contamination, ( 2 ) detection of mycotoxins in food materials and selective removal of the contaminated portion, and (3) destruction of any toxin present. OF MOLDGROWTH A. PREVENTION
To prevent initial mold contamination, it is necessary to correct the initial harvesting and storage procedures and to identify the point where mold spores gain access to the product. Austwick and Ayerst (1963) observed that in the ground the mature groundnut fruit is frequently invaded by a number of saprophytic fungi, including A. flavus, and that the kernels are resistant to attack at this time. After removal from the soil they begin to dry and may be mechanically damaged, becoming more susceptible to attack by a number of saprophytic and weakly parasitic fungi. Moisture becomes vne of the main factors limiting the growth of
344
BORKER, INSALATA, LEVI, AND WITZEMAN
TABLE VI MYCOTOXIN-SUSCEPTIBLE ANIMALS Aspergillus humans dogs horses swine cattle sheep turkeys ducks chickens guinea pigs rats mice
Penicillium humans
dogs horses swine cattle sheep rats pigeons Peking ducklings mice rabbits
Fusarium humans swine cattle Nocardia humans hogs laboratory animals Cluciceps humans Symnascus rats Pithom y ces humans sheep cattle
Dendrodochium humans horses Sclerotinnia human animal Gibberella swine cattle Sporodesium sheep guinea pigs cattle rabbits mice
Stachbotrys humans livestock
fungi. Invasion by A. fluvus presumably occurs while the nuts are drying in the field and is subsequently stopped by further drying. High moisture content in the stored groundnuts as a result of exposure to rain or condensation permits A. fEavus and other fungi to grow rapidly. Spensley (1963) considers the most critical stage for mold growth to be immediately following harvest. The nuts still have a high moisture content when lifted, and unless both atmospheric conditions and harvesting practices are good, drying to a safe moisture level may not take place quickly enough to avoid significant mold development. Irving (1965) reported that the development of aflatoxin in peanuts in windrows was quite variable. Under hot humid conditions (over 90°F. and 70% relative humidity) significant amounts of aflatoxin were found in a small percentage of samples. Under cool conditions (70" ) no aflatoxin development occurred, even after a week or more in the windrow, regardless of rain and high humidity. Again, aflatoxin was associated with broken pods. Although the preceding discussion relates primarily to groundnuts and is concerned with A. fEavus, the general control principles are applicable
MYCOTOXINS IN FEEDS AND FOODS
345
to all products in connection with mycotoxic fungi. Over all, this involves avoiding physical damage to the product itself and thus eliminating possible portals of entry for fungal contamination. Also, moisture must be kept to a minimum, a measure which involves dry storage in humid regions, rapid drying in the field, and prevention of exposure to rain. A direct approach to the control of mycotoxicoses would be the inclusion of a suitable compound in the field that would prevent fungal growth completely or at least so modify fungal metabolism as to prevent formation of mycotoxins. Forgacs et al. (1963) screened a number of compounds for antifungal and antimycotoxic properties and found that several were effective. Hydroxyquinoline is effective in suppressing fungal growth and consequently mycotoxicoses in chicks under field conditions. Another control approach uses the inhibition of mold growth in feed by y-radiation (Webb et al., 1959).
B. REMOVAL OF CONTAMINATED PRODUCTS If mold attachment occurs and toxin contaminates an agricultural commodity, much, if not all, of the toxin-containing portions of the crop can be removed by sorting out visible mold growth, Hand and electronic sorting of peanuts have dramatically reduced aflatoxin in shipments of edible peanuts. Where there is a sensitive analytical method, as in the case of aflatoxin, shipments or lots of products can be tested and prevented from entering commercial feed and food channels if contaminated. This is not a particularly effective or economic method of control but will serve if better field and toxin control measures are not available. C. INACTIVATION OF TOXIN In overcoming or destroying mycotoxins present in food products, few concrete measures have as yet been developed. Of course, such measures cannot be general but must be specific for each mycotoxin in specific foods. Under certain conditions of processing for specific foods, such as the alkali wash which is employed in the production of peanut oil for margarine, the toxin is destroyed. The Russians have noted that food made from grain definitely poisoned with the toxin of F . sporotrichoides is found to be nontoxic after processing in large commercial mills. Some of the mycotoxins have been found to be unusually stable physically and chemically. Stachybotrys toxin is resistant to sunlight, ultraviolet light, and X-rays; it is thermostable (withstanding temperatures of 120°C. for at least 1 hour), and it is not affected by 2% concentrations of inorganic or organic acids, but it is destroyed by alkalis (Forgacs,
346
BORKER,
INSALATA, LEVI,
AND WITZEMAN
1962). Aspergillic acid is stable under acid and alkaline conditions and can be distilled using steam without loss of activity (Forgacs, 1962). According to Carnaghan and Crawford (1964) aflatoxin is not inactivated by dry heating to 160°C. for 1hour nor by steam heating and is therefore likely to remain potent after cooking. Platt et al. (1962) report that moist heat treatment of partially purified aflatoxin (170°C. at 80-90 p s i . for 10-20 minutes) will inactivate the toxin. The toxin produced by F . sporotrichoides is also stable to acids, alkali, and temperatures of 200°C. (Anonymous, 1962b). Byssochalmic acid, which is produced by certain strains of Byssochalmic fluva ( a fungus similar, if not identical, to P . varioti), is remarkably stable to acid and alkali. This toxin has been reported to withstand treatment with hot dilute sodium hydroxide or mineral acids and dissolves readily in concentrated nitric or sulfuric acids without change in potency (Mayer, 1953). Forgacs (1962) notes that mycotoxins are not antigenic and therefore animals that have recovered from mycotoxicoses can again become affected. It would seem, then, that the most reliable area of control is the prevention of initial fungal contamination and toxin production. REFERENCES Adye, J., and Mateles, R. I. (1964). Biochim. Biophys. Acta 86, 418. Allcroft, R. (1965a). In “Mycotoxins in Foodstuffs” (G. N. Wogan, ed.), p. 153 ff. M. I. T. Press, Cambridge, Massachusetts. Allcroft, R., and Carnaghan, R. B. A. (1962). Vet. Record 74, 863. Allcroft, R., and Carnaghan, R. B. A. (1963a). Chem. b lnd. (London) No. 2, pp. 50-53. Allcroft, R., and Camaghan, R. B. A. (I963b). Vet. Record 75, 259-263. Allcroft, R., and Carnaghan, R. B. A. (1966). Nature 209, 154-7. Allcroft, R., and Lewib, G. (1963a). Biochem. J. 88, 58. Allcroft, R., and Lewis, G. (1963b). Vet. Record 75, 487. Allcroft, R., Carnaghan, R. B. A,, Sargeant, K., and O’Kelly, J. (1961). Vet. Record 73, 428-429. Allcroft, R., Rogers, €I., Lewis, G., Nabney, J., and Best, P. E. (1966). Nature (in press ) . Andrellos, P. J., and Reid, G. R. (1964). J. Ass. Ofic. Agr. Chemists 47, 801-803. Anonymous. (1962a). T. P. I. Rept. No. 25. Trop. Prod. Inst., London. Anonymous. (196213). Nutr. Reo. 20, 337-339. Anonymous. (1964a). Food Cosmet. Toxicol. 2, 487-488. Anonymous. ( 1964b). “Photorecording of Fluorescent Spots on Chromatoplates.” Trop. Prod. Inst., London. Anonymous. ( 1 9 6 4 ~ )T. . P. I. Rept., 1963. Trop. Prod. Inst. Comm. (with the report of the Director of the Trop. Prod. Inst.), London. Anonymous. ( 1965). Food Processing, Food Regulation Section, Armbrecht, R. H., and Fitzhugh, 0. G. (1964). Toxicol. Appl. Pharmacol. 6, 421-426.
MYCOTOXINS IN FEEDS AND FOODS
347
Armbrecht, R. H., Hodges, F. A., Smith, H. R., and Nelson, A. A. (1963). J. Assoc. O ~ CAgr. . Chemists 46, 805-817. Army Medical Service Graduate School. (1953). Med. Sci. Publ., Army Med. Sew. Grad. School, Walter Reed Army Med. Center 2. Asao, T., Buchi, G., Abdel Kader, M. M., Chang, S. B., Wick, E. L., and Wogan, G . N. (1963). J. Am. Chem. SOC. 85, 1706. Asao, T., Buchi, G., Abdel Kader, M. M., Chang, S. B., Wick, E. L., and Wogan, G. N. (1965). J. Am. Chem. SOC. 87, 882. Ashley, L. M. (1962). Proc. Working Conf. Trout Hepatoma, 1962 Natl. Inst. Health D.H.E.W., Rept., Bethesda, Maryland. April. Ashley, L. M., Halver, J. E., and Johnson, C. L. (1962). Federation PTOC.21, 304. Asplin, F. D., and Carnaghan, R. B. A. (1961). Vet. Record 73, 1215-1219. Austwick, P. K. C., and Ayerst, G. (1963). Chem. G Ind. (London) No. 2, pp. 55-61. Bailey, W. S., and Groth, A. H. (1959). J. Am. Vet. Med. Assoc. 134, 514-516. Barger, G. ( 1931 ). “Ergot and Ergotism.” Guerney and Jackson, Edinburgh. Berman, C. (1951 ). “Primary Carcinoma of the Liver. Lewis, London. Bilay, V. I., ed. (1%0). “Mycotoxicoses anf Man and Agricdtural Animals” (English transl.) Office Tech. Serv., U.S. Dept. Com., Washington, D.C. Black, H. S., and Altschul, A. M. (1965). Biochem. Biophys. Res. Commun. 19, 661-664. Blount, W. P. (1961). Turkeys 9, 52, 55-58, and 61-71. Brian, P. W., Dawkins, A. W., Grove, J. F., Hening, H. G., Love, D., and Norris, G. L. F. (1961). 1. Exptl. Botany 12, 1-12. Broadbent, J. H., Cornelius, J. A., and Shone, G. (1963). Analyst 88, 214-216. Brown, E. V., Novack, R. M., and Hamdan, A. A. (1961). J. Natl. Cancer Inst. 26, 1461-1464. Bullock, E., Robert, T. C., and Underwood, J. G. (1962). J. Chem. SOC. pp. 41794183. Bush, M. T., Goth, A,, and Dickinson, H. L. (1945). J . Pharmacol. Exptl. Therap. 84,262. Bush, M. T., Touster, O., and Brochman, J. (1951). J. BioZ. Chem. 188, 685. Butler, W. H., and Barnes, J. M. (1964). Brit. J. Cancer 17, 699. Butler, W. H., and Clifford, J. I. (1966). Nature (in press). Campbell, A. D., Dorsey, E., and Eppley, R. M. (1964). J. of Ass. Ofic. Agr. Chem. 47, 1002-1003. Campbell, A. D., and Funkhouser, J. T. (1966). J. Ass. Ofic. Agr. Chem. ( i n press). Campbell, A. D. (1966). J. Ass. Ofic. Agr. Chem. (in press). Campbell, J. G. (1956). Proc. Roy. SOC. Edinburgh B66, 111-129. Camaghan, R. B. A., and Allcroft, R. (1962). Vet. Record 74, 925-926. Camaghan, R. B. A., and Crawford, M. (1964). Brit. Vet. J. 120, 201. Camaghan, R. B. A., Hartley, R. D., and OKelly, J. (1963). Nature 200, 1101. Carnaghan, R. B. A., Lewis, G., Patterson, D. S. P., Allcroft, R. (1965). Private communication (will be offered for publication). Chang, L., Abdel Kader, M. M., Wick, E. L., and Wogan, G. N. (1963). Science 142, 1191-1192. Cheung, L. K., and Sim, G. A. (1964). Nature 201, 1185-1188. Codner, R. C., Sargeant, K., and Yeo, R. (1963). BiotechnoL Bioeng. 5, 185. Coomes, T. J., and Feuell, A. J. (1965). T. P. I. R e p . G 13. Trop. Prod. Inst., London.
348
BORKER, INSALATA, LEVI, AND WITZEMAN
Coomes, T. J., and Sanders, J. C. (1963). Analyst 88, 209-213. Coomes, T. J., Crowther, P. C.,Francis, B. J., and Shone, G. (1964). Analyst 89, 436-437. Coomes, T. J,, Crowther, P. C., Francis, B. J., and Stevens, L. (1965). Analyst 90, 492. Cucullu, A., Lee, L. S., Mayne, R. Y., and Goldblatt, L. A. (1966). J. Am. Oil Chem. SOC.43, 89. Daniel, M. R. (1965). Brit. J. Exptl. Pathol. 46, 183. Davidson, C. S. (1963). Med. Sci. 32. Davis, N. D., Diener, U. L., and Landers, K. E. (1966). Auburn University (to be published). de Iongh, H. (1965). In “Mycotoxins in Foodstuffs” (G. N. Wogan, ed.), p. 235. M. I. T. Press, Cambridge, Massachusetts. d e Iongh, H., Beerthius, R. K., Vles, R. O., Barrett, C. B.,and Ord, W. 0. (1962). Biochim. Biophys. Acta 65, 548. de Iongh, H., Vles, R. O., and Van Pelt, J. G. (1964). Nature 202, 466-467. Derzsy, D., Meszaros, J., Prokopovitsch, L., and Toth-Baranyi, I. (1961). Magy. All. Lapja 17, 49. de Vogel, P., von Rhee, R., and Koelensmid, W. A. A. B. (1965). 1. Appl. Bacteriol. 28, 213. Dickens, F., and Jones, H. E. H. (1961). Brit. J. Cancer 15, 85-100. Dollar, A. M., and Katz, M. (1962). Res. Fisheries 139, 23-25. Drobotko, V. G . (1945). Ann. Rev. Soviet Med. 2, 238. Engebrecht, R . H., Ayres, J. L., and Sinnhuber, R. 0. (1965). J. Ass. Ofic. Agr. Chem. 48, 815-818. Eschenbrenner, A. B., and Miller, E. (1946). J. Natl. Cancer Inst. 6, 325-341. Falk, R. L., Thompson, S. J., and Kotin, P. (1965). Proc. Am. Assoc. Cancer Res. 6, 18. Fialkov, Y. A., and Serebryanyy, S. (1949). “A New Fungus Disease of Horses and People.” Kiev. Fishbach, H., and Campbell, A. D. (1965). J. 08. Anal. Chem. 48, 128. Fitzhugh, 0. G., and Nelson, A. A. (1948). Science 108, 626-628. Forgacs, J. ( 1962). Foodstuffs 34, 124-134. Forgacs, J. (1965). In “Mycotoxins in Foodstuffs” (G. N. Wogan, ed.), p. 87. M. I. T. Press, Cambridge, Massachusetts. Forgacs, J. ( 1966). Bacteriol. Reu. (submitted for publication). Forgacs, J., and Carll, W. T. (1962). Aduan. Vet. Sci. 7. Forgacs, J., Carll, W. T., Herring, A. S., and Hinshaw, W. R. (1958). Trans. N . Y. Acad. Sci. [21 20, 787. Forgacs, J., Koch, H., and White-Stevens, R. H. (1963). Avian Diseases 7, 56-66. Friedman, L. (1964). Food Technol. 18, 1553. Funkhouser, J. T., and Campbell, A. D. (1966). J . Ass. Ofice Agr. Chem. (in press). Gabbai, Lisbonne, and Pourquier (1951). Brit. Med. J. 11, 650. Gajan, R. J., Nesheim, S., and Campbell, A. D. (1964). J. Assoc. Ofic. Agr. Chemists 47, 27-28. Gajdusek, D. C. (1953a). Med. Sci. Publ., Army Med. Sero. Grad. School, Walter Reed A m y Med. Center 2, 82-105. Gajdusek, D. C. (195313). Med. Sci. Publ., Army Med. Serv. Grad. School, Walter Reed Army Med. Center, 2, 107-111. Genest, C., and Smith, D. M. (1963). 1. Ass. Ofic. Agr. Chem. 46, 817-818. Ghittino, P., and Ceretto, F. ( 1962). Tumori 48, 393-410.
MYCOTOXINS IN FEEDS AND FOODS
349
Haddow, A., and Blake, V. (1933). I. Pathol. Bacteriol. 36, 41-47. Halver, J. E. (1962). Proc. Working Conf. Trout Hepatoma, 1961 Natl. Inst. Health, D.H.E.W., Rept., Bethesda, Maryland. Halver, J. E. (1965a). Personal communication. Ilalver, J. E. (1965b). In “Mycotoxins in Foodstuffs” ( G . N. Wogan, ed.), p. 209. M. I. T. Press, Cambridge, Massachusetts. Halver, J. E., Johnson, C. L., and Ashley, L. M. (1962). Federation Proc. 21, 390. Harding, J. D. J., Done, J. T., Lewis, G., and Allcroft, R. (1963). Res. Vet. Sci. 4, 217. Hartley, R. D., Nesbitt, B. F., and O’Kelly, J. (1963). Nature 198, 1056-1058. Heusinkveld, M. R., Shera, 0. C., and Baur, F. J. (1965). 1. Ass. Ofic. Agr. Chem. 48, 448. Higginson, J., and Oettle, A. G . (1960). I . Natl. Cancer Inst. 24, 587-671. Hodges, F., Allen, J. R., Zush, J. R., Nelson, A. A., Armbrecht, R. H., and Campbell, A. D. (1964). Science 145, 1439. Holding, A. S. (1964). Vet. Bull. (Commonwealth Bur. Animal Health) 34, 327. Holtka, D. T. S. E. (1964). Private communication. Hueper, W. C., and Payne, W. W. (1961). 1. Natl. Cancer Inst. 27, 1123-1143. Irving, G . W. (1965). I. Am. Oil Chemists’ Soc. 42, 466A. Joffe, A. Z. (1962). Mycopathol. Mycol. Appl. 16, 201. Joffe, A. Z. (1965). In “Mycotoxins in Foodstuffs” (G. N. Wogan, ed.), p. 77. M. I. T. Press, Cambridge, Massachusetts. Kawamata, J., Nakabayashi, N., Kawai, A., and Ushida, T. (1958). Med. J. Osaka Uniu. 8, 753-762. Kawamata, J., Nakabayashi, N., Kawai, A., Fujita, H., Imanishi, M., and Ikegmi, R. (1959). Biken’s J. 2, 106-112. Kinosita, R., and Shikata, T. (1965). In “Mycotoxins in Foodstuffs” ( G . N. Wogan, ed.), p. 111. M. I. T. Press, Cambridge, Massachusetts. Kohler, H., and Swaboda, R. (1962). Wien. Tieraerztl. Monatsschr. 49, 205. Kulik, Y. I. (1954). Reo. Med. Vet. Mycol. 3, 782. Lancaster, M. C., Jenkins, F. P., and Philp, J. Mcl. (1961). Nature 192, 1095-1096. LaRoche, G., Halver, J. E., Johnson, C. L., and Ashley, L. M. (1962). Federation Proc. 21, 300. LeBreton, E., Frayssinet, C., Lafarge, C., and Recondo, A. M. (1964). Food Cosmet. Toxicol. 2, 675. Lee, W. V. (1965). Analyst 90, 305-307. Legator, M. S., and Withrow, A. (1964). 1. Ass. Ofic. Agr. Chem. 47, 1007-1009. Legator, M. S., Zoffante, S. M., and Hart, A. Nature 208, 345-7 (1965). Loosmore, R. M., and Markson, L. M. ( 1961 ). Vet. Record 73, 813-814. Mateles, R. I., and Adye, J. C. (1965). Appl. Microhiol. 13, 208-211. Mayer, C. F. (1953). Military Surgeon 113, 173-189. Miller, M. W. (1961). “The Pfizer Handbook of Microbial Metabolites.” McGrawHill, New York. Miyake, M., and Saito, M. (1965). In “Mycotoxins in Foodstuffs” (G. N. Wogan, ed.), p. 133. M. I. T. Press, Cambridge, Massachusetts. Mikaye, M., Saito, M., Enomoto, M., Uraguchi, K., Tsukioka, M., and Ikeda, Y. (1955). Acta. Pathol. Japon. 5, 208. Miyake, M., Saito, M., Enomoto, M., Shikata, T., Ishiko, T., Uraguchi, K., Sakai, F., Tatsuno, T., Tsukioka, M., and Noguchi, Y. (1959). Gann 50, 117-118. Miyake, M., Saito, M., Enomoto, M., Shikata, T., Ishiko, T., Uraguchi, K., Sakai, F., Tatsuno, T., Tsukioka, M., and Sakai, Y. (1960). Acta. Pathol. Japon. 10, 75-123.
350
BORKER, INSALATA, LEVI, AND WITZEMAN
Moody, D. P. (1964). N(iture 202, 188. Nabney, J., and Nesbitt, B. F. (1964). Nature 203, 862. Nabney, J., and Nesbitt, B. F. (1965). Analyst 90, 155-160. Nesbitt, B., O’Kelly, J., Sargcant, K., and Sheridan, A. (1962). Nature 195, 10621063. Nesheim, S. (1964). J. Ass. 0ff;c. Agr. Chem. 47. 1010-1017. Nesheim, S., Banes, D.. Stoloff, L., and Campbell, A. D. (1964). J. Ass. Ofic. Agr. Chem. 47, 586. Newberne, P. M., and Carlton, W. W. (1963). Federation Proc. 22, P.1, 262 (abstr. 610). Newberne, P. M., Carlton, W. W., and Wogan, G. N. (1964). Pathol. Vet. (Basel) 1, 105. Ninard, B., and Hintermann, J., (1946). Bull. Inst. Hyg. Maroe. 5, 49. Oettle, A. G. (1956). J. Natl. Cancer Inst. 17, 249-280. Paget, G. E. (1954). J. Pathol. Bacteriol. 67, 393-400. Patterson, J. S., Crook, J. C., Shano, A., Lewis, C., and Allcroft, R. (1962). “Groundnut Toxicity as the cause of exudative hepatitis (Oedema disease) of guinea pigs.” The Vet. Rec. 74, 639-640. Perkel, N. V. (1960). In “Mycotoxicoses of Man and Agricultural Animals” (English transl.) ( V . I. Bilay, ed.), p. 117. Office Tech. Serv., U. S. Dept. Com., Washington, D.C. Philp, J. McL. (1964). Food Cosmet. Toxicol. 2, 674-675. Philps, R. H. (1964). Texas Nutr. Conf., October I964 (abstr.). Platonow, N. (1964). Vet. Record 76, 589. Ylatt, B. S., Stewart, R. J. C., and Gupta, S. R. (1962). Proc. Nuk. Soc. (Engl. Scot.) 30, 21. Pons, W. A., and Goldblatt, L. A. (1965). J. Am. Oil Chemists’ Soc. 42, 471-475. Quass, F. W. (1959-1960). Ann. PTOC. Assoc. Sci. Tech. SOC. S . Africa pp. 43-59. Raper, K. B., and Fennell, D. I. (1965). “The Genus Aspergillus.” Williams & Wilkins, Baltimore, Maryland. Rhodes, A., Boothroyd, B., McGonagle, M. P., and Sommerfield, C. A. (1961). Biochem. J. 81, 28. Robertson, T. A., Lee, L. S., Cucullu, A. F., and Coldblatt, L. A. (1905). Journal Ann. Oil Chem. SOC. 42, 467-471. Rubinshteyn, Yu. I. ( 1960). In “Mycotoxicoses of Man and Agricultural Animals” (English transl.) (V. I. Bilay, ed.), p. 89. Office Tech. Serv., U. S. Dept. of Com., Washington, D. C . Salmon, W. D., and Newberne, P. M. (1963). Cancer Res. 23, 571-575. Salmon, W. D., Newberne, P. M., and Prickett, C. 0. (1963). Federation Proc. 22, 262. Sargeant, K., O’Kelly, J., Carnaghan, R. B. A., and Allcroft, R. (1961a). Vet. Record 73, 1219-1223. Sargeant, K., Sheridan, A., O’Kelly, J., and Carnaghan, R. B. A. (1961b). Nature 192, 1096. Sargeant, K., Carnaghan, R. B. A., and Allcroft, R. (1963). Chem. G Ind. (London) pp. 53-55. Schoental, R. (1961). Brit. 1. Cancer 15, 812. Schoental, R., and White, A. F. (1965). Nature 205, 57-58. Shank, R. C., and Wogan, G. N. { 1964). Federation Pruc. 23,200. Shank, R. C., and Wogan, G. N. (1965). Federation Proc. 24, 627. Shimkin, M. B., and Kraybill, H. F. (1964). Aduan. Cancer Res. 8, 1991.
MYCOTOXINS IN FEEDS AND FOODS
351
Shui-Chin Chen, and Friedman, L. (1966). J. Ass. Ofic. Anal. Chem. 49, 28. Siller, W. G., and Ostler, D. C. (1961). Vet. Record 73, 134-138. Sinnhuber, R . 0. ( 1965). Private communication. Smith, R. H. (1963). Biochem. J. 88, 5OP-51P. Spensley, P. C. (1963). Endeauor 22, 75. Stoloff, L., Graff, A., and Rich, H. (1966) AOAC 79th Ann. Meeting Assoc. Ofic. Anal. Chem. (in press). Tilden, E. B., Halton, E. H., Freeman, S., Wilhamson, W. M., and Koenig, V. L. (1961 ). Mycopathol. Mycol. Appl. 14, 325. Trager, W. T., Stoloff, L., and Campbell, A. D. (1964). J . Ass. Ofic. Agr. Chem. 47, 993-1001. Tulpole, P. Q. et al. (1964). Lancet 1, 962-963. Uraguchi, K., Tatsuno, T., Sakai, F., Tsukioka, M., Sakai, Y., Yonemitsu, O., Ito, H., Miyake, M., Saito, M. Enomoto, M., Shikata, T., and Ishiko, T. (1961). Japan. J. Exptl. Med. 31, 19. Uraguchi, K., Tatsnno, T., Tsukioka, M., Sakai, Y., Sakai, F., Kobayashi, Y., Saito, M., Enomoto, M., and Miyake, M. (1964). Japan. J. Exptl. V e d . 31, 1. van der Linde, J. A,, Frens, A. M., van Esch, G. J. (1965). In “Mycotoxins in Foodstuffs” (G. N. Wogan, ed.), p. 247 ff. M, 1. T. Press, Cambridge, Massachusetts. van der Merwe, K. J. (1965). Nature 205, 1112. van der Merwe, K. J., Fourie, L., and Scott, de B. ( 1963). Chem. G Znd. (London) pp. 1660-1661. van der Zijden, A. S. M., B. Koelensmid, W. A. A. B., Boldingh, J., Barret, C. B., Ord, W. O., and Philp, J. (1962). Nature 195, 1060-1062. van Dorp, D. A., van der Zijden, A. S. M., Beerthuis, R. K., Sparreboom, S., Ord, W. O., de longh, K., and Keunning, R. (1963). Rec. Trav. Chim. 82, 587-592. Verret, M. J., Marliac, J. P., and McLaughlin, J., Jr. (1964). J. Ass. O ~ CAgr. . Chem. 47, 1003-1006. Webb, B. D., Thiers, H. D., and Richardson, L. R. (1959). Appl. Microbiol. 7, 329-333. White, E. C., and Hill, J. H. (1943). J. Bacteriol. 45, 433. Wilson, B. J. (1966a). Proc. Natl. Acad. Sci. U . S. (in press). Wilson, B. J. (1966b). Bacterial. Rev. (in press). Wilson, B. J., and Wilson, C. H. (1964). Science 144, 177. Wogan, G. N. (1963). Contract Progr. Rept. ( 1 and 2 ) Ph. 43-62-468 to Natl. Cancer Inst. Natl. Inst. Health, D.H.E.W. Wogan, G. N., ed. (1965). “Mycotoxins in Foodstuffs.” M. I. T. Press, Cambridge, Massachusetts. Wogan, G. N. (1966). Bacteriol. Reu. (in press). Wogan, G. N., and Friedman, M. A. (1965). Federation Proc. 24, 627. Wogan, G. N., Wick, E. L., Dunn, C. G., and Scrimshaw, N. S. (1963). Federation Proc. Abstr. No. 2696. Wood, E. M., and Larson, C. P. (1961). Arch. Pathol. 71, 471-479. Wynston, L. K., and Tilden, E. B. (1963). Mycopathol. Mycol. Appl. 20, 272.
This Page Intentionally Left Blank
AUTHOR INDEX Numbers in italics refer to pages on which the complete references are listed,
A Abd-El-MaIek, Y., 272, 283 Abdel Kader, M. M., 321, 322,347 Abe, S., 8, 26 Abelson, P. H., 56, 56 Abraham, E. P., 27 Ackerman, T. V., 120, 139 Adams, A. B., 230, 243 Adams, J. N., 38, 56 Adelberg, E. A., 15, 17, 25, 27, 33, 49, 56 Adiga, P. R., 52, 56 Adye, J. C., 319, 330, 334, 346, 349 Agranoff, B. W., 53, 56 Aitchison, W. S., 147, 193 Alacevic, M., 39, 56 Alexander, L. J., 109, 111, 139 Alexander, M., 42, 56 Alikhanian, S. I., 33, 38, 39, 44, 46, 55, 56, 58 Allcroft, R., 317, 319, 324, 325, 326, 327, 329, 331, 334, 335, 346, 347, 349, 350 Allen, J. R., 319, 349 Allen, L. A., 82, 83, 85, 101, 258, 265, 280, 283, 284 Allen, R. S., 270, 285 Altemeier, W. A., 277, 284 Altschul, A. M., 347 Amberg, H. R., 121, 139 Amdur, B. H., 230, 245 Ames, B. N., 4 , 25, 5Q58 Anderson, D. L., 38,42, 43, 57 Anderson, G. W., 147, 192 Andrellos, P. J., 322, 334, 346 Andrewes, F. W., 196, 243 Angelotti, R., 263, 264, 266, 281, 282, 285 Angus, T. A., 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 307, 308, 309, 312, 313 Angyal, J., 238, 243 Anthony, D. H., 225, 243 Aoki, K., 291,512 Appleman, M. D., 208, 209, 218, 221,
243, 249, 264, 265, 266, 267, 275, 279, 284, 288 Appleton, J. L. T., 227, 235, 243 Arakawa, K. Y., 304, 313 Armbrecht, R. H., 319, 320, 334, 346, 347, 349 Armbruster, E. H., 129, 142 Amdt, J., 231, 235, 244 Arnim, S. S., 207,220, 243 Amstein, H. R. V., 19, 25 Amstein, M. G., 147, 191 Arvidson, M., 234, 243 Asao, T., 321, 322, 347 Ashe, W. K., 220, 243 Ashley, L. M., 316, 347, 349 Asplin, F. D., 317, 326, 327, 347 Austin, L. B., 230, 243 Austwick, P. K. C., 318, 343, 347 Avery, 0. T., 42, 56 Ayerst, G., 318, 343, 347 Ayres, J. C., 270,281,282, 284 Ayres, J. L., 317, 333, 348
B Bach, M. K., 53, 58 Backer Dirks, O., 208, 251 Backus, M. P., 30, 56 Bader, H. I., 215, 243 Baer, P. N., 218, 243 Bagdasaryan, G. A., 177, 191 Bagger, S. V., 276, 283 Bahn, A. N., 205, 206,243,249 Baich, A., 17, 25 Bailey, W. S., 327, 347 Bailit, H. L., 243 Baines, S., 89, 101 Baker, S. K., 287 Balassa, G., 42, 56 Baldini, I., 273, 286 Baldridge, G. D., 219, 244 Baldwin, D. C., 243 Ballinger, D. G., 134, 141 Balogh, K., 238, 243 Bamba, K., 255, 284 Bancroft, P. M., 164, 191
353
354
AUTHOR INDEX
Banes, D., 333, 350 Bar-Eli, A., 52, 56 Barger, G., 315, 342, 347 Barker, A. N., 88, 97, 101 Barksdale, W. L., 45, 5fi Barnes, E. M., 256, 259, 269, 270, 271, 277, 283, 286 Barnes, J. M., 322, 324, 347 Barrett, C. B., 319, 320,322, 348, 351 Barritt, N. W., 81, 101 Barron, A. L., 162, 192 Bartels, H. A., 220, 231, 243, 244 Barth, E. F., 95, 102 Bartley, C. H., 255, 258, 262, 267, 279, 281, 288 Bartsch, A. F., 116, 139 Batchelor, F. R., 19, 25 Batty, I., 206, 248 Baty, J. B., 165, 168, 176, 187, 193 Baur, F. J., 333, 349 Bautz, E., 36, 56, 57 Baylis, J. R., 109, 113, 121, 139 Beadenkopf, W. G., 164, 193 Beadle, G. W., 36, 56 Beard, J. W., 232, 250 Beard, P. J., 115, 139 Beck, A. J., 93, 103 Becks, H., 237, 244 Beckwith, J. D., 114, 143 Beckwith, J. R., 23 Beckwith, T. D., 217, 244 Beerthius, R. K., 319, 322, 348, 351 BBguin, S., 293, 305, 306, 312, 313 Bejuki, W. M., 107, 108, 139 Benarde, M. A., 129, 139 Bender, I. B., 205, 244, 250 Bennett, J. C., 54, 57 Bent, D. F., 258, 262,288 Berg, G., 147, 164, 169, 170, 172, 173, 176, 177, 181, 191 Berger, U., 200, 201, 222, 231, 235, 239, 244 Berke, J. D., 212, 244 Berliner, E., 291, 312 Berman, C., 328, 347 Berndt, A. L., 226, 244 Bernfield, M., 51, 58 Berrah, G., 277, 283 Berry, G. P., 230, 245 Best, P. E., 325, 346
Beust, T. B., 221,244 Beyer, O., 303, 313 Bhat, J. V., 83, 84, 85, 100, 102 Bibby, B. G., 202, 217, 218, 220, 230, 231, 244, 245 Billingham, R. E., 53, 56 Bils, R. F., 275, 288 Biondi, D., 197, 244 Bissada, N. F., 216, 244 Bisset, K. A., 243, 244 Bjiirn, H., 208, 209, 244 Black, H. H., 117, 137,139 Black, H. S., 347 Blahd, M., 242, 246 Blair, G. Y., 128, 139 Blake, V., 316, 349 Blank, H., 218, 244 Blayney, J. R., 211, 234, 247 Blechman, H., 231, 244 Bloom, H. H., 171, 178, 180, 181, 191, 192 Bloomfield, A. L., 199, 200, 220, 221, 222,227,228,240, 244,250 Blount, W. P., 317, 326, 347 Blum, H. L., 215, 248 Bonicke, R., 231, 235, 244 Bohnhoff, M., 235,236,244,248 Boldingh, J., 320, 351 Boling, L. R., 207, 249 Bolton, E. T., 41, 58 Bonnefoi, A., 294, 305, 306, 312, 313 Bonner, D., 18, 25 Boothroyd, B., 332, 350 Boring, W. D., 120, 135, 141 Borisova, I. N., 38, 39, 56 Borisova, L. N., 39, 56 Bornstein, S., 274, 283 Bortnick, L., 216, 250 Bouma, C., 254, 286 Bovee, C. W., 164, 165,193 Bowditch, H. I., 196, 244 Bowen, W. H. T., 205,213,227, 244 Boyd, D. A., 204, 244 Boyd, W. C., 222, 244 Boyd, W. S., 216, 244 Boyer, A., 201, 245 Boyle, W. J., 97, 102 Bracco, R. M., 42, 56 Bradel, S. F., 211, 247 Bradley, J. L., 218,219,244
355
AUTHOR INDEX
Bradley, S. G., 30, 37, 38, 39, 40, 41, 42, 43, 48, 50, 56, 57, 58, 59 Brandtzaeg, P., 242, 244 Braun, W., 254, 283 Brent, L., 53, 56 Brewer, J. H., 277, 286 Brian, P. W., 66, 75, 318, 347 Briggs, J. D., 303, 304, 312 Brill, N., 215, 244 Brimacombe, R., 51, 58 Brink, J. J., 53, 56 Britt, E. M., 257, 284 Broadbent, J. H., 332, 347 Brochman, J., 319, 347 Brock, T. D., 63, 75 Brody, H., 232, 244 Brooke, J. W., 220,221, 244 Brooke, M., 130, 139 Brookes, P., 35, 36, 58 Brooks, E., 265, 283 Broquist, H. P., 20,27, 263, 289 Brown, D. F., 263, 264, 266, 281, 282, 285 Brown, E. A., 227,244 Brown, E. V., 322, 347 Brown, L. R., 270, 285 Brown, L. R., Jr., 206, 244 Browning, C. H., 267, 283 Bryan, C. S., 260, 283 Bryant, A. R., 118, 119, 130, 131,140 Buchi, G., 321, 322, 347 Buck, R., 226, 244 Bulloch, W., 196, 243, 245 Bullock, E., 330, 347 Bunting, R. W., 237, 247 Burbank, R., 277, 283 Burden, R. P., 123, 125,126,136,140 Burg, R. W., 24, 26 Burgerjon, A., 304, 308, 311, 313 Burges, H. D., 293,294,295,311,313 Burgoon, C. F., 219, 244 Burgwald, L. H., 108, 139 Burkholder, W. H., 107, 139 Burkwall, M. K., 255, 262, 263, 265, 266, 270, 271, 272, 276, 277, 280, 282, 283, 285 Burman, N. P., 265, 283 Burmeister, H. R., 270, 283 Burnet, F. M., 55, 57 Burnett, G. W., 254, 283
Burns, R. O., 6, 14, 26 Burstone, M. S., 218, 243 Burton, K., 49, 57 Bush, M. T., 319, 347 Butler, W. H., 322, 324, 325, 347 Butterfield, C. T., 84, 102, 115, 123, 124, 125, 126, 127, 128, 140 Buxton, C. L., 239, 248 Buxton, E. W., 57 Bywaters, A., 103
C Calam, C. T., 29, 57 Calaway, W. T., 89, 102, 115, 140 Caldwell, A. L., 108, 131,142 Calonius, P. E. B., 204, 218, 223, 245, 251 Calvert, C. K., 113, 140 Calvo, J. M., 16, 25 Campbell, A. D., 319, 333, 334, 335, 347, 348, 349, 350, 351 Campbell, A. M., 45, 57 Campbell, J. G., 326, 347 Campbell, J. J. R., 272, 283 Campo, G., 199, 245 Cantwell, G. E., 303, 304,313 Caplow, M., 238, 245 Carll, W. T., 343, 348 Carlson, H. J., 159, 171, 175, 181,191 Carlsson, J., 208, 209, 243 Carlton, W. W., 322, 350 Carnaghan, R. B. A., 317, 319, 320, 322, 324, 325, 326, 327, 329, 331, 334, 346, 346, 347, 350 Carr, H. S., 277, 287 Casas Campillo, C., 65, 75 Caswell, M., 259, 278, 284 Catlin, B. W., 42, 57 Caulet, M., 255, 263, 264, 282, 287 Ceretto, F., 316, 348 Chaloupka, J., 23, 25 Chamberlain, C. R., 236, 249 Chamberland, 197, 249 Chambers, C. W., 105, 109, 110, 111, 114, 118, 124, 126, 127, 128, 130, 131, 132, 134, 135, 136, 138, 140, 142, 143 Chambers, L. A., 131, 140 Chang, L., 321, 347 Chang, S. B., 321,322,347
356
AUTHOR INDEX
Chang, S. L., 118, 119, 123, 125, 126, 130, 136, 140, 146, 164, 167, 170, 173,175, 178, 179,181, 182,191 Changeux, J. P., 4, 25, 26 Chaplin, C. E., 122, 140 Chapman, G. H., 255, 257, 262, 275, 277, 283, 284 Chernosvitova, V. I., 55, 56 Chesbro, W. R., 259, 263,277, 284 Cheshire, F. R., 291, 313 Chesney, W. E., 134, 142 Cheung, L. K., 347 Cheyne, V. D., 218,245 Cheyne, W. W., 291, 313 Chibata, I., 7, 26 Chigasaki, Y., 291, 312 Chi& E., 258,265, 280, 284 Chin, T. D. Y., 172, 173, 192 Chirnside, I. M., 206,207, 245 Chodkowski, A., 271, 288 Christenson, C. W., 112, 140 Christie, R., 271, 287 Churchman, J. W., 260, 284 Clark, E. M., 171, 175, 191, 193 Clark, F. Y., 196, 245 Clark, H. F., 110, 111, 140, 255, 259, 263,264, 281,282,285 Clark, M. E., 171, 173, 174, 177, 178, 179, 187, 192 Clark, W. S., Jr., 255, 263, 265, 270, 272, 280, 282, 287 Clarke, N. A., 120, 135, 141, 146, 155, 164, 167, 170, 171, 173, 175, 176, 178, 179, 181, 182, 185, 186,191 Clarke, P. H., 131, 142 Clarke, S. K. R., 240, 246 Clauss, E., 199, 245 Clayton, R. K., 23, 25 Clegg, L. F. L., 131, 138,140 Clifford, J. I., 325, 347 Clough, 0. W., 230, 231, 235, 244, 245 Codner, R. C., 318, 319,347 Coffey, J. H., 171, 173, 192 Cohen, G . N., 14, 15,25,27 Cohen, S., 162, 192 Cole, M., 19, 25 Coleman, M. B., 171, 173, 174, 177, 178, 179, 187, 192 Collins, A. A., 216, 246
Colmer, A, R., 273, 286 Colohert, L., 256, 266, 284, 286 Commoner, B., 49, 57 Conradi, H., 260, 274, 284 Cooke, W. B., 86, 87, 102 Coolidge, T. B., 237, 248 Coombs, R. R. A., 222, 250 Coomes, T. J., 332, 334, 347, 348 Cooper, K. E., 259, 260, 267, 268, 269, 270,271,274,278,279,284 Cope, E. J., 276, 288 Connack, J. F., 121, 139 Cornelius, J. A., 332, 347 Costello, R., 203, 245, 254, 264, 287 Courant, P. R., 245 Cousins, C. M., 131, 138, 140 Cox, G. J., 198, 200, 245 Cox, R. R., 53, 57 Crabtree, K., 97, 102 Crawford, J. J., 207, 245 Crawford, M., 346, 347 Crawford, R., 131, 142 Creamer, H. R., 230, 243 Criep, L. H., 241, 245 Croft, C. C., 265, 281, 284 Crook, J. C., 324, 350 Crowe, H. W., 260,284 Crowther, P. C., 332, 334, 348 Cruickshank, C. A., 227, 244 Cucullu, A. F., 331, 333, 348, 350 Culatta, C. S., 171, 193 Cunningham, L. S., 42, 57 Curds, C. R., 88, 102 Curnen, E. C., 173, 192 Custer, R. P., 225, 226, 245 D Dahiya, R. S., 234, 245 Dale, A. C., 216, 250 Dammann, C., 256, 258, 266, 273, 278, 285 Damminger, R., 200, 245 Damon, S. R., 274, 288 Dancis, J., 201, 245 Daniel, M. R., 335, 348 Danielian, A,, 223, 247 Daoust, D. R., 12, 25 Darby, C. W., 262, 279, 284 Darke, B. H., 205, 246 Datta, P., 12, 13, 25, 27
357
AUTHOR INDEX
David, T., 197, 245 Davidson, C. S., 348 Davie, E. W., 50, 59 Davies, 0. L., 36, 57 Davis, B. D., 9, 25 Davis, G. H . G., 243, 244 Davis, N. D., 318, 348 Davis, R. E., 53, 56 Dawes, C., 214, 245 Dawkins, A. W., 318, 347 Day, H. G., 204,249 Deakins, M., 218, 245 de Araujo, W . C., 211, 216,245, 246 deBarjac, H., 294, 304, 308, 312, 313 de Bruin, A. S., 256, 263, 265, 287 de Castro, C., 216, 245 Dedonder, R., 304, 313 Deibel, R. H., 254, 273, 275, 277, 278, 280, 284 Deindoerfer, F. H., 117, 140 de Iongh, H., 319, 322, 325, 332, 348, 351 Delaney, W. J., 133, 141 Delaporte, B., 293, 313 Delbruck, M., 31, 58 Demain, A. L., 8, 18, 19, 20, 21, 24, 26, 27 Dennis, E. G., 242, 247 Dennis, J. M., 168, 191 DeRobichon-Szulmajster, H., 14, 27 Derzsy, D., 317, 348 de Stoppelaar, J. D., 231, 235, 245 DeValeria, H., 19, 26 de Vogel, P., 319, 348 Diamant, H., 226, 245 Dias, F. F., 83, 84, 85, 100, 102 Dick, L. A., 260, 284 Dickens, F., 323, 338, 348 Dickinson, H. L., 319, 347 Diener, U. L., 318, 348 Diernhofer, K., 261, 284 Dietz, A. K., 235, 243 Disraely, M. N., 238, 245 Dobell, C., 196, 245 Dogon, I. L., 230, 245 Dold, H., 200, 245 Dollar, A. M., 316, 348 Dondero, N. C., 86, 102, 108,140 Done, J. T., 325, 349 Donohue, J., 275, 288
Donohue, W. L., 171, 175,191,193 Dorsey, E., 335, 347 Dostal, K. A., 118, 142 Doty, P., 41, 59 Dougherty, E. C., 232, 245 Dougherty, M. H., 93, 102 Douglas, S. R., 196, 243 Dowell, V. R., Jr., 277, 284 Drabble, W. T., 32, 59 Drake, B. D., 309, 313 Drake, C. S., 137, 140 Dreizen, S., 217, 219, 239, 245 Dreyer, G., 196, 243 Dreyer, W. T., 54, 57 Driak, F., 198, 245 Drigalski, V., 260, 284 Drobotko, V. G., 343, 348 Dubos, R., 61, 75, 203, 245, 254, 264, 287 Dugan, P. R., 97, 102 Dujardin-Beaumetz, E., 236, 245 DuIaney, E. L., 33, 57 Dunlop, S. G., 107, 140, 258, 288 Dunn, C. G., 318, 351 Dunne, H. W., 254, 286 du Pan, R. M., 239, 248 Duttweikr, D., 164, 193
E Eckenfelder, W. W., 79, 102 Edwards, S. J., 258, 260, 263, 264, 271, 276, 284 Eichel, B., 224, 226, 245 Eickenberg, C. F., 203, 251 Eisman, P. C., 110, 112, 140 Elander, R. P., 53, 57 Elliker, P. R., 108, 122, 128, 131, 141, 142 Elliot, E., 259, 278, 284 Ellsworth, S . M., 164, 191 Emmons, J., 171, 173, 192 Engebrecht, R. H., 317, 333, 348 Englehard, W. E., 164, 191 Engstrom, B., 208, 245 Ennever, J., 214, 245 Enomoto, M., 343, 349, 351 Ensign, P. R., 107, 141 Eppley, R. M., 335, 347 Erdl, 198, 245 Erebo, L., 37, 57
358
AUTHOH INDEX
Ericsson, H., 234, 243 Eschenbrenner, A. B., 322, 348 Esty, J. R., 116, 140 Ettinger, M. B., 94, 95, 102, 103 Ettinger, M . , 131, 140 Evans, C . A., 164, 191 Evans, J. B., 259, 263, 277, 284 Ezekiel, H. D., 17, 26 F Fair, G. M., 120, 123, 125, 126, 136, 140, 143, 175, 191 Falk, R. L., 330, 348 Fanelli, M. J., 270, 281, 282, 284 Fantini, A. A., 37, 57 Fnrkas-Himsley, H., 126, 140 Fast, P. G., 302, 307, 313 Favero, M. S., 137, 140 Federoff, S., 222, 251 Feingold, M., 240, 245 Fencl, Z., 15, 26 Fennell, D. I., 318, 350 Ferraro, F. M., 264, 265, 279, 284 Feuell, A. J., 332, 347 Feuss, T. V., 134, 140 Fialkov, Y . A., 343, 348 Ficinus, R., 198, 246 Fifield, C. W., 258, 260, 264, 273, 280, 286 Fildes, P., 196, 243 Finland, M., 53, 57 Fish, E. W., 217, 246 Fish, K. H., Jr., 112, 141 Fishbach, H., 335, 348 Fisher, B. J., 238, 247 Fisher, D. F., 225, 243 Fitzgerald, R. J., 212, 246, 255, 284 Fitzhugh, 0. G., 322, 334, 346, 348 Fitz-James, P. C., 292, 296, 297, 299, 300, 313 Flanagan, J. B., Jr., 228, 248 Fleming, A., 230, 246, 274, 275, 284 Fleming, W. C . , 221, 247 Flewett, T. H., 240, 246 Florey, B. M., 203, 251 Florman, A. L., 239, 240, 246 Flu, P. C., 115, 140 Flynn, E. H., 19, 26 Folinazzo, J. F., 107, 129, 143 Forbes, E., 38, 58
Forgacs, J., 315, 343, 345, 346, 348 Forget, A., 262, 266, 267, 284 Formal, S . B., 218, 248 Forsberg, O., 246 Foster, E. M . , 271, 284 Fothergill, L. D., 240, 246 Fourie, L., 322, 351 Fousek, M. D., 240, 246 Fowler, E. B., 112, 140 Fraenkel, A., 197, 246 Frame, J. D., 91, 98, 102 Francis, B. J., 332, 334, 348 Francis, T., Jr., 231, 246 Frank, B. B., 222, 250 Frank, I., 242, 246 Frank, R., 215, 246 Franklin, J. G., 256, 270, 284 Frayssinet, C., 329, 349 Frazier, W. C., 254, 271, 284 Fredette, V., 262, 266, 267, 284 Freeman, S., 319, 351 Freeman, V . J., 44, 57 Freese, E., 35, 36, 56, 57 Freese, E. B., 36, 57 Freese, J. A., 208, 209, 218, 243 Frens, A. M., 325, 351 Freundlich, M., 6, 14, 26 Frey, J. R., 178, 192 Friedman, L., 333, 336, 348, 351 Friedman, M. A., 329, 351 Frisbie, H. E., 206, 246 Frontali, L., 50, 57 Frostell, G., 208, 245, 246 Fry, R. M., 270, 284 Fndenberg, H. H., 54, 57 Fuhs, G. W., 38, 57 Fujita, C., 26 Fujita, H., 322, 349 Fujiwara, K., 255, 284 Funkhouser, J. T., 333, 347, 348
G Gabbai, 342, 348 Gajan, R. J., 334, 348 Gajdusek, D. C., 342, 343, 348 Gale, D., 233, 249 Galpin, J. V., 255, 263, 264, 282, 287 Card, S., 170, 171, 193 Gardener, J. F., 55, 58 Gardner, A. D., 196, 243
AUTHOR INDEX
Gardner, A. F., 205, 246 Carey, J. C., 271, 284 Garmise, L., 45, 56 Garrod, L. P., 261, 275, 284 Gaston, C., 203, 248 Gaudy, A. F., 92, 102 Gavin, J. B., 216, 246 Gear, J., 159, 171, 177, 192 Geldreich, E. E., 110, 111, 140 Gellis, S . S., 240, 245 Gellman, I., 92, 102 Gelpi, A. G., Jr., 278, 286 Genest, C., 332, 348 Gentelli, E. J., 87, 102 Georgi, C. E., 275, 287 Gerencser, V. F., 256, 266, 267, 284 Gerhart, J. C., 3, 26 Germaine, G. R., 43, 57 Gest, H., 12, 13, 25, 27 Chittino, P., 316, 348 Gibbons, R. J., 201, 210, 212, 216, 220, 226, 227, 231, 235, 239, 245, 246, 250 Gibson, J., 90, 102 Gibson, T., 272, 283 Gibson, W. A., 217, 246 Gilbert, R., 274, 284 Gilchrist, R. K., 225, 246 Gilcreas, F. W., 109, 117, 140, 176, 186, 192 Gillett, W. A., 38, 58 Gilley, E. J., 217, 219, 245 Gilmour, M. N., 205, 213, 244 Gilvary, C., 6, 27 Glauert, A. M., 41, 57 Clock, G. E., 218, 246 Godzeski, C. W., 19, 26 Goetz, A., 269, 284 Going, D. H., 216, 245 Goldblatt, L. A., 331, 333, 334, 348, 350 Goldblith, S. A., 263,264,282,287 Goldgraber, M. B., 242, 247 Goldhaber, P., 215, 243 Goldstein, M., 116, 117, 136, 140 Gonzales, F., 214, 246 Goodenough, R. D., 130, 141 Goodfellow, A. M., 171, 175, 193 Goodgal, S. H., 34, 59 Goodman, N. S., 298, 309, 313
359
Cordon, D. F., Jr., 220, 226, 227, 246 Cordon, M. H., 234, 246 Gorini, L., 49, 57 Goth, A., 319, 347 Cots, J. S., 15, 26 Gould, I. A., 108, 139 Gowans, C. S., 15, 26 Graff, A., 333, 351 Gram, A. L., 79, 88,102, 130 Granstrom, M . L., 129, 139, 141 Gravelle, C. R., 172, 173, 192 Gray, E., 12, 27 Green, G. E., 205, 246 Green, M., 240, 246 Green, R. H., 232, 246 Greenberg, A. E., 91, 98, 102, 114, 141 Greer, J. E., 257, 284 Gregory, K. F., 33, 57 Grich, E. R., 93, 102 Griffin, A. E., 128, 141 Grobow, E., 201, 245 Groesbeck, W. M., 277, 284 Groman, N. B., 45, 57 Gros, F., 50, 59 Grossman, L. I., 208, 246 Croth, A. H., 327, 347 Grove, J. F., 318, 347 Gruby, D., 196, 246 Guillot, N., 233, 246 Gulbransen, R., 267, 283 Gunsalus, I. C., 272, 283 Gupta, S. R., 346, 350 Gurbaxani, M., 88, 102 Gurley, W . B., 229, 246 Guthof, O., 256, 258, 266, 273, 278, 285 Gutverg, M., 217, 246 Gyllenberg, H., 267, 271, 285 Gyorgy, P., 240, 247
H Haherman, S., 217, 246 Haddow, A., 316, 349 Hadley, F. P., 237, 247 Hafer, H., 242, 248 Hajna, G. A., 259, 265, 285 Hall, H. E., 263, 264, 266, 281, 282, 285 Hall, I. M., 304, 313 Hall, J. B., 52, 57
360
AUTHOR INDEX
Halner, J. E., 349 Halton, E. H., 319, 351 Halver, J. E., 316, 327, 336, 347, 349 Hamdan, A. A., 322, 347 Hammer, B. W., 108, 142 Hammond, B. F., 237, 247 Hann, V. A., 130, 135, 141 Hannay, C. L., 258, 285, 292, 296, 297, 313 Hansen, P. A., 255, 285 Harding, J. D. J., 325, 349 Hare, G. C., 207, 248 Harford, C. G., 198, 250 Harper, W . J., 107, 116, 117, 141, 142 Harris, W . C., Jr., 128, 141 Harrison, A. P., Jr., 255, 285 Harrison, F. C., 272, 279, 285 Harrison, M., 108, 141 Harrison, R. W., 211, 234, 247 Hart, A., 328, 349 Hartley, R. D., 319, 320, 322, 347, 349 Hartman, P. A., 254, 255, 256, 262, 263, 265, 266, 270, 271, 272, 273, 275, 276, 277, 280, 282, 283, 285, 286 Hartmann, G., 261, 262, 285 Haseltine, T. R., 93, 102 Hauge, S., 268, 271, 272, 285, 287 Haus, E., 216, 244 Hawkes, H. A., 80, 82, 85, 86, 87, 89, 91, 94, 101, 102 Haxthausen, H., 260, 285 Hayes, W., 48, 57 Hays, H., 128, 141 Hazey, G. J., 126, 141 Heathman, L. S., 138, 141 Hebert, T. T., 107, 141 Hegemann, F., 235, 247 Heimpel, A. M., 294, 295, 299, 300, 301, 302, 303, 304, 305, 306, 307, 309, 313 Helmers, E. N., 91, 98, 102 Hemmens, E. S., 211,234,238,247, 250 Henderson, D. A,, 159, 164, 169, 192 Hendlin, D., 8, 24, 26 Hening, H. G., 318, 347 Henomatsu, K., 237, 251 Herring, A. S., 348 Herriot, R. H., 34, 59 Herrmann, W., 278, 285
Heukelekian, H., 80, 84, 86, 87, 88, 91, 92, 102, 103, 108, 141 Heusinkveld, M . R., 333, 349 Hewitt, C. H., 89, 101 Hidaka, Y., 237, 251 Higginson, J., 328, 349 Hill, E. O., 277, 284 Hill, J. H., 273, 285, 319, 351 Hill, T. J., 228, 247 Hine, M. K., 204, 223, 230, 231, 244, 247, 249 Hinshelwood, C. N., 79, 102 Hinshaw, W . R., 348 Hintermann, J., 325, 350 Hirch, A., 254, 257, 261, 268, 272, 275, 288 Hirsch, A., 235, 251 Hlavac, C., 33, 57 Hoadley, A. W., 107, 141 Hodges, E., 233, 247 Hodges, F., 319, 320, 347,349 Hohnl, G., 94, 97, 102 Hoerman, K. C., 231, 247 Hoffman, H., 210, 247 Holding, A. S., 327, 349 Holliday, R., 38, 57 Holman, W. L., 232, 247 Holtka, D.T.S.E., 330, 349 Honeywell, G. E., 107, 114, 128, 141 Hoover, J. R. E., 240, 247 Hoppert, C. A., 237, 250 Hopwood, D. A., 38,41,57 Horibata, K., 45, 56 Horie, S., 256, 266, 285 Horiuchi, T., 23, 26 Hombrook, M., 242, 247 Horvath, B., 232, 247 Horwood, M . P., 85, 86, 103 Howell, A., Jr., 213, 247 Howell, K. M., 242, 246 Howitt, B. F., 221, 247 Huang, H. T., 14, 26 Huang, J. C. C., 33, 57 Huang-Lo, L., 39, 57 Huber, D. A., 262, 264, 289 Hucker, G. J., 258, 259, 260, 261, 264, 265, 273, 274, 284, 288 Hueper, W . C., 316, 349 Huff, C . B., 120, 135, 141 Hugenschmidt, 218, 247
AUTHOR INDEX
Hughes, M., 13, 27 Hulse, E. C., 271, 288 Hummeler, K., 240, 247 Humphrey, A. E., 117, 140 Humphreys, E. M., 274, 284 Hunt, E. E., Jr., 243 Hunt, H. R., 237, 250 Hunter, C. A., 107, 141, 259, 273, 287 Huntsberger, D. V., 270, 285 Hurst, V., 199, 200, 201, 247 Hurwitz, E., 93, 103 Hussong, R. V., 255,269,270, 272, 287 Husz, B., 293, 313 Hutchinson, H., 115, 141 Hutchinson, J. M., 38, 58
I Ikeda, Y., 38, 57, 343, 349 Ikegmi, R., 322, 349 Iljina, T. S., 44, 56 Imanishi, M., 322, 349 Ingols, R. S., 129, 141 Ingraham, J. L., 109, 141 Ingram, G. C., 269, 283 Ingram, M., 108, 142, 269, 283 Ingram, W. M., 116, 139 Irving, G. W., 336, 344, 349 Isaacs, R., 223, 247 Isenberg, E., 80, 92, 103 Ishiko, T., 343, 349, 351 Ishioka, K., 249 Ishitani, C., 38, 57 Ishizaka, K., 242, 247 Israel, B. M., 129, 139 Itami, T., 237, 251 Ito, H., 343, 351 Ito, M., 14, 26 Ivakina, N. S., 39, 56 Iya, K. K., 269, 286 J Jackson, M., 8, 26 Jacob, F., 4, 26, 47, 57 Jacob, T. A., 8, 26 Jacobs, S . E., 293, 313 Jacobson, N. L., 270, 285 James, L. V., 55, 58 Jarai, M., 43, 58 Jarrett, J. M., 108, 141 Jasewicz, L., 83, 84, 85, 103
361
Jaulmes, C., 219, 250 Jay, P., 237, 247 Jeffrey, L. P., 112, 141 Jenkins, D., 87, 100, 103 Jenkins, F. P., 317, 323, 324, 349 Jenkins, G. D., 224, 251 Jenkins, G. N., 228, 247 Jenkins, S. H., 89, 101, 103 Jensen, A. L., 237, 244 Jerne, N. K., 54, 58 Jerrel, E. A., 293, 313 Jewell, A. B., 137, 141 Joffe, A. Z., 342, 349 Johanesson, J. K., 129, 130, 134, 141 Johnson, A., 235, 250 Johnson, C. L., 316, 347, 349 Johnson, H . G., 53, 58 Johnson, H. N., 197, 247 Johnson, R. H., 270, 285 Johnsson, H., 37, 57 Jones, F. E., 114, 141 Jones, H. E . H., 323, 338, 348 Tones, L. A., 30, 33, 42, 57, 58 Jones, M., 203, 249 Jones, P. H., 87, 103 Jukes, T. H., 51, 58 Jungeblut, C . W., 232, 247 Jurney, E. T., 112, 140
K Kabler. P. W., 107, 109, 110, 14, 1 8, i i g , 130,. 131, 136, 138, 140, 141, 142, 155, 164, 170, 171, 173, 175, 176, 178, 179, 181, 182, 185, 186, 191, 255, 259, 263, 264, 281, 282, 285 Kachmar, J. F., 86, 94, 99, 103 Kahler, D., 118, 132, 142 Kakudo, Y., 237, 251 Kalle, G. P., 15, 26 Kallings, L. O., 240, 247 Kameneva, S. V., 38, 56 Kapavets, M., 200, 201, 244 Kappy, M. S., 15, 26 Kapsinialis, B., 201, 216, 220, 246 Karelitz, S., 240, 246 Karlstrom, O., 49, 58 Karzon, D. T., 162, 192 Kasai, G. J., 238, 247 Kasper, J. A., 276, 288
362
AUTHOR INDEX
Katchalski, E., 52, 56 Kato, J., 7, 26 Katz, M., 316, 348 Kauffman, A. L., 238, 247 Kaufman, I. J., 205, 244, 250 Kaufman, W . T., 98, 102 Kawai, A., 322, 349 Kawamata, J., 322, 349 Keary, G. T., 205, 246 Keefer, C. E., 93, 94, 103 Kelly, S. M., 109, 117, 140, 151, 164, 171, 172, 173, 174, 176, 177, 178, 179, 180, 181, 183, 184, 186, 187, 188, 192 Kelman, A., 107, 141 Kempf, J. E., 185, 192 Kenner, B. A., 255, 259, 263, 264, 281, 282, 285 Kereluk, K., 255, 281, 286 Kerr, A. C., 230, 234, 245, 247 Kessel, J . F., 159, 192 Keunning, R., 322, 351 Keyes, P. H., 254, 255, 284, 286 Khorana, H . C., 51, 58 Kidder, C. W., 116, 141 Kihlman, B. A., 33, 58 Kimball, R. F., 34, 58 King, R. M., 208, 247 Kinnear, J., 267, 271, 286 Kinoshita, S., 8, 11, 14, 26, 27 Kinosita, R., 349 Kirk, E . C., 214, 247 Kirsner, J. B., 242, 247 Kisumi, M., 7, 26 Kitada, S., 11, 14, 26 Kjellander, J., 256, 257, 285 Klebanoff, S. J., 230, 247 Klein, G., 98, 102 Klein, H., 218, 227, 243, 247 Kleinberg, I., 288, 247 Kleineberger-Nobel, E., 286 Klencke, H., 198, 247 Kletzinsky, D., 229, 247 Kling, C., 185, 192 Klinkhamen, J. M., 226, 247 Knapp, E. P., 87, 102 Kneeland, Y., Jr., 199, 247 Knighton, H. T., 203, 235, 247 Knowles, D. S., 171, 175, 191, 193
Knox, A. W., 232, 247 Kobayashi, Y., 343, 351 Koch, F. E., 256, 261, 273, 276, 286 Koch, H., 343, 345, 348 KZhler Ellingsen, J., 268, 271, 272, 285, 286 Koelensmid, W. A. A. B., 319, 320, 348, 351 Koenig, V. L., 319, 351 Kofoid, C. A., 196, 247 Kohler, H., 317, 349 Kolesnikov, G. P., 240, 247 Konetzka, W. P., 277, 283, 288 Konno, J., 241, 248 Konyouklova, M. V., 39, 56 Koser, S. A., 233, 238, 247 KosteEka, F., 200, 201, 202, 218, 247 Kotin, P., 330, 348 Kozinn, P. J., 219, 250 Kramer, E., 261, 273, 286 Kramer, H. P., 134, 141 Kramer, S. D., 159, 192 Krantz, G. E., 254, 286 Krasilnikav, N. A., 29, 58 Krasner, R. I., 233, 251 Krasse, B., 216, 220, 223, 226, 234, 248, 250 Kraus, F. W., 203, 235, 241, 242, 244, 248, 249 Kraus, H., 15, 26 Kraus, L. S., 80, 103 Krauss, M. R., 42, 56 Kraybill, H. F., 316, 318, 322, 323, 326, 350 Kreckova, P., 23, 25 Kriieger, B. J., 171, 178, 180, 181, 191, 192 Krumwiede, C., 260, 286 Kulik, Y. I., 319, 349 Krup, M., 93, 103 Kryiasides, K., 116, 141 Krywienczyk, J., 298, 308, 313 Kull, F. C., 110, 112, 140 Kunn, R., 240, 247 Knroda, S., 43, 58 Kurtz, H. M., 221, 249 Kushner, D. J., 305, 313 Kuwagata, M., 237, 251 Kuznetsov, V. D., 55, 58
AUTHOR INDEX
L Labrec, E. H., 218, 248 Lachica, 17. F., 255, 256, 263, 270, 273, 286 Lackey, J. B., 86, 87, 99, 103, 115, 140 Lacy, I. O., 129, 141 Lafarge, C., 329, 349 Lagergren, c., 246 Lake, D. E., 280, 284 Lammers, T., 202, 223, 236, 242, 248 Lampen, J. O., 23, 26 Lancaster, M. C., 317, 323, 324, 349 Landers, K. E., 318, 348 Langelier, W. F., 118, 141 Langston, C. W., 254, 286 Lanni, F., 232, 250 LaRoche, G., 316, 349 Larson, C. P., 316, 351 Lathrop, F. D., 225, 226, 248 Law, A. B., 134, 141 Lawley, P. D., 35, 36, 58 Laxminarayana, H., 269, 286 Lazareva, M. F., 83, 84, 85, 103 Lear, C. S. C., 228, 248 Leber, T., 198, 248 LeBras, G., 14, 27 LeBreton, E., 329, 349 Leder, P., 51, 52, 58 Lederberg, E. M., 46, 58 Lederberg, J., 38, 42, 46, 57, 58 Lederrr, S. J., 133, 141 Ledingham, J. C. G., 196, 243 Lee, G. F., 129, 141 Lee, L. S., 331, 333, 348, 350 Lee, W. V., 332, 349 Leeffang, K. W. H., 114, 141 Legator, M. S., 328, 335, 349 Lehner, T., 219, 248 Leidy, G., 42, 56 Leifson, E., 108, 110, 141 Lengyel, P., 51, 58 Lensen, J. G., 185, 192 Lenz, H., 215, 250 Leoni, L., 50, 57 Lepine, P., 185, 192 Lerman, L. S., 43, 58 Lester, M. R., 147, 191 Leung, S. W., 212, 248 Levaditi, S. G., 185, 192
363
Levy, B. M., 212, 213, 214, 218, 248 Lewis, C., 324, 350 Lewis, D., 58 Lewis, G., 325, 326, 329, 346, 347, 349 Lewis, R. F., 108, 141 Lichstein, H. C., 255, 262, 266, 286, 288 Lightfoot, L. H., 237, 248 Lilienthal, B., 204, 215, 234, 248 Lilley, B. D., 277, 286 Lingens, F., 15, 26 Lingens, S., 15, 26 Linton, A. H., 260, 267, 268, 269, 271, 284 Lisanti, V. F., 224, 226, 245 Lisbonne, 342, 348 Liston, J., 258, 287 Liu, S. F., 225, 226, 248 Litsky, W., 258, 260, 264, 273, 280, 286, 287 Littmann, M. L., 84, 102 Lonnerblad, T., 246 Loew, O., 261, 286 Loosmore, R. M., 325, 349 Love, D., 318, 347 Lu, K. H., 204, 248 Lubinskaya, S. I., 55, 56 Ludwig, H. F., 118, 141 Ludzack, F. J., 87, 91, 94, 102, 103 Luebke, R. G., 230, 247 Lungren, D. G., 97, 102 Luria, S. E., 15, 26, 31, 58 Lynde, E. J., 120, 139 367
M Maas, W. K., 4, 15, 26, 46, 58 McAndrew, J, R., 225, 246 McCabe, J., 79, 102 McCabe, L. J., 116, 117, 136, 140 McCarthy, B. J., 41, 58 McCarthy, C., 200, 201, 248 McCarthy, P. L., 219, 244 McCarty, M., 42, 56,254,286 MacConkey, A,, 272, 276, 286 McConnell, E., 303, 304, 313 McCormick, M. H., 19, 26 McCoy, E., 97, 102, 107, 141 McCulloch, E. C., 122, 130, 137, 141 McDermott, G. M., 95, 102 McDonald, I. j.,273, 286
364
AUTHOR INDEX
Macdonald, J. B., 207, 216, 228, 245, 246, 248, 250 Macdonald, K. D., 38, 58 MeDougall, W. A., 208, 209, 210, 213, 218, 248 McEachen, D. C., 224, 248 McEwen, F. L., 311, 313 McFall, E., 4, 26, 46, 58 McGonagle, M. P., 332, 350 MacGregor, A., 206, 248 MacGregor, D. R., 122, 141 Mach, B., 52, 58 Mack, W . N., 171, 178, 180, 181, 191, 192 McKenzie, D. A., 256, 258, 259, 261, 262, 267, 268, 270, 271, 286 McKeown, J. J., 121, 141, 142 McKhann, C . F., 159, 171, 175, 181, 191 McKinney, R. E., 82, 83, 84, 85, 86, 88, 89, 103 McLanghlin, J., Jr., 335, 351 McLean, D. M., 151, 192 MacLeod, C. M., 42, 53, 56, 57 MacLeod, R. A., 272, 273, 286 McNamee, P. D., 84, 102 McNary, R. R., 93, 102 McNulty, P. J., 134, 141 Macphee, G. G., 229, 248 Magnani, T . J., 218, 248 Malaney, G. W . , 117, 130, 140, 142 Mallmann, W . L., 118, 132, 142, 171, 178, 180, 181, 191, 192, 255, 258, 260, 262, 264, 273, 278, 279, 280, 281, 284, 286 Mandel, I. D., 212, 213, 214, 216, 218, 248, 251 Manson, E . M., 224, 248 Markson, L. M., 325, 349 Marliac, J. P., 335, 351 Marmur, J., 41, 59 Marsland, E. A., 206, 248 Martin, G. R., 214, 249 Martin, R. G., 4, 25, 50, 58 Martin, W . J., 227, 250 Martin, W . R., 235, 236, 244 Martin, W . R., 278, 286 Mashimo, A., 237, 251 Massler, M., 206, 215, 248, 249 Mastromatteo, L., 258, 273, 286 Mateles, R. I., 319, 330, 334, 346, 349
Matselyukh, B. P., 43, 58 Matt, M. M., 239, 248 Matter, L. D., 164, 192 Mattes, O., 291, 313 Matthaei, J. H., 51, 58 Mattick, A. T. R., 235, 251 Maxcy, K. F., 162, 164, 192 Mayer, C. F., 342, 346, 349 Mayeux, J. V., 273, 286 Mayne, R. Y., 331, 348 Mead, C. C., 256, 265, 268, 286 Measrock, V., 159, 171, 177, 192 Mechalas, B. J., 303, 309, 313 Medawar, P. B., 53, 56 Medrek, T. F., 269, 277, 286 Megna, J. C., 309, 313 Megregian, S., 124, 126, 127, 128, 140 Meisel, I., 94, 103 Melnick, I. L., 171, 173, 174, 192 Menkin, V., 224, 248 Mergenhagen, S . E., 214,242, 249 Merrifield, R. B., 56, 58 Merrill, J. P., 53, 58 Meszaros, J., 317, 348 Metzenberger, R. L., 15, 26 Meyer, K. F., 116, 140 Meyer, P., 273, 288 Meyer, R. L., 110, 112, 140 Meyers, F. P., 23, 27 Mickelson, M. N., 278, 286 Middleton, J. D., 214, 248 Mieth, H., 256, 258, 266, 270, 271, 276, 282, 286 Mikhailova, G. R., 39, 58 Miles, W. T., 198, 251 Millarr, C., 237, 244 Miller, C . P., 234, 235, 236, 244, 248 Miller, E., 322, 348 Miller, H. I., 241, 242, 248 Miller, M. W., 87, 102, 340, 349 Miller, W. D., 197, 198, 227, 248 Millward, E., 227, 250 Mindlin, S. Z., 38, 39, 56, 58 Minuse, E., 231, 246 Misawa, M., 8, 26 Mitchell, C. T., 220, 243 Miyake, M., 343, 349, 351 Mizukami, I., 50, 58 Mocquot, C., 255, 263, 264, 282, 287 Mdler, A., 37, 57
365
AUTHOR INDEX
Monod, J., 4, 26, 47, 57, 79, 103 Monro, R. E., 297, 298, 299, 313 Montgomery, P. W., 218, 248 Moody, D. P., 330, 350 Moore, B., 172, 192 Moore, D. F., 224, 248 Moore, D. H., 239, 248 Moore, E. W., 175, 191 Moore, F. J., 159, 192 Moore, N. A., 233, 236, 250 Moore, W. A., 134, 141 Moorrees, C. F. A., 228, 248 Morelis, P., 256, 266, 284, 286 Mori, M., 237, 251 Morowitz, H. J., 95, 103 Morris, D., 19, 25 Morris, J. C., 123, 125, 126, 130, 136, 140 Morris, R. G., 220, 249 Morris, W., 262, 286 Morrison, H. B., 108, 142 Morrison, L. F., 224, 249 Morse, M. L., 46, 58 Mosely, J. W., 164, 192 Moser, R. H., 164, 193 Mossel, D. A. A., 108, 142, 256, 263, 265, 282, 286, 287 Moyed, H . S., 4, 17, 26 Miihlemann, H. R., 209, 210, 215, 249, 250 Mueller, J. H., 277, 287 Mueller, W. S., 128, 142 Muhler, J. C., 204, 249 Mulder, E. G., 108, 142 Munch-Petersen, E., 271, 287 Mundt, J. O., 254, 268, 287 Murphy, J. M., 207, 249 Murphy, W. T., 159, 192 Murray, M. M., 218, 246 Musilkova, M., 15, 26 Myers, H. I., 233, 236, 250
N Nabney, J., 325, 332, 346, 350 Naeslund, C., 213, 249 Nakabayashi, N., 322, 349 Nakamura, K., 15, 26, 38, 57 Nakamura, T., 249 Nakayama, K., 8, 11, 14, 26 Nankivell, A. T., 109, 142
Nara, T., 8, 14, 26 Nash, L., 224, 249 Nason, H. K., 128, 133, 142 Neefe, J. R., 165, 168, 175, 176, 187, 193 Neidhardt, F. C., 24, 26 Nelson, A. A., 319, 320, 322, 347, 348, 349 Nelson, R. R., 37, 58 Nesbitt, B. F., 319, 320, 322, 332, 349, 350 Nesheim, S., 333, 334, 348, 350 Neter, E., 273, 287 Nevin, T. A., 221, 249 Newberne, P. M., 322, 324, 350 Nicholls, E. E., 277, 288 Nicholls, P., 267, 287 Nickerson, J. F., 235, 249 Niedl, C., 178, 180, 183, 184, 187,192 Niemala, S., 267, 285 Ninard, B., 325, 350 Nirenberg, M., 51, 58 Nirenberg, M. W., 51, 52, 58 Nissle, A., 236, 249 Niven, C. F., Jr., 254, 255, 278, 280, 284, 286, 287, 288 Noguchi, Y., 349 Nordbring, P. W., 240, 249 Nordstrom, K., 37, 57 Norman, N. N., 107, 142 Norris, G. L. F., 318, 347 Norris, J. R., 293, 294, 295, 296, 298, 300, 307, 313 Norton, I. L., 258, 285 Novack, R. M., 322, 347 Novick, A., 23, 26, 32, 58 Nuckolls, I., 206, 246 Nunheimer, T. D., 19, 27
0 Oakberg, E. F., 15, 26 Obiger, G., 271, 272, 288 Ochoa, S., 51, 58 Oe, M., 237, 251 Oettle, A. G., 328, 349, 350 O’Kelly, J., 317, 319, 320, 322, 324, 331, 334, 346, 347, 349, 350 Okuda, K., 50, 59 Olivieri, V. P., 129, 139 Olson, J. C., Jr., 128, 142
366
AUTHOR INDEX
O’Neal, C., 51, 58 ONeil, F. W., 143 O’Neil, R., 221, 249 Onise, M., 249 Orban, B., 207, 219, 221, 249 Ord, W. O., 319, 320, 322, 348, 351 Orford, H. E., 80, 92, 103 Orgel, L. E., 33, 58 Ormsby, H. L., 147, 193 Oshrian, H., 216, 251 Ostrolenk, M., 259, 273, 287 Ostler, D. C., 326, 351 Ostroukhov, A. A., 55, 58 Ouchterlony, O., 234, 243 P Packer, R. A., 256, 259, 261, 262, 263, 264, 287 Page, N. A., 225, 226, 248 Paget, G. E., 317, 350 Painter, H. A,, 103 Palin, A. T., 135, 142 Palmer, C. E., 227, 243 Palmer, C. M., 108, 142 Papazian, H. P., 36, 58 Pappas, G. D., 216, 251 Pardee, A. B., 3, 4, 17, 23, 26, 27 Parikh, S. R., 206, 249 Parker, R. B., 108, 128, 131, 142, 200, 201, 248 Parson, T. W., 15, 26 Pasteur, L., 197,249, 291, 313 Patterson, D. S. P., 326, 329, 347 Patterson, J. S., 324, 350 Paul, F., 213, 247 Paul, J. R., 170, 171, 193 Paulus, H., 12, 27 Paunio, I., 245 Paxton, J. A., 108, 139 Payne, W. W., 316, 349 Perkel, N. V., 342, 350 Perry, C . A,, 259, 265, 274, 285, 287 Perry, W. I., 235, 249 Person, L. H., 107, 141 Petran, E., 274, 287 Petrie, L. M., 159, 192 Petrucz, K., 238, 243 Pettenella, G., 273, 287 Pfaundler, M., 199,200, 249 Pfeifer, G., 200, 201, 244
Phaff, H. J., 87, 102 Phillips, R. A., 86, 102 Phillips, R. W., 205, 250 Phillips, V. A., 114, 142 Philp, J. McL., 317, 320, 323, 324, 349, 350, 351 Philps, R. H., 329, 350 Perce, G. O., 138, 141 Pierce, M. A. F., 265, 283 Pierce, M. E., 185, 192 Pierson, D. J., 17, 25 Pike, R. M., 258, 259, 266, 275, 287 Pillai, S. C., 88, 99, 103 Pipes, W. O., 87, 100, 103 Pisu, I., 258, 286 Platonow, N., 324, 350 Platt, B. S., 346, 350 Poczenik, A., 164, 193 Pollock, M. R., 30, 58 Pons, W. A., 333, 334, 350 Pontecorvo, G., 38, 58 Porges, N., 83, 84, 85, 103 Porter, J. R., 23, 27 Poskanzer, D. C., 164, 193 Pourquier, 342, 348 Powlen, D. O., 237, 251 Pratt, J. S., 201, 249, 260, 286 Prescott, S. C., 287 Prickett, C. O., 324, 350 Prinz, H., 229, 249 Proctor, B. E., 269, 288 Proctor, C. M., 130, 134, 136, 140 Prokopovitsch, L., 317, 348 Prophet, A. S., 206, 249
Q Quasso, F. W., 328, 350 Quillman, P., 205, 243 Quinley, R. L., 258, 287
R Rafelson, M. E., 15, 27 Rafelson, M. E., Jr., 33, 59 Rahn, O., 116, 142 Raibaud, P., 255, 263, 264, 282, 287 Rainbow, C., 254, 287 Raj, H., 258, 287 Rakus, L. M., 134, 141 Ramadan, F. M., 259, 268, 270, 274, 278, 279, 284 Ramakrishnan, T., 17, 27
AUTHOR INDEX
Rammell, C. G., 273, 287 Ramstrom, G., 208, 245 Randall, G. B., 137, 140 Randel, H. W., 164, 165, 193 Rantasalo, I., 256,267, 268, 287 Raper, K. B., 58, 318, 350 Rasmussen, A. F., 197, 249 Rawls, W. B., 277, 284 Rayman, M. M., 262, 264, 289 Recondo, A. M., 329, 349 Reed, A. I., 239, 245 Reese, M. K., 199, 200,251 Reid, G. R., 322, 334, 346 Reif, W., 231, 235, 244 Reimold, G., 200, 245 Reinbold, G. W., 254, 255, 263, 265, 269, 270, 272, 275, 280, 282, 285, 287 Reinhold, J. G., 165, 168, 176, 187, 193 Reiss, F., 219, 250 Reitter, R., 142 Renkonen, K. O., 204, 251 Renn, C. E., 134, 142 Reynoldson, T. B., 89, 103 Rheins, B. T., 132, 138, I42 Rhian, M., 185, 192 Rhines, C. E., 110, 142 Rhodes, A., 332, 350 Rhodes, A. J., 146, 147, 150, 151, 153, 154, 155, 156, 157, 158, 159, 161, 162, 163, 169, 171, 173, 175, 191, 193 Rich, H., 333, 351 Richards, A. G., 303, 304, 313 Richards, T., 262,267,268,287 Richardson, L. R., 345, 351 Richardson, R. L., 203, 213, 249 Ridenour, G. M., 129, 141, 142, 159, 171, 175, 181, 191 Riegle, B. J., 178, 192 Riehl, M. L., 132, 138, 142 Riera, M., 208, 209, 218, 243 Rihova, L., 23, 25 Riley, M., 4, 17, 27 Ritchie, R. C., 171, 175, 191, 193 Ritter, C., 258, 262, 287 Ritz, H. L., 210, 249 Rivera, R., 45, 56 Rivers, M. R., 121, 139
367
Rizzo, A. A., 213, 214, 220, 242, 243, 247, 249 Roach, A. W., 115, 137,142 Robeck, G. G., 118, 142 Robert, T. C., 330, 347 Robertson, T. A., 333, 350 Robin, C., 249 Robinson, H. B. G., 207, 249 Robinson, J. R., 226, 249 Rochaix, A., 271, 287 Roe, A. S., 42, 56 Rolla, G., 231, 232, 249 Rogers, H., 325, 346 Rogers, H. J., 239, 249 Rogoff, M. H., 298, 309, 313 Rogovskaya, T. I., 83, 84, 85, 103 Rohlich, G. A,, 97, 102 Roine, P., 271, 285 Roper, J., 38, 58 Rose, A. H., 254, 287 Rose, E. J., 217, 244 Rose, H. M., 277, 287 Rose, K. D., 275, 287 Rose, M., 236, 249 Rose, R. E., 280, 287 Rose, S. S., 225, 249 Rosebury, T., 232, 233, 242, 249, 254, 287 Rosen, S., 237, 250 Rosenkranz, H. S., 277, 287 Rosenthal, E., 216, 250 Rosenthal, L., 222, 249 Rosenthal, S. L., 216, 244 Rothe, W. C., 258, 262, 287 Rottenstein, J. B., 198, 248 Rottman, F., 51, 58 Rouf, M. A., 97, 103 Roux, 197, 249 Rovelstad, G. H., 224, 249 Rowbury, R. J., 15,22, 23, 27 Rubbo, S. D., 55, 58 Rubinshteyn, Yu. I., 350 Ruchhoft, C. C., 84, 86, 94, 99, 102, 103 Ruchman, I., 197, 249 Rudolph, C. E., Jr., 206, 244 Rueber, F. M., 107, 114,128, 141 Ruefenacht, W. G., 236, 249 Ruger, M., 33, 57 Rupert, C. S., 34, 59 Rushton, M. A., 204, 249
AUTHOR INDEX
Rutter, R. R., 236, 249 Ryan, F. J., 36, 59 Rycroft, J. A., 259, 277, 287
S Sabin, A. B., 170,193, 197,249 Saheki, K., 256, 266, 285 Saito, M., 343, 349, 351 Sakai, F., 343, 349, 351 Sakai, Y., 343, 349, 351 Sakellariou, E., 93, 103 Saleh, B. A., 263, 264, 282,287 Salkind, A., 216, 251 Salmon, W. D., 324, 350 Salomon, R., 199, 200, 249 Salotto, B. V., 95, 102 Salviolo, J. A., 207, 249 Samejima, H., 14, 26 Sandborn, J. R., 127, 128, 142 Sanders, J. C., 332, 348 Sanderson, W. W., 151, 171, 172, 173, 174, 178, 179, 180, 181, 183, 184, 186, 187, 188, 192 Sandholzer, L. A., 255, 256, 258, 276, 289 Sandine, W. E., 128, 141 Sandvik, O., 287 Saraswat, D. S., 254, 255, 263, 265, 270, 272,275,280,282,285,287 Sargeant, K., 317, 318, 319, 324, 331, 334, 346, 347, 350 Sato, Z., 8, 26 Saul, R. A., 270, 283 Saunders, J. B. de C. M., 206,246 Sawyer, C. N., 78, 91, 98,102,103 Sawyer, S., 216, 246 Scalettar, E., 239, 246 Scarlett, C. A., 131, 142 Schaedler, R. W., 203, 245, 254, 264, 287 Schafer, W., 257, 274, 288 Schafer, W. D., 112, 140 Schaffer, E. M., 216,217,221,244,250 Schaffer, R.B., 91,94,103 Schattenfroh, A., 288 Scherago, M., 115, 141 Scherp, H. W., 202,226,250, 254,283 Scherr, G. A., 33, 59 Scherr, G. H., 15, 27 Schick, B., 239, 246
Schildkraut, C. L., 41, 59 Schlessinger, D., 50, 59 Schlossmann, A., 199, 200, 249 Schneider, H., 218, 248 Schneider, U. K., 209, 210, 249 Schneyer, L. H., 229, 250 Schoental, R., 324, 330, 350 Schoof, H. F., 171, 173,192 Schrack, W. D., 164, 192 Schroeder, A., 207, 250 Schroeder, H. E., 215, 250 Schroff, J., 225, 250 Schulhoff, H. B., 84, 102 Schweitzer, B., 199, 200, 250 Scott, D. B., 214, 249 Scott, de B., 322, 351 Scrimshaw, N. S., 318, 351 Scrivener, C. A., 233, 236, 249, 250 Sedat, J. W., 52, 57 Seelemann, M., 271, 272, 288 Segerstad, L. H. A., 208, 245 Seiberling, D. A., 107, 116, 142 Seifert, E., 225, 250 Sekiya, K., 255, 284 Seligmann, E. B., Jr., 262, 286 Seligmann, R., 142 Seltsam, J. H., 232, 250 Seltzer, S., 205, 244, 250 Semans, H. M., 199, 250 Serebryanyy, S., 343, 348 Sermonti, G., 38,57, 58, 59 Setlow, J. K., 34, 59 Setlow, R. B., 34, 59 Sevag, M. G., 32, 59 Severens, J. M., 264, 284 Shank, R. C., 329, 350 Shankle, R. J., 207, 245 Shannon,A. M., 113, 121,142 Shannon, I. L., 217, 246 Shano, A., 324, 350 Shapleigh, E., 222, 244 Sharpe, M. E., 256, 267, 268, 269, 270, 284, 288 Sharry, J. J., 223, 250 Shattock, P. M. F., 254, 288 Shaw, E. W., 173, 192 Shay, E. G., 131, 142 Shellenberger, P. R., 270, 285 Sheppard, D. E., 17, 27 S h n , 0. C., 333, 349
AUTHOR INDEX
Sheridan, A,, 317, 319, 331, 350 Sherman, J. M., 255, 265,273,276,288 Shibuya, M., 234, 250 Shifrine, M., 87, 102 Shikata, T., 343, 349, 351 Shimada, F. T., 171, 175,191,193 Sdvey, J. K. G., 115,137,142 Shimkin, M. B., 316, 318, 322, 323, 326, 350 Shiota, T., 238, 245 Shone, G., 332, 347, 348 Shui-Chin Chen, 333, 351 Shull, I. F., 258, 287 Suer, W. G., 326, 351 Silverman, G. J., 263, 264, 282, 287 Sim, G. A,, 347 Simonton, G. W., 217, 244 Sims, W., 278, 288 Singer, S., 298, 309, 313 Sinnhuber, R. O., 317,333,334, 348,351 Skriabin, G. K., 29, 58 Slack, G. L., 227, 250 Slanetz, L. W., 255, 258, 262, 267, 279, 281, 288 Slavin, G., 271, 289 Slobodkin, L. B., 95, 96, 99,103 Small, W., 259, 278, 284 Smiley, K. L., 255, 288 Smimoff,W. A., 294,299, 303,305,313 Smith, B., 22, 27 Smith, D. M., 332, 348 Smith, G. H., 250 Smith, H. F., 120, 135, 141 Smith, H. M., 265, 283 Smith, H. R., 320, 347 Smith, J. F., 274,275, 288 Smith, J. W., 199, 250 Smith, L., 239, 250 Smith, L. W., 232, 244 Smith, M. H., 214, 250 Smith, R. H., 329, 351 Smuckler, S. A., 266, 267, 288 Smythe, C.V., 309, 313 Snell, E. E., 263,272,273,286, 289 Snow, W. B., 142 Snyder, M. L., 200, 201, 248, 255, 257, 262, 266, 273, 286, 288 So, A. G., 50,59 Socransky, S. S., 201, 210, 212, 216, 220, 226, 227, 239, 246, 250
369
Sognnaes, R. F., 214, 246 Sohier, R., 219, 250 Solberg, M., 269, 288 Somerson, N. L., 19, 27 Sommerfield, G. A., 332, 350 Soule, M. H., 185, 192 Soulides, D. A., 262,267,268, 287 Soulides, E., 262, 267,268,287 Sparreboom, S., 322, 351 Spaulding, C. H., 117, 137, I39 Spaulding, C. W., 113, 142 Speck, M. L., 234,245 Spensley, P. C., 318, 335,344,351 Speyer, J. F., 51, 58 Spies, T.D., 217, 219, 239, 245 Spino, D. F., 107, 142 spizizen, J., 42, 59 Splittstoerser, C. M., 311, 313 Splittstoesser, D. F., 258, 259, 261, 264, 265, 273, 274, 288 Spratt, J. S., Jr., 225, 250 Springer, G. F., 222, 250 Srere, P. A., 273, 288 Stableforth, A. W., 271, 288 Stadler, L. J., 34, 59 Stadtman, E. R., 5, 6, 14,27 Stainsby, W. J., 277, 288 Stamper, M. C., 19, 26 Stapert, E. M., 107, 114, 128, 141 Starkey, R. L., 121, 142 Stauffer, J. F., 30, 56 Stebbins, M. R., 185, 192 Steed, P. D. M., 221, 250 Steen, E., 216, 251 Steinhaus, E. A,, 293, 313 Stephan, R. M., 208,211,220,238,250 Sternberg, G. M., 197, 250 Stevens, L., 334, 348 Stevenson, L. G., 250 Stevenson, R. E., 118, 119, 140, 173, 176, 178, 179, 181, 182, I91 Stewart, G. G., 208, 246 Stewart, R. J. C., 346,350 Stocker, B. A. D., 70, 75 Stokes, J., Jr., 165, 168, 175, 176, 187, 193 Stokes, J. L., 97, 103, 109, 141 Stoloff, L., 333, 350, 351 Stoudt, T. H., 12, 25 Stuart, P., 271, 288
370
AUTHOR INDEX
Stnedell, J. T., 233, 247 Sturani, E., 13, 27 Subrahmanyan, V., 88, 99, 103 Sugimoto, S., 237, 251 Sulkin, S. E., 198, 250 Suznki, T., 8, 26 Swaboda, R., 317, 349 Swan, A., 271, 288 Swartz, M. L., 205, 250 Swern, M., 255, 269, 270,272, 287 Swinburne, L. M., 222, 250 Sylvester, C. J., 237, 250 Szilard, L., 53, 59 Szita, J., 276, 288
T Tabak, H. If., 109, 110, 114, 140, 142 Tachibana, Y., 249 Takakura, S., 249 Talbot, H. S., 225, 250 Taschdjian, C. L., 219, 250 Tasman, A., 239, 250 Tatsuno, T., 343, 349, 351 Tatum, E. L., 52, 58 Taylor, D. F., 233, 249 Tecce, G., 50, 57 Tennenbaum, S., 112, 116, 142 Terner, C., 242, 250 Teteryatnik, A. F., 46, 56 Tewfik, E. M., 41, 59 Thiercelin, M. E., 253, 288 Thiers, H. D., 345, 351 Thomas, J. W., 275, 288 Thomas, R. C., 115, 143 Thomassen, P. R., 236, 249 Thompson, M. H., 303, 304, 313 Thompson, R., 234, 235,250 Thompson, S. J., 330, 348 Thorne, C. B., 45, 59 Tilden, E. B., 319, 351 Tissieres, A., 50, 59 Tolmach, L. J., 43, 58 Tomaszewski, W., 223, 251 Torrey, J. C., 199, 200, 251 Toth-Baranyi, I., 317, 348 Toumanoff,C., 293,297,300,305,313 Touster, O., 319, 347 Trager, W. T., 333, 351 Trask, J. D., 170, 171,193 Traxler, R. W., 114, 142, 143
Treece, E. L., 262, 287 Treick, R. W., 277, 288 Tribby, I., 233, 247 Triolo, G., 230, 251 Troch, P., 274, 284 Trott, J. R., 215, 251 Trown, P. W., 22, 27 Trupin, J., 51, 58 Trupin, J. S., 20, 27 Tsukioka, M., 343, 349, 351 Tsuneishi, N., 269, 284 Tucker, F. L., 275, 288 Turner, B. G., 92, 102 Turner, R. O., 117, 142
U Uchida, K., 38, 57 Udagawa, K., 8, 26 Udaka, S., 8, 27 Uemura, I., 50, 52, 56, 59 Umbarger, H. E., 4, 6, 14, 16, 17, 25, 26, 27 Umemoto, Y., 237, 251 Underwood, A. S., 198, 251 Underwood, J. G., 330, 347 Uraguchi, K., 343, 349, 351 Urbach, F., 219, 244 Ushida, T., 322, 349
V Vallquist, B., 234, 243 Valude, 230, 251 Vanderleck, J., 272, 279, 285 van der Linde, J. A., 325,351 van der Merwe, K. J., 322, 340, 351 van der Zijden, A. S. M. B., 320, 322, 351 vanDiepen, H. M. J., 256,263,265,287 van Dorp, D. A., 322, 351 van Esch, G. J., 325, 351 Van Horn, H. H., Jr., 270, 285 Van Horn, M., 117, 142 van Houte, J., 211, 246 Van Kesteren, M., 218, 245 Van PeIt, J. G., 325, 332, 348 VanRooyen, C. E., 146, 147, 150, 151, 153, 154, 155, 156, 157, 158, 159, 161, 162,163,169, 173, 193 Vera, H. D., 257, 270, 288 Verret, M. J., 335, 351 VignaI, G., 197, 251
AUTHOR INDEX
Viney, M., 103 Vitali, R. A., 8, 26 Vladimirov, A. V., 39, 58 Vles, R. O., 319, 322, 325, 332, 348 170ge1, H. J., 4, 27 Vogt, J. E., 164, 193 Voltonen, V., 204, 251 von L. Buxton, R., 226, 244 von Rhee, R., 319, 348 Voreadis, E. G., 213, 251
W Waerhaug, J., 216, 251 Wagg, B. J., 202, 204, 251 Wahl, R., 273, 288 Wainwright, S. D., 50, 59 Waksman, S. A., 65, 75 Wallace, W. M., 113, 121, 142 Walper, J. F., 275, 288 Wang, W.-L. L., 258, 288 Warin, R. P., 240, 246 Warner, B. W., 233, 236, 250 Wasserman, B. H., 212, 214, 218, 248 Wasserman, R. H., 231, 243 Watkins, J. H., 86, 103 Watson, D. H., 296, 298, 313 Watson, T. A., 224, 248 Wattie, E., 84, 86, 87, 103, 123, 124, 126, 127, 128, 132, 140, 143 Weaver, R. H., 115, 141, 256, 262, 266, 267, 284, 286 Webb, B. D., 345, 351 Webb, S . J., 222, 251 Wedderhurn, D. L., 234, 247 Weichlein, R. G., 82, 83, 84, 85, 103 Weil, I., 123, 125, 126, 136, 140 Weinherg, E. D., 288 Weindling, R., 115, 143 Weinmann, J. P., 219, 221, 249 Weinstein, E., 216, 251 Weinstein, P. R., 205, 246 Weisberg, E., 86, 102 Weisberger, D., 238, 251 Weiser, H. H., 117, 132, 138, 142 Weissenbach, R.-J., 259, 276, 288 Welch, J. L., 107, 129, 143 Welch, W. H., 197, 251 Welsch, M., 30, 44, 59 Wertheimer, F. W., 217, 251 Wheater, D. M., 235, 251
371
Whipple, G. C . , 120, 143 Whipple, M. C., 120, 143 White, A. F., 330, 351 White, E. C., 273, 285, 319, 351 White, J. C., 255, 276, 2SS White, M. F., 276, 288 White-Stevens, R. H., 343, 345, 348 Whitlenhury, R., 280, 288 Whitley, 0. R., 274, 288 Whitman, L., 277, 287 Wick, E. L., 318, 321, 322,347,351 Wiehe, W. J., 258, 287 Wilhamson, W. M., 319, 351 Wilkinson, E. G., 216, 251 Williams, I. L., 265, 283 Williams, N. B., 203, 237, 239, 251 Williams, R. E. O., 254, 257, 261, 268, 272, 275, 288 Williams, W. L., 263, 289 Willis, A. T., 111, 143 Wilson, B. J., 319, 336, 351 Wilson, C., 120, 121, 143 Wilson, C . D., 271, 288, 289 Wilson, C . H., 319, 340, 351 Wilson, M. G . , 185, 192 Winkelstein, W., Jr., 162, 171, 174, 178, 180, 187, 192 Winkler, F., 278, 285 Winkler, K. C . , 208, 251 Winnick, T., 50, 52, 56, 59 Winsser, J., 171, 174, 178, 180, 187, 192 Winter, C . E., 255,256, 258, 276, 289 Withrow, A., 328, 349 Wittea, L. D., 108, 143 Wixom, R. L., 22, 27 Wogan, G. N., 318, 321, 322, 329, 330, 347, 350, 351 Wolf, C . G. L., 196, 243 Wolff, W. I., 232, 244 Wood, A. W. S., 207, 248 Wood, E. M., 316, 351 Wood, N., 259, 278, 284 Woodruff, H. B., 65, 75 Woods, D. D., 22, 23, 27 Woodward, R. L., 116, 117, 118, 119, 131, 136, 140, 142, 143 Woolfolk, C . A., 6, 27 Woolley, D. W., 232, 246 Wright, D. E., 223,224, 251, 278, 289 Wright, J., 240, 246
372
AUTHOR INDEX
Wright, R., 258, 259, 261, 264, 265, 273, 274, 288 Wuhrmann, K., 78,92, 103, 130,143 Wyman, M., 241, 251 Wynston, L. K., 319, 351 Wyss, O., 126, 143
Y Yamvrais, C., 308, 313 Yea, R., 318, 319, 347 Yonemitsu, O., 343, 351 Young, F. G., 196, 251 Young, G., 233, 251 Young, I. E., 297, 299, 300, 313 Young, M. Y., 274, 275, 284
Yudkofsky, P. L., 233, 251 Yugari, Y., 6, 27 Yukioka, M., 52, 56
Z Zabel, R. A., 133, 143 Zaborowski, H., 262, 289 Zander, H. A., 213, 251 Zebow, B. J., 230,243,251 Zimmerman, W., 130, 143 Zinke, G., 197, 251 Zobell, C. E., 114, 143, 222, 251 Zobrist, F., 130, 143 Zoffante, S. M., 328, 349 Zush, J. R., 319, 349
SUBJECT INDEX A Actinomycetes, 40, 68 Actinomycin, 74 Actinophages, 44 Activated sludge. 77, 180 approach to research on kinetics of, 79 bacteria in, 81, 82, 84 ecology of, 95 effect of, temperature upon, 93 Active oral immunity, 241 Adenovirus, 146 types, 152 Adsorption, 183 Aeration, 184 Aflatoxin, 315, 316 effects, biochemical, 329 biological, 322 physical and chemical properties, 319 Aflatoxin-producing organisms, 318 A. flavus-oryzae group, 319 Aftergrowth, 111, 113 Agglomeration, 81 Agglutination, 222 Aleukia, 342 Alkaline earth compounds, 236 Alkaline phosphatase, 329 Alkylating agents, 36 Allergic rhinitis, 241 Allosteric inhibition, 3 Amino adipyl-cysteinylvaline, 19 Aminopurine, 35 Amperometric titrator for chlorine, 133 Amphibiosis, 232 Anaerobes, 201, 236 Anagasta kuhniella, 307 Antibiosis, 232, 234 Antibiotic-producing organisms, kinds of, 67 Antibiotics, 63, 64 biosynthesis, 17 detection of, 69 new, 66 patents, 64 role of, 64, 66 search for new, 63 synthesis, 48 Antibodies, 240, 276 Antibody synthesis, 53
Antilactobacillus factor, 230 A.P.C. viruses, 147 Aporepressor, 4 Arsenicals for water purification, 133 Arthus phenomenon, 242 Aspergillus flauus, 318 Azide dyes plus, 264 elevated incubation temperatures plus, 265 esculin plus, 266 function, 267 precautions, 266
B Bacillus thuringiensis, 293 Bacitracin, 299 Bacteria in water count, 136 sample collection, 136 Bacteriocines, 73 Bacteriological requirements, nondomestic waters, 106 Bacteriophage, 115 temperate, 4 5 Batch method of activated sludge treatment, 182 Benzylpenicillin, 1, 17 Bile salts, 276 Bioassay, 311 aflatoxin, 334 Biogeochemical agents, 62 Biological antagonists, 184 Blood group A substance, 232 BOD, 78, 79, 178 Borrelia, 209 Bromine, 129 Bromouracil-induced mutations, 35 Bulking, 82, 86
C Canclida albicans 204
Cell-free systems for protein synthesis, 50 Cephalosporin C, 2, 17, 20 Chemical assays, aflatoxin, 330 Chlorination, 185 water, 123
373
374
SUBJECT INDEX
Chlorine, problcms of analytical control, 133
Chlorine dioxide, 129 analysis, 134 Chromatography, 331 Citrate, 272 Code, amino acid, 51 Coliform, 174 Column chromatography, 319 Comamoms, 85 Commensalism, 232 Competence, 43 Competition, activated sludge, 96 Continuous flow method of activated sludge treatment, 183 Control mechanisms for mycotoxicoses, 343 Co-repressor, 23 Coumarins, 322 Countercurrent distribution, 319 Coxsackie group A, 157 Coxsackie group B, 158 Coxsackie viruses in sewage, 173 Crystal-forming bacilli, toxic materials, 294 Crystal-forming bacteria, taxonomy, 293 Crystalline protein, physiochemical nature, 296 Crystal protein, toxic action, 300 Crystal violet, 260 Crystals, cell physiology, 298 Cumulative feedback, 6
D Degradability of wastes, 91 Dental calculus, 212 Dental caries, 198 Dental plaque, 208 Dental pulp, 206 Dentine, 205 Desquamated cells, 217 Diffusible factor, 234 Diphtheroids, 231 Direct selection, 30 Disinfection 185 DNA replication, 329 Dose rate, 311 Drug resistance, 30 Drug-resistant organisms, 53
E ECHO viruses, 180 Ecological approach to antibiotic search, 72 Ecological factors in sludge, 89 Ecological studies of activated sludge, 81 Ecology, 61, 77 End-product inhibition, 2, 3, 49 Enteric viruses in feces, 159 Enteric viruses in sewage, 145 Enterococci, 253 analysis in dairy products, 282 in intestinal contents, 282 in nondairy foods, 281 in water, 280 isolation and enumeration, 253 Enterovirus, 146 in water, 162 Enterovirus group definition, 151 properties, 156 Enzyme, 230 Enzyme induction, 4 Esculin, 288 Exotoxins, water-soluble, 303 F Feedback control, 2 3 Feedback inhibition, 3 Fermentation, 309 Filters, bacterial growth, 111 Filtration of bacteria, 117 Floc, 183 Flocculation, 81 reducing bacterial content, 118 Floc-forming bacteria, 84 Fluorescence, 332 Fly toxin, 303 Folk acid, 238 Freundlich isotherm, 182 Fumagillin, 74 Fungi causing mycotoxicoses, 336 in activated sludge, 86 Furane ring, 320 Fusiform bacilli, 200, 231 G
fl-Galactosidase synthesis, 47 Gauze pad sampling method, 171
375
SUBJECT INDEX
Genetic control of biosynthesis, 46 Genetic homology, Actinomycetes, 40 Genetic transformation, 42 Genetics, 29 Gentian violet, 260 Geotrichum, 94 Germicides in water, 122 Gingival crevice, 215 Gingival tissue, 215 Growth in water, 109
H Hemagglutination, 232 Hemocele, 300 Hemolymph, 300 Hepatitis virus, 146 Hepatocarcinogens, 322 Hepatomas, 316 Hepatotoxic feeds, 316 Histidine, 6 Host susceptibility, 306 Human saliva, 227 Hybridization, 39 Hydrogen peroxide, regulatory importance, 235 Hydroxyapatite, 214 Hydroxyquinoline, 345 Hypersensitivity, 241
I Imhoff tank, 177 Impoundment, surface waters, 120 Induction, 46 Industrial waste, 91 Infectious hepatitis in water, 164 Inhibition of E. coli growth, 235 Inosinic acid fermentation, 8, 10 Insect pathogens, 291 Iodine, 129 Ion exchange media, bacterial growth, 112 Ion exchange units, bacterial growth, 111 Ionizing radiation, 34 Irrigation water, 107 Isoelectric point, 297 Isoenzymes, 5 Isoleucine, 16
J Jute packing, 113
L Laboratory animals, 323 Lactobacilli, 205 Lactones, 338 Lecithinases, 305 Lepidoptera, 300 Leukocytes, 212 Lime, water purification, 131 Lysine fermentation, 8, 11 Lysogenic conversion, 44 Lysogenization, 4 3 Lysogeny, actinomycetes, 44 Lysozyme, 230
M McConnell-Richards factor, 305 Media, 254 Metabiosis, 232 Metallic ions, activated sludge, 95 Methionine, 16, 20 Mevalonate, 330 Microbial ecology, 61 Microhial enzymes, 23 Microhial genetics, 53 Minimal flow areas, bacterial growth, 113 MPN, 174 Mucin, 214 Multiple antibiotics, 70 Multiple enzymes, 5 Multivalent feedback, 6 Mutagens, 33 Mutants analog-resistant, 15 relaxed, 24 Mutation, 30 Mutation rate, 31 Mutational program, 36 Mutualism, 232 Mycotoxins, 315
N Nematodes, 89 Neutralizing antibodies, 240 Nitrification in sludge, 85 Nocardia, 210 Nondomestic water supplies, 105 Nucleic acids, 24 Nuisance organisms, 82 Nutritional factors in microbial ecology, 90
SUBJECT INDEX
0 Odontoblastic processes, 205 Oral bacteria, 234 Oral cavity, 228 Oral immunology, 239 Oral leukocytes, 234 Oral microbiology, 195 history, 195 Oral microbiota, biological control, 227 child and adult, 202 infant, 199 Oral mucous membrane, 217 Oral streptococci, 231 Oral vestibule, 228 Organic matter in sewage, 90 Ornithine fermentation, 8 Ortho-Tolidine test for chlorine, 133 Oxidation lagoons, 188 Oxygen, dissolved, 92 Oxygen supply, activated sludge, 92 Ozone, 130
P Parasexuality, 37 Parotid gland, 229 Parotitis, 225 . .’ oral immunity, 239 p assive Pathology of mycotoxicoses, 342 Penicillin, 17, 276 biosynthesis, lysine, 18 Penicillitim puberulum, 319 Pentachlorophenate, 132 PFU, 174 pH, 186, 236, 277, 300 activated sludge, 94 Phage-host interactions, 43 Phenethyl alcohol, 277 Phospholipase enzymes, 305 Plant pathogens, 107 Poliomyelitis in water, 162 Poliovirus, 159, 162 in sewage, 170 Precursor addition, 2 Predation, sludge, 99 watcr, 115 Prcdators, 88 primary, 82 Prcdatory fungi, 87
Prey-predator relationships, 99 Primary sedimentation, 177 Probiosis, 232 Protein synthesis, 49, 329 Protozoa of sludge, 88 I’seudomonads, prevalent in water, 109 Pseudomonas, 107 Psychrophilic bacteria, 107 Pyridoxine, 238
Q Quaternary ammonium compounds, 131
R Rabies, 197 Radiation, 34 Ragweed-sensitive individuals, 242 Rational screening, 32 Reaginic activity of saliva, 242 Receptor sites, 222 Recombination bacterial, 41 in Actinomycetes, 38 sexual, 36 syncytic, 38 Recombinational mechanisms, 36 Regulatory mechanisms, 1, 17 Replica plating, 32 Repression, 3, 46 Resident flora, 200 Kesistance, 30 to antimetabolites, 1 4 to phages, 43 Revcrse mutations, 17 Rinsing effect of saliva, 227 Ropy saliva, 229 Rotifers, 89
S Saliva, 197, 226 antibacterial effects, 229 antiviral effects, 231 medium supplement, 237 microbial interactions in, 232 microbial nutrient mcdium, 236 Salivary agent, 230 Salivary amino acids, 239 Salivary carbohydrates, 239 Salivary corpuscles, 224 Salivary glands, 224
377
SUBJECT INDEX
Salivary leukocytes, 223 Saprophytes, primary, 81 Screening, 32 antibiotic, 71 Selection, 30 Selective markers, 37 Selective techniques, 33 Selenite, 278 Self-inhibition, 30 Settled sludge, 188 Sewage domestic, 91 storage, 175 treatment methods to remove viruses, 175 Shwartzman reaction, 242 Silver, in water, 130 Sludge assimilating, 84 endogenous, 84 rising, 82 settling characteristics, 82 transfer of organic matter to, 93 Sodium azide, 261 Sodium chloride, 273 Soil formation, 62 Solubility characteristics, 297 Sphaerotilus, 86, 121 Sporulation, 298 Stachybotryotoxicosis, 343 Staphylococci, 200 Submaxillary gland, 229 Symbionts, 62 Symbiosis, 232 Synergisms in sludge, 100 Synthesis of macromolecules, 23
T Taurocholate, sodium, 276 Teeth, 204 Tellurite, 274 Temperature, 265 control of water bacteria, 116 sludge process, 93 Tetracycline, 215 Tetrathionate, 278 Tetrazolium salts, 269 Thallium salts, 267 plus other ingredients, 271
Thallous acetate, 269 Thin-layer chromatography, 319 Thiocyanate, 229 Threonine fermentation, 13 Thymine dimers, 35 Toxicity of organic compounds, activated sludge, 95 Transduction, 45 Transformation, 42 Trickling filters, 179 Turkey X disease, 317
U Ultraviolet radiation, 34 Ureolytic bacteria, 205
V V a h e fermentation, 13, 14 Van Leeuwenhoek, 195 Veillonella, 200 Viruses, 146 in sewage, 170 Virus infections of man, 148 Vitamin A, 329 Vitamin B,, 238 Vitamins, salivary, 238
W Water, 105 antibiotic effects on, 115 as bacteriological medium, 108 as source of bacterial contamination, 106 coliform index, 137 economics of control, 135 ultraviolet light, 118 Waterborne bacteria, 107 Weathering, 62 X Xanthylic acid fermentation, 8 X-Rays, 34 Y
Yeasts in sludge, 87
Z Zoogloea, 84
This Page Intentionally Left Blank