Rapid Microbiological Methods for Foods, Beverages and Pharmaceuticals

THE SOCIETY FOR APPLIED BACTERIOLOGY TECHNICAL SERIES NO. 25 Rapid Microbiological Methods for Foods, Beverages and Pha...

Author: C. J. Stannard | S. B. Petitt | F. A. Skinner

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THE SOCIETY FOR APPLIED BACTERIOLOGY TECHNICAL SERIES NO. 25

Rapid Microbiological Methods for Foods, Beverages and Pharmaceuticals kv C.]. STANNARD Edited

Applied Researdl Department, Pedigree PeJjoods, iHelton Mowbray, Leicestershire LEl3 lBE

S. B. PETITT U.B. (Ross Youngs) Ltd, Ross House, Wickham Road, Grimsby, South Humberside DN31 3SW'

F. A. SKINNER Harpenden, /fens

BLACKWELL SCIENTIFIC PUBLICATIONS OXFORD LONDON EDINBURGH BOSTON MELBOURNE

RAPID MICROBIOLOGICAL METHODS FOR FOODS, BEVERAGES AND PHARMACEUTICALS

A complete list of titles in the Society for Applied Bacteriology Technical Series appears at the end of this volume

THE SOCIETY FOR APPLIED BACTERIOLOGY TECHNICAL SERIES NO. 25

Rapid Microbiological Methods for Foods, Beverages and Pharmaceuticals kv C.]. STANNARD Edited

Applied Researdl Department, Pedigree PeJjoods, iHelton Mowbray, Leicestershire LEl3 lBE

S. B. PETITT U.B. (Ross Youngs) Ltd, Ross House, Wickham Road, Grimsby, South Humberside DN31 3SW'

F. A. SKINNER Harpenden, /fens

BLACKWELL SCIENTIFIC PUBLICATIONS OXFORD LONDON EDINBURGH BOSTON MELBOURNE

©

1989 by the Society for Applied Bacteriology and published for them by Blad.-wcll Scientific Publications Editorial offices: Osney Mead, Oxford OX2 OEL 8 John Street, London WCIN 2ES 23 Ainslie Place, Edinburgh EH3 6AJ 3 Cambridge Center, Suite 208 Cambridge, Massachusetts 02142, USA 107 Barry Street, Carlton Victoria 3053, Australia

British Library Cataloguing in Publication Data

AU rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without the prior permission of the copyright owner.

ISBN 0·-632-02629-4

First published 1989

Rapid microbiological methods for foods, beverages and pharmaceuticals. L Industrial microbiology. Laboratory techniques I. Stannard, CJ. II. Petitt, S.B. III. Skinner, F.A. (Frederick Arthur, 1919~) 1I1l. Series

660'.62'028

Library of Congress Cataloguing-in-Publication Data Rapid :\1icrobiological methods for foods, beverages, and pharmaceuticals/edited by C. J. StaIUlard, S. B. Petitt, F. A. Skinner. p. em. - (Technical series; flO. 25) Papers presented at the Society for Applied Bacteriology Demonstration Meeting, University of Bath, 30th September 1987. ISBN 0-632-02629-4

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1. Food - .1\1icrobiology - Technique Congresses. 2. Beverages - .Mkrobiology Technique - Congresses. 3. Drugs Microbiology - Technique - Congresses. I. Stannard, c.]. (Catherine].) II. Petitt, S. B. Ill. Skinner, F. A. (Frederick Arthur), 1919- . IV. Society for Applied Bacteriology. Demonstration Meeting (1987: University of Bath) V. Series: Technical series (Society for Applied Bacteriology) no. 25. QRl15.R36 1990 664'.07 - dc20 89-17715

eIP

Contents

Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

x

Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

xiii

The Use of ATP Bioluminescence for the Analysis of Beer in Polyethylene Terephthalate (PEl) Bottles and Associated Plant. ]. W. AVIS AND P. SMITH

I

Materials and methods, 2 Results, 6 Discussion, 9 Acknowledgements, 10 References, 10

Rapid Assessment of the Bacterial Content of Milk by Bioluminescent Techniques. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . M. W. GRIFFITHS AND]. D. PHILLIPS

13

lv1aterials and methods, 14 Results, 17 Discussion, 26 References, 29

DEFT': Recent Developments for Food and Beverages. . . . . . . . . . L. PETTIPHER, R. G. KROLL, L. J. FARR AND R. P. BETTS

G.

General principles, 33 Apparatus, reagents and methodology, 33 Selective pre-incubation for the detection of low levels of spoilage bacteria by the DEFT, 36 Osomotolerant yeasts in confectionery products, 38 Selective enumeration of bacteria by the DEFT, 39 Use of DEFT for irradiated foods, 42 References, 44

v

33

vi

CONTENTS

The Rapid Estimation of Bacterial Counts on Meat and Poultry by the Direct Epifluorescent Filter Technique. . . . . . . . . . . . . . . . . B. G. SHAW AND L. J. FARR

47

DEFT methodology for meat and poultry, 47 Comparison of DEFT and plate counts, 52 Applicability of DEFT to meat and poultry, 5S Acknowledgement, 56 References, 56

Medical and Pharmaceutical Applications of the Direct Epifluorescent Filter Technique (DEFD

S. P.

DENYER,

R. A. P.

LYNN AND Urine examination by the DEFT, 59 Analysis of intravenous fluids by the DEFT, 64 Conclusion, 69 References, 70

P. S.

,..

59

POVER

The Use of Image Analysis for MIC Determination and Bioassay B. J. BROOKS AND K. COLEMAN

73

What is image analysis?, 73 Image analysis at Brockham Park, 74 Application to bioassay, 75 Application to MIC determination, 78 References, 85

Optimization of Automated Electrometric Methods. . . . . . . . . . . .

D. M.

87

GIBSON

General hints, 89 Curve quality, 89 Comments on some conventional assays, 90 Temperature, 91 Calibration curves, 91 Practical experiments on fish, 93 Pathogen detection, 96 Inorganic constituents, 96 Conclusions, 98 References, 98

Conductance Techniques for the Detection of Contaminants in Beer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. L. KYRIAKIDES AND P. A. THURSTON General procedures, 102 Screening of non-selective media, 103 Detection routine evaluation, 104 Development of selective media, 105

101

CONTENTS

VII

Field trial, 109 Discussion, 115 References, 117

Electrical Methods for Water Quality Testing T. E. IRVING, G. STANFIELD AND B. W. T.

..........

119

HEPBURN

Conventional water tests, 120 Use of the Bactometer M123 for environmental and recreational water samples, 120 Use of the Bactometer M123 for potable water samples, 123 Use of the Malthus microbial growth analyser for potable water analysis, 125 Acknowledgement, 130 References, 130

A Conductance Screen for Enterobacteriaceae in Foods S. B. PETITT

131

Rapid tests for the Enterobacteriaceae, 132 Materials and methods, 133 Result~, 137 Discussion, 139 Acknowledgements, 140 References, 140

Electrical Screening of Powdered Dairy Products . . . . . . . . . . . . . . SHEILA M. FRYER AND KATE Materials and methods, 144 Result~, 147 Discussion, 151 Acknowledgement, 153 References, 153

143

FORDE

An Inter-Laboratory Evaluation of an Electrometric Method for Detection of Salmonellas in Milk Powders. . . ... . . . . . . . . . . ... . G. A. PRENTICE, P. NEAVES, D. I. JERVIS AND M. C. EASTER Participating laboratories, 156 Materials and methods, 157 Comparison of the British Standard method v.ith the Easter-Gibson method, 159 Results and discussion, 160 Acknowledgements, 164 References, 164

155

viii

CONTENTS

Rapid Salmonella Detection by a Combination of Conductance and Immunological Techniques JULIE A. BIRD, M. C. EASTER, S. GAYE IIADFIELD, E. MAY AND M. F. STRINGER

165

Materials and methods, 166 Results, 172 Discussion, 179 Acknowledgement, 182 References, 182

Automated Conductimetric Detection of Salmonellas in Confectionery Products. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S.]. PUGH AND M. L. ARNOTT

185

Description and principles, 186 Method evaluation, ] 95 Conclusion, 199 Acknowledgement, 200 References, 200

A Medium for Detection of Lancefield Group D Cocci in Skimmed Milk Powder by Electrometric Methods ..... . . . . . . . . P. NEAVES, M. J. WADDELL AND G. A. PRENTICE

203

Preparation of skimmed milk powder contaminated with Lancefield Group D cocci, 203 Use of conventional selective media in elcctrometric instruments, 205 Medium development for electrometric detection, 206 Effect of medium composition on curve quality, 207 Calibration of Malthus and Bactometer instruments, 209 Acknowledgements, 211 References, 211

BIOCHECK - a Mediated Amperometric Microbial Activity Monitor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. S\VAIN, M. ALLEN, B. H. SCHNEIDER, F. TAYLOR AND A. P. F. TURNER Requirements for amperometric biomass sensing, 214 Evaluation of suitable mediators, 217 Assessment of electrodes, 219 Prototype development, 220 Considerations for the examination of real samples, 224 Acknowledgements, 225 References, 225

213

CONTENTS

Detection of Electron Transfer for the Assessment of Bacterial Contamination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . R. G. KROLL, R. A. PATCHETT, STEPHANIE E. LEAROYD AND C. F. THURSTON

~

227

Materials and methods, 229 Results and discussion, 233 References, 239

Computer-Assisted Identification of Moulds. . . . . . . . . . . . . . . . . .

241

A. P. WILLIAMS AND ANIA BIALKOWSKA The evolution of mould identification, 242 Methods, 243 Conclusion, 248 References, 248

Immunological Detection Methods for Salmonellas in Foods. . . . C. DE W. BLACKBURN AND CATHERINE J. STANNARD

249

Bio-Enzabead Screen Kit, 249 TECRA Salmonella Visual Immunoassay, 253 Kirkegaard and Perry Salmonella ELISA, 256 Salmonella 1-2 Test, 259 Discussion, 262 References, 263

Immunoassay Kits for the Detection of Toxins Associated with Foodbome Illness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . SALLY A. ROSE, N. P. PATEL, A. O. SCOTT AND

265

M. F. STRINGER Materials and methods, 266 Results and discussion, 267 Concluding remarks, 280 Acknowledgements, 280 References, 280

Rapid Detection of Viruses in Water and the Water Environment H. MERRETT AND C. E. STACKHOUSE

283

lVlaterials and methods, 284 Results, 289 Discussion, 289 References, 290,

Index

293

Contributors

M. ALLEN, Bioelectronics Division, Biotechnology Centre, Cranfield Institute of Technology, Cranfield, Bedfordshire MK43 OAL, UK M. L. ARNOTT, Cadbury Schweppes PLe, Group Research, The Lord Zuckerman Research Centre, The University of Reading, Reading RG6 2LA, UK J, W. AVI5, Research and Development Laboratory, Allied Breweries Ltd, 107 Station Road, Burton-on-Trent, Derbyshire DEJ4 1HZ, UK R, P. BE TT 5, Campden Pood (5 Dn'nk Research Association, Chipping Campden, Gloucestershire GL55 6LD, UK ANIA BIALKOWSKA, Leatherhead Food RA" Randal/s Road, Leatherhead, Surrey KT22 7RY, UK JULIE A. BIRD, Microbiology Department, Unilever Research and Engineering, Colworth Laboratory, Co/worth House, Sharnbrook, BedjOrdshire MK44 1LQ UK C. DE W. BLACKBURN, Applied Microbiology Section, Leatherhead Food RA., Randalls Road, Leatherhead, Surrey KT22 7RY, UK B. J. BROOKS, Beecham Phannaceuticals Research Division, Chemotherapeutic Research Centre, Brockham Park, Betchworth, Surrey RH3 7A], UK K. COLEMAN, Beecham Phannaceuticals Research Division, Chemotherapeutic Research Centre, Brockham Park, Betehworth, Surrey RH3 7AJ, UK S. P. DENYER, Department ofPh annaceuticaI Sciences, University ofNottingham, University Park, Nottingham NG 7 2RD, UK M. C. EASTER, Express Foods Group LtdJ 430 Viaoria Road, South Ruislip, Middlesex I/A4 OHF, UK L. J. F ARR, Foss Elean'c (UK) Ltd, The ChantryJ Bishopsthorpe, York Y02 IQf, UK KATE FORDE, Unigate Foods Ltd, Station Road, Wincanton, Somerset BA99ED UK SHEILA M. FRYER, St Ivel Technical Centre, Abbey J/ouse, Church Street, BradfOrd-on-Avon, Wiltshire BAIS JDH, UK D, M. GIBSON, Ministry of Agriculture, Fisheries and Food, Torry Research Station, J35 Abbey Road, Aberdeen AB9 8DG, Scotland, UK M, W. GRIFFITHS, Hannah Research Institute, Ayr KA6 SHL, Scotland, UK S. GA YE HADFIELD, Wellcome Research Laboraton'es, Langle,.y Court, Beckenham, Kent BR3 3BS, UK J

x

CONTRIBUTORS

xi

B. W. T. HEPBURN, Wessex Water, Regional Scientific Centre, Mead Lane, SaltfOrd, Bristol BS18 3ER, UK T. E. IRVING, Water Research Centre, Henley Road, Medmenham, PO Box 16, Marlow, Buckinghamshire SL7 2l/D, UK D. I. JERVIS, St Ivel Technical Centre, Abbey House, Church Street, Bradfordon-Avon, Wiltshire BA15 1DH, UK R. G. KROLL, Department of Microbiology, AFRC Institute of Food Research, Reading Laboratory, Shinfield, Reading RG2 9AT, UK A. L. KYRIAKIDES, Grand Metropolitan Brewing Ltd, Stag Brewery, 91 Brick lane, London E1 6QN, UK STEPHANIE E. LEAROYD, Department of Microbiology, King's College, University of London, Campden Hill Road, London W8 7AH, UK R. A. P. LYNN, Department of Pharmaceutical Sciences, Universi~v of Nottingham, Universi~y Park, Nottingham NG7 2RD, UK E. MAY, School of Biological Sciences, Portsmouth Po~ytechnic, King Henry I Street, Portsmouth, Hampshire POI 2DY, UK H. MERRETT, Virology Unit, Welsh Water PLC, Engineering and Environment Ltd, Bridgend Ojfice and Laboratory, Tremains House, Tremains Court, Bridgend, Mid Glamorgan CF31 2AR, Wales, UK P. NEAVES, Technical Division, Milk Marketing Board, Thames Ditton, Surrey KT70EL, UK R. A. PATCHETT, Department ojMicrobiology,AFRC Institute ofFood Research, Reading Laboratory, Shinfield, Reading RG2 9AT, UK N. P. PATEL, Campden Food (5 Drink Researth Association, Chipping Campden, Gloucestershire G155 6LD, UK S. B. PETITT, U.B. (Ross Youngs) Ltd, Ross House, Witkham Road, Gn'ms~v, South Humberside DN31 3SW, UK G. L. PETTIPHER, CadburySchweppesPLC, Group Research, TheLordZuckerman Research Centre, The University of Reading, Reading RG6 2LA, UK J, 0, PHILLIPS, Hannah Research Institute, 4vr KA6 SHL, Scotland, UK P. S. POVER, Ana{ytical Measun'ng s.ystems, London Road, Pampis.fOrd, Cambridge, CB2 4EF, UK G. A. PRENTICE, Milk Marketing Board, Thames Ditton, Surrey KT7 OEL, UK S. J. PUG II, Cadbury Ltd, Technical Laboratories, Bournville, Birmingham B30 2LU, UK SALL Y A. ROSE, Campden Food (5 Drink Research Association, Chipping Campden, Gloucestershire GLSS 6LD, UK B. H. S C H N EID ER, Bioelectronics Division, Biotechnology Centre, Cranfield Institute of Technology, Cranfield, Bedjordshire MK43 OAL, UK A. O. SCOTT, CampdenFood (5 Drink Research Association, ChippingCampden, GlouteStershire GL55 6LD, UK

xii

CONTRIBUTORS

B. G. S HA W, AFRC Institute of Food Research Bristol Laboratory, LangfOrd, Bristol BS18 7DY, UK P. SMITH, Research and Development Laboratory, Allied Breweries Ltd, 107 Station Street, Burton-an-Trent, Derbyshire DE14 1BZ, UK C. E. STACKHOUSE, Virology Unit, Welsh Water PLC, Engineen'ng and Environment Ltd, Bridgend Office and Laboratory, Tremains House, Tremains Court, Bridgend, Mid Glamorgan CF3 J 2AR, Wales, UK G. STANFIELD, Water Research Centre, Henley Road, jWedmenham, PO Box 16, Marlow, Buckinghamshire SL 7 2HD, UK C. ]. STANNARD, Applied Research Department, Pedigree Petfoods, Me/ton Mowbray, Leicestershire LE13 7BR, UK ~1. F. S T RING ER, Department ofA1icrobiology, Campden Food (5 Drink Research Assoa"ation, Chipping Campden, Gloucestershire GLSS 6LD, UK A. SWAIN, Bioelectronics Division, Biotechnology Centre, Cranfield Institute of Technology, Cranfield, Bedfordshire MK43 OAL, UK F. TAYLOR, Bioelectronics Division, Biotechnology Centre, Cranfield Institute of Technology, Cranfield, BedjOrdshire MK43 OAL, UK C. F. THURS TON, Department of Microbiology, King's College, Um'versity of London, Camden Hill Road, LontkJn SW14 7ET, UK P. A. THURSTON, Grand Metropolitan Brewing Ltd, Mortlake, London SW14 7ET, UK A. P. F. TURNER, Bioelectronics Division, Biotechnology Centre, Cranfield Institute of Technology, Cranfield, BedjOrdshire MK43 OAL, UK M. J. WADDELL, Technical Division, Jl1ilk Marketing Board, Thames Ditton, Surrey K17 OEL, UK A. P. WILLIAMS, Leatherhead Food R.A., Randal/s Road, Leatherhead, Surrey KT22 7RY, UK

Preface

This book is the 25th in the Technical Series of the Society for Applied Bacteriology. Each chapter is the written version of a practical contribution given at the Demonstration Meeting of the Society, held at the University of Bath on 30 September, 1987. For many years, more rapid and labour-saving methods have been sought as alternatives to conventional microbiological techniques. Many of the pioneering studies have taken place in clinical laboratories. For foods, beverages and pharmaceuticals, the materials tested and the organisms sought are more varied. The contributions to this book illustrate the wide variety of approaches that workers in these industries have taken in order to solve the particular problems associated with their own products. The methods described in this book include electrometric techniques, ATP assay, and immunological methods for a wide range of organisms from salmonellas to viruses. We feel that it is apparent that the choice of a rapid method for industry depends upon the equipment available and the accuracy required. This book should be useful to those in the food, beverage and pharmaceutical industries, or in research or teaching, who require a practical guide to the use of rapid microbiological methods. We should like to thank the contributors for all their hard work in preparing the demonstrations and contributions for the book, and Dr Ron Board and his staff at the University of Bath for the organization of the meeting.

J. Stannard S. B. Petitt F. A. Skinner

Catherine

Xlll

The Use of ATP Bioluminescence for the Analysis of Beer in Polyethylene Terephthalate (pET) Bottles

and Associated Plant J. W. AVIS" AND P. SMITH Research and Development Laboratory, Allied Breweries Ltd, 107 Station Street, Burton-on-Trent, Der~yshire DEl4 IBZ, UK

The growing demand for products packaged in polyethylene terephthalate (PET) bottles (which cannot be pasteurized) has led to increased emphasis on plant hygiene and end-product quality assurance. Rapid methods of detecting microbial contamination are especially useful for these products and this work concentrates on the rapid membrane filtration of products directly from PET bottles and the analysis of membrane filters and production plant swabs by adenosine triphosphate (ATP) bioluminescence. Many changes are currently taking place in microbiology. Micro-organisms are being studied increasingly as part of their natural environment rather than in isolation; automation has allowed large numbers of 'routine' samples to be examined and there is increased use and development of rapid methods of detection. In the brewing industry rapid methods would be particularly helpful to detect process failure, assess the quality of pitching yeast and the microbiological status of packaged products (Hope & Tubb 1985). Two electrical methods which have shown promise are impedence measurement as a rapid forcing test for beer (Evans 1982) and conductance measurement for the rapid detection of both lactobacilli in beer (Evans 1985) and Obesumbacterium proteus in pitching yeast (Kilgour & Day 1983). The sensitivity of the method is low, however, because cells are detected only when they reach a level of 105 -1 0 6/ ml and incubation times can be in excess of 48 h for some organisms.

" Present address: Group Quality Control Laboratory, Bass Brewing Ltd, 137 High Street, Burton-on-Trent, Derbyshire DE14 IJZ, UK. Copyright Rapid fvlicrobiological .~lethods for Foods, Beverages and Pharmaceuticals

©

1989 bJ' the Society jiJr Applied /JaaerioloJ{)' All rights of rtpmdlletirffl in any jimn reserved

0-632-02629-4

2

J.

W. AVIS AND P. SMITH

The Direct Epifluorescent Filtration Technique (DEFT) has been used with success for milk (Pettipher et al. 1980) and to a more limited extent for beer (Kilgour & Day 1983) where the method for heat-treated samples was improved by counterstaining with methylene blue. Counterstaining with Janus Green B was found to give more consistent results than methylene blue (Rodrigues & Kroll 1986) but DEFT preparations on heat-treated beverages were still found to be unreliable in differentiating between viable and nonviable yeast cells. Adenosine triphosphate bioluminescence has been used to detect microbial contamination in carbonated beverages (Littel & La Rocco 1986), wine (Lonvaud-Funel & Joyeux 1982) and beer (Hyserl et al. 1976; Kilgour & Day 1983; Dick et al. 1986). The methods detect only viable organisms at levels of the order of 100 yeast cells or 1000 bacterial cells/ml suspension. In the work to be described ATP bioluminescence was used specifically for the problems of PET bottled beer. The PET bottle is a popular package in the UK for many bottled drinks and sales arc continuing to increase. The package cannot be pasteurized or heat-treated, however, so plant hygiene is particularly important. The ATP method has a role in assuring the quality of both packaging plant and finished product. In developing the method we have concentrated on the detection of yeasts rather than bacteria, following our findings that yeasts were the cause of over 95 % of contamination problems examined over an 8-month period (Avis 1988).

Materials and Methods

ATP analysis The work was carried out using the Lumac Biocounter 2010 (Lumac BV, Schaesberg, The Netherlands). Samples are contained in disposable plastic cuvettes which are inserted into a light-tight chamber for reading by a sensitive photomultiplier tube. The amount of light emitted is displayed as a digital readout and is expressed in Relative Light Units (RLUs). The absolute amount of ATP contained in a sample may be found by injecting an ATP standard into the sample and taking a second reading. To be accurate the RLU value of the standard should be two to five times that of the sample so a range of standards are made up from a stock solution and the RLU values of these are checked before samples arc analysed. The reagents and standards required for the analyses were supplied by Lumac, and were: 'Somase' (a non-microbial ATP-ase), 'F-NRS' (used here as a buffer), 'NRB' (a nucleotide-releasing agent for the extraction of ATP from microbial cells) and 'Lumit-PM' (luciferin-Iuciferase reagent). ATP standards were made up from a stock solution of 1.65 x 10- 6 moll I ATP. All

ATP ANALYSIS FOR PET BOTTLED BEER

3

powdered reagents were stored between 0° and 2°C and, when reconstituted, Lumit-PM, Somase and ATP standard stock solution were split into 1- or 2-ml aliquots which were stored for a maximum of 4 weeks at -18°C. Temperature-sensitive reagents were kept on ice during analysis.

PET bottle filtration device Filtration methods currently used for PET bottled products suffer from two disadvantages. Firstly, the majority of commercial membrane filtration units have only a 250-ml reservoir for beer, which is inconvenient if beer has to be continually poured into the unit, particularly if a laminar flow cabinet is not available. Secondly, there exists the possibility of contamination of the beer from the outside of the neck of the bottle as the beer is poured into the filter funnel. This has led to a piercing technique in which a portion of the bottle is swabbed with 70% methylated spirit and then pierced with a hot needle; the beer is then poured into the filter. This technique is also inconvenient. The device which will be described here was developed to avoid these problems by allowing the entire contents of the bottle to be taken directly from an upright bottle without the necessity of pouring or piercing. The device is shown in Fig. 1 and is in two parts: a quick-release clamp and the gas inlet and beer outlet tube assembly. The 1/4 -inch National Pipe Thread fitting on the beer outlet tube accepts a conventional 47-mm membrane filter holder (e.g. Swinnex) and the whole device can be autoc1aved with the membrane in place, after which it can be attached very quickly to the bottle to be sampled. All the bottle contents are then passed through the membrane by applying a top pressure of gas which is filtered in-line. The membrane is then removed for plating on agar or ATP analysis.

Filters Conventional cellulose acetate (0.45 [lm) and Vltipore (1.2, 0.8, 0.65 [lm) nylon membrane filters (Pall Process Filtration Ltd, Portsmouth) were used. The latter type are electrostatically charged, the magnitude and polarity of the zeta potential being dependent on the pH. At pH 4 (approximately the pH of beer) the filter has a positive charge.

Swabs Plain, cotton wool sterile swabs (Northern Media Supply Ltd, Hessle) were used to assess the cleanliness of plant associated with PET bottling. Charcoalimpregnated swabs must not be used as these affect the results of ATP analysis.

4

J. W. AVIS AND P. SMITH

FIG. 1. PET filtration device. A, quick release damp; B, bras inlet and beer outlet tubes; C, NPT fitting to accept Swinnex-typc filter holder; D, in-line filter for top pressure gas.

1/4 -in

il1edia Cotton wool swabs wefC incubated after usc in 'Lumaculf, a pre-sterilized, low-ATP growth medium, at pI! 7.0 (Lumac BY). Yeast E>..1ract, Nialt Extract

Broth (Difco) adjusted to plI 4.5 was used for all other membrane incubations involving ATP analysis. \Ve have found that this broth is particularly suitable for ATP work because it has low background ATP and does not quench the luminescence too greatly. Batches do vary, however, and it is advisable to test the nledium before use. Plate counts were performed either on \Vallcrstein Laboratories Nutrient (\VLN) Agar (Difeo) for yeast spp. or on Raka Ray No. 3 Agar (Difeo) tor Pediococcus spp. and Lattohacillus spp.

ATP ANALYSIS FOR PET BOTTLED BEER

5

Organisms The organisms used in this work were taken either from the Allied Breweries culture collection, where they are stored as lyophilized cultures, or from contaminated products. In both cases the organisms were sub-cultured on Malt Extract, Yeast Extract Glucose Peptone (MYGP) Agar (Difco) and into MYGP broth (Difco) to prepare a working culture for dilutions. Ana~ysis

of swabs

Before swabbing, 1 ml of Lumacult was dispensed into disposable plastic, sterile, 30-ml screw-capped bottles. These were found to be most suitable because of their conical bases which allowed maximum coverage of a swab tip with the minimum volume of medium. Swabs for ATP analysis were broken olf into these bottles for incubation whilst swabs for conventional analysis were broken off into 10 ml of 1/4 - strength, sterile Ringer solution. The Ringer solution was agitated on a vortex mixer for 30 s before membrane filtration. The membrane was placed on WLN agar plates. The swabs in Lumacult were incubated overnight (16 h minimum) at 27°C before examination by the following method: tubes were agitated for 30 s on a vortex mixer and a 100 I-ll sample of the Lumacult transferred to a cuvette. 100 I-li of NRB were added to the cuvette and after 60 s, 100 I-ll of Lumit PM were added and, without further mixing, the cuvette was placed in the Lumac Biocounter. The number of RLUs was read after a lO-s integration period. The whole procedure took place at ambient temperature.

Product

ana~ysL~

PET-bottled beer was membrane-filtered using either the conventional apparatus or the PET filtration device and the membranes examined by conventional plating procedures or by ATP analysis. The ATP analysis was carried out as follows: after membrane filtration, the membrane was removed from the holder and placed in a 50-mm Petri dish. Yeast-malt extract broth (YMB), 500 I-lI, at pH 4.5 was added to the membrane which was incubated at 27°C for 20 h. FNRS buffer and 20 I-ll V4 strength Somase were added and the mn-lure allowed to stand for 30 min at room temperature. ATP was extracted with 500 I-ll NRB for 60 sand 200 I-ll of the extract was transferred to a sample cuvette which was placed in the Lumac Biocounter. Lumit-PM (100 ~ll) was added and integration started. The sensitivity of the method was tested by spiking l00-ml volumes of beer with 1 ml of several serial dilutions of contaminant yeasts. The beer

6

J. W. AVIS AND P. SMITH

was membrane-filtered and the membrane examined by the bioluminescence method above. The original serial dilutions were also spread on WLN agar for conventional plate counts.

Detection level A blank or control RLU value was obtained by testing an uninoculated broth or membrane before each series of tests. Both swab and membrane cultures were considered to be positive when the RLU value of the sample exceeded the blank value plus three times the standard deviation for this value. 'I'his threshold value was arrived at from a consideration of normal distribution theory. The standard deviation for blank tests was found to be c. 8 RLU. For example, a blank value of 15 RLU would give a detection level of 40 RLU.

Filtration times Filtration times of different membranes were found by timing the filtration of 2 I of beer by the PET filtration device. Top gas pressures from 15 to 50 Ibl in2 gauge pressure (psig) were provided from a nitrogen cylinder.

Filtration efficiencies The effidency of filters was tested by filtering 100 ml of beer containing

different numbers of organisms through the filter of interest. The filtrate was then passed through a second filter of 0.22 ~m pore size. Filters were plated on appropriate media. The range of organisms used for these tests was chosen for their beer spoilage potential and were as follows: Acetobacter rancens NCIB 6429; Laaobacillus brevis NCIB 8847; Pediococcus acidilactici NCIB 6990; Pediococcus cerevisiae NeIB 8066; Obesumbaaerium proteus AB68; Zymomonas anaerobia NCIB 8227; Zymomonas mobilis NeIB 3938; Saccharomyces cerevisiae strains AB7, AB70, AB140; and wild yeasts XY69, XY70, XY71, XY72, XY73, XY74, XY75.

Results Swabs Of 400 swabs taken routinely from different parts of a working bottling machine 66 % of ATP results agreed with the plate count taken at the same time, 27% were falsely positive (positive result by ATP testing without a corresponding plate count), and 9% were falsely negative (negative result by ATP testing with a corresponding plate count).

7

ATP ANALYSIS FOR PET BOTTLED BEER

Product

An example of filtration rates for various filters is seen in Fig. 2 for 2-1 PET bottles of lager (original gravity I032°Sacch.). The efficiencies of the filters are shown in Table 1. The mean values for retention at four different levels of scven ycast strains and seven bacterial species were greatcr than 95% and there was no significant difference between filter types at the 5% level for yeast or bactcria. Figure 3 shows the log molar concentration of ATP plotted against log number of colony forming units for inocula of three yeasts and two filter types.

.

10.0

.~

~~

8.0

~

E 6.0 E .;:; c

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LL

;~

~.•

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!

!

20

30

!

10

c:i

40

50

Pressure (psig)

2. Filtration times for a 2-1 PET bottle of lager expressed in min/I. Membrane filter types: ., 0.45 11m cellulose acetate; A, 0.65 11m Ultipore N66; ., 0.80 11m Ultipore N66; +, 1.20 [Tm Ultipore N66.

FIG.

TABLE

I. Efficiency of various membrane filters fOr different levels of bacteria and yeast % Retention" (membrane type and pore size)

Nominal loading (cells/membrane)

I 10 100 1000

Sartorius (0.45 [tm) 100 97.6 98.1 96.4

(I DO)t (97.0) (94.0) (93.3)

Ultipore (0.65 fUn) 91.7 96.9 96.1 95.3

(100) (99.9) (95.2) (92.9)

Ultipore (0.8 f!.m) 93.2 94.6 97.8 96.7

(100) (94.1) (96.6) (97.1)

" Mean retentions for four replicates of seven different strains or species. t Figures in brackets indicate efliciencies for yeast.

Ultipore (1.2 f!.m) 100 96.8 97.1 97.5

(100) (97.3) (97.6) (94.8)

8

J.

W. AVIS AND P. SMITH

-4 c

2

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1

0

~

C m ()

c 0

-6

4 3

u Cl.

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m

"0

E

+

-8

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en

X 0

0 ....J

-10 0

0.5

1.5

2

2.5

Log 10 plate count/ml FIG. 3. Correlation curves between ATP concentration and number of CFUs for different filters and for inocula of three strains of contaminant yeast. Strain XY70: 0,0.45 !-lm filter; *, 1.20 11m filter (combined for regression line 2, r = -0.99). Strain XY71: 6, 0.45-(!m filter; +, 1.20-jJ.m filter (combined for regression line 1, r = -0.97). Strain XY72: 0, 0.45-flm filter (regression line 3, r = -0.91); x, 1.20-jJ.m filter (regression line 4, r = -0.97).

TABLE

2. Detection ofyeasts in 250-ml volumes of beer* (Yo Detection

Yeast

!VIean no. cells

Lumac

Plate

Difference

XY73

5

100 29

100 57

n.s. n.s.

7 1

100

57

100 29

n.s. n.s.

5 1

100

100

0

43

n.s. n.s.

2

XY74 XY75

n.s., not significant. '*' Based on a comparison of seven replicates at each level.

For two of the yeasts shown there was no significant difference between the slopes of the lines, so a single regression line has been drawn in this case. The difference between the intercepts for each yeast can be ascribed to differences in their ATP content per cell and growth rate. Table 2 compares the sensitivity for three contaminant yeasts with detection by conventional membrane filtration and plating. It was possible to detect one cell per 250 ml by ATP biolumi-

ATP ANALYSIS FOR PET BOTTLED BEER

9

nescence but correlation with the plate method was not 100% until a mean level of five cells per 250 ml was reached. At levels lower than this there was no significant difference between the two methods. Discussion ATP bioluminescence combined with positively charged membrane filtration can provide an integrated approach to the rapid monitoring of PET-bottled beer and associated plant. One should exercise some caution, however, when comparing results with conventional plating procedures which are notorious for their poor correlation with other procedures (Sharpe 1980). The comparison of two methods will often give rise to false positive and false negative results. When considering a false positive by the ATP method one should allow for the possibility that it is a false negative by the plate method and vice versa. For example, if the results for swabs were examined in this light then the actual situation existing on the plant could be that 91 % (66 % + 25 %) of swabs were a true reflection of the state of the plant by the ATP method compared with 75% (66% + 9%) of swabs being a true reflection by the plate count method. We must also be cautious in comparing the results obtained from swabs taken from adjacent areas on the machine because they could be considered as two separate samples. There is little doubt, however, that the ability to obtain a result after 20 h led to improved cleaning and attention to hygiene. Rapid testing and reporting also allowed trends in contamination to be built up more quickly with persistent problem areas being identified and receiving closer attention. The effectiveness of conventional cellulosic and polycarbonate filters has been compared for brewery bacteria by Lin (1976) and the use of various large-porosity membrane filters has been described by Zierdt (1979) who ascribed their effectiveness to electrostatic charge. Kroll (1985) used electropositively-charged filters to concentrate bacteria from foods and subsequently to elute them from the membrane. The increasing availability of charged membranes is likely to increase their use for samples which take a long time to filter. In previous work, positively charged 'Posidyne' filters (Pall Process Filtration Ltd) were found to inhibit the growth of some brewery contaminants (Avis 1988) and the Ultipore filters were sought as an alternative. The inhibition was ascribed to the presence of the quaternary ammonium compounds which are used to enhance the electrostatic charge that is present on nylon filters. The results presented here have shown that large porosity filters can be used for conventional and bioluminescence procedures \vithout compromising on the numbers of micro-organisms which are retained on the filter. When used in combination with the PET filtration device the filters allow more beer t() be examined with, consequently, a greater inoculum for

J. W. AVIS AND P. SMITH

10

ATP analysis. The lowest flow rate consistent with a reasonable filtration time should be sought because filtration can be a source of physiological stress which may be exacerbated at high pressures and flow rates. The rapid detection of microbial contamination in beer is not an easy task when the levels are low, typically only a few cells/I, particularly if cells have been sub-lethally damaged or stressed by pasteurization. The results here show that it is possible to detect cells at a level of 4 cells/l in 20 h provided that concentration and pre-incubation steps are included in the test. The combination of large-porosity filters with bioluminescence has an advantage over the current method for PET bottles because the whole package can be assessed rather than a 2S0-ml portion. \\'here organisms afe unevenly distributed in the bottle, which is often the case with flocculant yeast, there is a greatcr probability of detecting contamination if the levels arc also low. One must recognize, however, that the detection of any organism by ATP bioluminescence ultimately depends on its ATP content, its specific growth rate and the lag time before exponential growth begins. For example, with wild yeast cells that have doubling times of 1.02 to 1.83 h and ATP contents of 103 to 320 fg per cell we calculated that the time to detection for an inoculum of one cell before enrichment could range from 11 to 19 h under ideal conditions (Avis 1988). Alternatively) Dick et ai. (1986) concluded that after an enrichment time of 48 h and before concentration, Sacch. cerevisiae must be present at c. 2 cells/l in order to detect contamination with certainty.

Acknowledgements We wish to thank the directors of Allied Breweries for permission to publish this paper.

References

J.W. 1988. The use of ATP bioluminescence for the quality assurance of PET bottled beers. In Confermce proceedings of tlte Associaziotle Sodeta Italiatla di A1icrobiologia Applicata, ]\,letodi rapidi cd automatizziati nella nticrobiologia applicata, pp. 39-45, Societa Editoriale Farmaceutica Milan. DICK, E., WrEDMAN, R., LEMPART, K. & HAI\lMES, W.P. 1986. SchncUnachweiss mikrobeiller lnfektionen im Bier. Clmnie A1ikrohiologie Teclmologie der LebensmitteI 10, 37-41. EVANS, H.A.V. 1982. A note on two uses for impedimetry in brewing microbiology. Joumal of Applied Bacteriology 53, 423-426. EVANS, II.A.V. 1985. A note on the use of conductimetry in brewery microbiological controL Food Microbiology 2, 19-22. f lOPE, C.F.A. & TUBE, R.S. 1985. Approaches to rapid microbial monitoring in brev.ing. Journal AVIS,

of the Institute oj Brewing 91, 12-15. D.W., KOVECSES, F. & NloRR1SON, N.M. 1976. A firefly bioluminescence ATP assay method for rapid detection and enumeration of brewery microorganisms. Journal of the ,1merican Socie~y of Brewing Chemists 34, 145 - 150.

HYSERT,

ATP ANALYSIS FOR PET BOTTLED BEER

11

KILGOUR, W.J. & DAY, A. 1983. The application of new techniques for the rapid detennination of microbial contamination in brewing. In Proceedings of the 19th Congress of the European Bmvery Convmtion, pp. 177 -184. Oxford: IRL Press Ltd. KROLL, R.G. 1985. Electropositively charged filters for the concentration of bacteria from foods.

Food Microbiolot,'Y 2, 183-186. LIN, Y. 1976. Use of various brands of membrane filters for the detection of brewery bacteria.

]oun!al of the American Society of Brcwing Chemists 34, 141-144. LITTEL, K.J. & LA Rocco, K.A. 1986. ATP screening method for presumptive detection of microbiolob>1Cally contaminated carbonated beverages. ]oumal of Food Scimce 51,474-476. LO[\'Vl\UD-FuNEL, A. & ]OYEUX, A. 1982. Application de la bioluminescence au denombrement des microorganismes vivants dans les "ins. Connaissance Vigue Vin 16, 241- 256. PETTlI'HER, G.L., MANSELL, R., McKINNON, C.H. & COUSINS, C.M. 1980. Rapid membrane filtration epilluorescent microscopy technique for direct enumeration of bacteria in raw milk.

Applied aud Enviroumental Microbiolot,'Y 39,423-429. RODRIGUES, U.M. & KROLL, R.G. 1986. Use of direct epilluorescent filter technique for the enumeration of yeasts. Joumal ofApplied Bacteriology 61, 139-144. SIIARI'E, A.N. 1980. Food Microbiolo&'Y - A Framework jilr the Future. Springfield, Illinois: Charles C. Thomas. ZIERDT, CJ I. 1979. Adherence of bacteria, yeast, hlood cells and latex spheres to large-porosity membrane filters. Applied and Euviro1l1nClltai Microbiology 38, 1166-1172.

Rapid Assessment of the Bacterial Content of Milk by Bioluminescent Techniques M. W. GRIFFITIIS AND J. D. PHILLIPS Hannah Research Institute, Ayr KA6 5HL, Scotland, UK

The use of bioluminescent ATP assay for the rapid detection of bacteria in foods has recendy been reviewed (Stannard & Gibbs 1986). The method relies on the fact that all living cells contain adenosinc 5-triphosphate (ATP). This can be readily assayed by measurement of the light emitted when ATP is reacted with the luciferin -Iuciferase enzyme complex. The amount of light generated is proportional to the ATP concentration which, in turn, is proportional to the number of cclls present. The detection of bacteria in milk by this method is complicated by the presence of free ATP associated with the colloidal calcium phosphate-citrate complex of the casein micelle (Richardson et al. 1980) and also presence of somatic cell ATP, both of which must be removed before meaningful estimates of bacterial ATP can be obtained. Bossuyt (1981) described a method for the estimation of bacterial numbers in milk using a bioluminescent assay system which involved extraction and removal of non-bacterial ATP prior to quantification of bacterial ATP. The test in this form takes c. 45 min to complete. For some time, the dairy industry has required a rapid bacterial counting procedure to monitor tanker milks arriving at processing sites. Ideally, this test should take less than 10 min to provide results before the tanker unloads. Bossuyt (1982) described a modification of his original test which allowed completion within 5-1 0 min: this revised procedure involved the addition of a Ca 2 + sequestrant to aid release of free ATP and an increase in the concentration of the apyrase enzyme required to hydrolyse the non-bacterial ATP. Using this system allowed the detection of milks with a bacterial count greater than 1 x 106 cfu/ml with 90% accuracy (Bossuyt 1982). However, with the introduction of payment schemes to farmers based on the total bacterial count (TIlC) of milks, the hygienic quality of farm bulk tank milks has improved dramatically (Harding 1987). The average TBC for milks produced in the

Cop)"ight Rapid lVlicrobiological Methods for Foods, Beverages and Pharmaceuticals

13

©

/989 by the Sodel)' jiJr Applied Btuuri%!'J' All righls of reproduction in any jimn reserved 0-632-02629-4

14

M. W. GRIFFITHS AND ]. D. PHILLIPS

UK is now about 2 X 104 cful m!. Thus, in order to be efIective, the sensitivity of the bioluminescent bacterial assay system must be improved. Techniques for achieving this have been investigated. These have included modifications to the assay procedure and removing and concentrating bacterial cells from raw milks. If dle bacteria from a relatively large volume of milk can be removed, a greater concentration of bacterial ATP can be presented to the assay system. Also, the quenching effect of milk on the light emission by luciferase will be absent. This should improve the sensitivity of the test. Various methods have been investigated for the removal of bacteria from milk and include electrostatic charge and affinity binding techniques. The use of the bacterial ATP assay for the assessment of the quality of pasteurized milks and for the monitoring of processing plant hygiene will be described.

Materials and Methods Samples Samples of ex-farm bulk tank milks wefe obtained from the Scottish Milk Marketing Board. In some cases where high bacterial counts were required these were incubated at 10°C for 48 h. Retail packs of freshly pasteurized milks were obtained from two creameries in south-west Scotland and transported to the laboratory in insulated containers. Samples were transferred to sterile containers (Sterilin Ltd, Hounslow, Middlesex) and stored in a thermostatic water bath (Grant Instruments (Cambridge) Ltd) at 6°C. Sub-samples were removed at intervals and the psychrotrophic count determined. The shelf-life of the samples was defined as the time in days for the psychrotrophic count in the milk to reach 1 x 107 cful m!. This is the count at which organoleptic changes in IDe product are usually detected (Muir & Phillips 1984). Samples were inoculated on Milk Agar plates (Oxoid) using a Spiral Plate Maker (Don Whitley Scientific Shipley, Yorks). When required, dilutions were made with Maximum Recovery Diluent (Oxoid CM733). Psychrotrophic counts were determined after incubation of the plates at 21°C for 25 h (Griffiths et af. 1980) and mesophile (total bacterial) counts after incubation at 30°C for 3 days.

Bacterial ATP assay in raw milk The reagents used were supplied as a Milk Bacteria Kit (Lumac BV, Schaesberg, The Netherlands). To NRS reagent (Lumac BY) were added

BACTERIAL ATP IN MILK

15

Somase (0.5 units/ml) (Lumac BV) and a chelating agent. Somase is the trademark for the ATP-hydrolysing enzyme, apyrase. In some experiments a different source of apyrase (Sigma Chemical Co. Ltd, Poole, Dorset) was used. The chelating agents studied were EDTA (ethylenediaminetetraacetic acid, disodium salt; 10 mmo1/I), EGTA (ethyleneglycolaminoacetylether tetraacetic acid; 80 mmol/l) and trisodium citrate (40 mmol/l). In one experiment, Triton X-lOO at a final concentration of 0.1 % was added with the citrate. NRS containing apyrase (500 !AI) and chelator were added to milk (500 !AI). Following incubation at room temperature for 5 min, 50 !AI were removed to a Lumacuvette (Lumac BV) and L-NRB (150 !!I; Lumac BV) were added. The cuvette was placed in a Lumac Biocounter M2010 and after 30 s, Lumit PM (100 !AI; luciferin-luciferase, Lumac BV) reconstituted in Lumit buffer (0.025 mo1/1 HEPES, pH 7.75; Lumac BV) according to the manufacturer's instructions, was added manually or using the automatic dispensing system of the instrument. The light emitted after lOs integration was read from the digital display as relative light units (RLU). The use of different luminometers to measure light output was also assessed. The machines used included Lumac Biocounter models M2010 and M2500, an LKB 1250 and a Turner Designs model 20-000. The wearing of disposable gloves was essential throughout the assay procedure to avoid contamination with ATP from the operator's skin. It was also important that reagents were allowed to reach room temperature slowly before use. Using cold reagents led to artificially low ATP levels being recorded. The modified procedure was compared with the method described by Bossuyt (1982). In this case 50 I-tl of milk sample were treated with 100 !AI NRS containing EDTA and apyrase at the concentrations described above. After 5 min continuous shaking at room temperature 150 !AI of L-NRB were added and after a further 15 s, 100 !AI of luciferin-Iuciferase solution were added. The generated light signal was integrated for lOs by the Biocounter and the result expressed in RLU.

Removal of bacteria from milk

By electrostatic charge Charged matrix (1.0 g) was placed in a chromatography column (1.5 X 30 em; Biorad Laboratories Ltd, Walford, Herts) and milk (10 ml) was added with the column outlet closed. In some experiments, nutrient broth cultures of bacteria were substituted for milk. The column was shaken at room temperature for 5 min using a wrist action shaker (Stuart Scientific Ltd, Croydon, Surrey) at maximum speed. After shaking, the column outlet was opened and the milk

16

M. W. GRIFFITHS AND J. D. PHILLIPS

removed. The column contents were washed with 10 ml of freshly distilled water, The compounds studied were magnetite (iron 2,3 oxide) and Celite (diatomaceous silica; Koch Light Ltd, Haverhill, Suffolk). Alternatively, the removal of bacteria from milk using magnetite was carried out in a test-tube. The magnetite was retrieved by placing a magnet at the bottom of the tube and the fluid decanted off. Being magnetic, the magnetite was retained in the tube. For removal of cells using Zeta plus filters (05S grade, 45 mm diameter; Gelman Sciences Ltd, Brackmills, Northarts), the filters were held in a 47-mm Swinnex filter holder (Millipore Ltd) and milk or nutrient broth cultures of bacteria (:0::;50 ml) were passed through the filter using a 60-ml syringe. The filter was washed with 10 ml of freshly distilled water.

By affinity techniques The lectins used in this study were Concanavalin A, Helix pomatia and Triticum vulgaris, immobilized on cross-linked 4% beaded agarose (Sigma Chemical Co. Ltd). Care was exercised in the choice of lectins used as some are extremely hazardous. Lectin (equivalent to I mg) was added to a chromatography column (l.5 x 30 cm) and milk (l0 ml) added with the column outlet closed. The column was shaken for 5 min at room temperature as described previously and subsequently the milk was removed from the column via the column outlet tap. The matrix was washed with 10 ml of freshly distilled water.

Bacterial ATP estimation on concentrated cells The matrix-bound cells obtained by the above procedure were washed with NRS (500 f.tl) with shaking for c. 5 min. The NRS was drained from the column and the matrix washed through with 10 ml of freshly distilled water. With the column outlet closed, L-NRB (200 f.tl) was added and the column contents shaken for 30 s. The L-NRB was collected and a sample (usually 100 f.tl) assayed for ATP with luciferin -luciferase in the manner already described.

Pasteurized milk testing Samples of freshly pasteurized milk (l0 ml) were pre-incubated at 21°C for 25 h in the presence of a sterile solution (0.1 ml) containing crystal violet (2 mg/ml), penicillin (20000 U/mi) and nisin (40000 U/mi) to prevent Grampositive bacterial growth (Griffiths et al. 1984a, b; Phillips et al. 1984). Following the pre-incubation procedure, the milk was assayed for bacterial ATP using the Milk Bacteria Kit (Lumac BY). NRS (500 ftl) was added to

BACTERIAL ATP IN MILK

17

milk (500 Ill) with the subsequent addition of Somase (20 Ill; one vial of somase reconstituted with 1 ml Lumit buffer). After incubation at room temperature for 45 min a sample (50 Ill) was removed and added to a Lumacuvette. L-NRB (150 Ill) was added and, after 30 s incubation, Lumit PM (100 Ill) was injected into the cuvette. The light emission following a lO-s integration time was noted.

Variation in pasteurized milk processing A series of experiments was performed using the processing facilities at the Institute which allowed production of pasteurized milks on plant with different degrees of sanitization. Milks were pasteurized using an APV Junior paraflow cream pasteurizer (10-15 gallons/h) (APV Co. Ltd, Derby). Inadequate cleaning was obtained when cold ODC (Reddish Savilles Ltd, Cheadle, Cheshire) (1 % w/v) was circulated through the plant using the product pump at a maximum flow rate of 20 gallons/h. A more effective cleaning regime was achieved by circulation of ODC (1 % w/v) at 75°C and 60 gallons/h for 0.5 h by means of a centrifugal pump (Parsilac type ZMH No.1). Pasteurized milk samples were collected into sterile containers and subjected to the pre-incubation-ATP assay procedure outlined above. Shelf-life of the milks was determined by following bacterial growth in the product at 6°C and lO°C. Results

Removal offree AT? by use ofsuquestrants The effect of EDTA, citrate, EGTA and citrate - Triton X-I 00 additions prior to extraction of nucleotide with NRS are shown in Fig. 1. There was little difference in the results obtained with all four chelating systems. The background readings appeared to be lowest in the presence of citrate but maximum light output was achieved with the assay system containing EDTA. The sensitivity of the assay remained the same regardless of the nature of the sequestrant.

Comparison of assay s.ystems jar use with raw milks There was a significant difference in results achieved using the 5-min bacterial ATP assay as described by Bossuyt (1982) and the modification in which an aliquot was removed before L-NRB extraction (Fig. 2). Substantial scatter of points was obtained using the original method, but, with the modification, a significant curvilinear relation (r = 0.84) was obtained between TBC and the bacterial count by bioluminescence. This may in part be due to the influence

18

M. W. GRIFFITHS AND ]. D. PHILLIPS

4.5

::> ...J

3.5

~ iJ

c:

:1 0

• •

U

a.. f-

« ~

0)

0

....J

log,o total bacterial count Icfu/ml)

I. Effect of chelating agents on the bacterial ATP assay in milk. The non-bacterial ATP was extracted with NRS containing: trisodium citrate, 40 mmol/l (0); EGTA, 80 mmol/l (.); EDTA disodium salt, 10 mmol/l (-); trisodium citrate (40 mmol/J) together with Triton X-100, 0.1% (A).

FIG.

3.2 ::> ...J

~

E :::)

0

3.0 2.8

u

a..

f-

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01 0 ...J

2.6 2.4 4

FIG.

••

~

2.2 a

•• . • • ••• • •• • . .. ,.• • • I~

••

5

•• •

••

•

6

7

:3

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:::)

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..~

Q)

...J

Log lO total bacterial count (cfu/mll

• •• • • ••

5

6

7

Log 10 total bacterial count (cfu/ml)

2. Comparison of the 5-min bacterial ATP assay procedure: (a) according to Bossuyt

(1982); (b) the modification of that procedure whereby a sample of the reaction mixture was removed following extraction with NRS, for rea.ction with luciferase-luciferin.

BACTERIAL ATP IN MILK

19

of shaking during incubation with NRS which was found in some instances to reduce the light output of the reaction mixture. However, there was a good correlation between results obtained when the assay mixture was shaken and unshaken (r = 0.89; n = 55). There was some variation in results obtained using the modified 5-min ATP test on different batches of milk. The main differences were observed for milks with counts below 1 x 105 cful mI. The variation \vas due to changes in background counts noted with these milks. The variation in ATP readings obtained for milks with counts in excess of 1 x 10 6 /ml was acceptable. There was no increase in sensitivity of the assay regardless of the photometer used. Comparison of luciftrin -luciftrase reagents

As well as the luciferin -luciferase supplied as Lumit PM (Lumac BV), an alternative source of the enzyme (Sigma Chemical Co. Ltd) was also studied. The light output in the assay system containing Sigma enzyme was c. 20 times that of the Lumit luciferase. However, there was little or no improvement in the sensitivity of the assay using Sigma enzyme (Fig. 3). The second-degree polynomial regression coefficients were 0.78 and 0.72 for the Sigma and Lumit enzymes, respectively. There was a strong correlation (r = 0.92) between results obtained using the luciferase supplied by Sigma and Lumac. Removal of bacteria from milk

~y

electrostatic interaaion

Bacteria usually carry a net negative charge on their surface, and use can be made of this fact to remove bacteria from suspension (Daniels 1972; Wood 1980). A number of charged matrices have been examined for their ability to remove bacteria from suspension. The efficiency of removal of some bacteria commonly found in milk is shown in Table 1. In general, Gram-positive bacteria were more readily adsorbed on to the charged matrices tested than Gram-negative organisms. However, certain species of Gram-negative bacteria were effectively adsorbed. Bacteria could be removed from milk by electrostatic adsorption with an efficiency of between 25 and 90'10 depending on the milk sample and matrix type (Table 2). Zeta plus 05S grade filters were most effective at removing bacteria from milk. Celite also proved reasonably effective. Both the Zeta plus filters and Celite removed cells by a combination of electrostatic interaction and entrapment. Removal of bacteria from milk ~y affini~y techniques

Lectins are proteins which selectively bind carbohydrates but do not exhibit enzymic activity. They have been shown to react with a wide range of bacteria including species commonly found in milk (Pistole 1981).

20

,•

M.W. GRIFFITHS AND ]. D. PHILLIPS 5.2

• • ••• •

•

4.7

• 4.2 ~

...J

~

C ~

0

()

a..

~

3.7

I-

0

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0 ...J

0

0

3.2

0

0

0

0 0

2.7

0

2.2

3

4

5

6

7

8

Log 10 total bacterial count (cfu/ml) FIG. 3. Comparison of luciferase-luciferin obtained from Lumac BV (0) and Sigma (e) for estimating the bacterial ATP content of raw milk.

Immobilized lectins were studied to ascertain whether they could bind bacteria present in milk (Table 3). Of the lectins studied, Concanavalin A (Con A) proved the most effective and was able to remove a significant proportion of the milk microflora. The Triticum vulgaris lectin was much less effective than Con A.

ATP assay of bacterial cells concentrated by electrostatic interac#on The use of Celite to entrap bacterial cells in milk followed by a bioluminescent assay may be a way of improving the sensitivity of the 5-min assay procedure. Figure 4 shows a good correlation (r = 0.90) between the RLU output during

21

BACTERIAL ATP IN "vIILK TABLE

1. EjfiCiL7llJ oj'removal oj' baclaia Fom sUSf!L7lSif!l1 by electrostatic dlarge '7,. Removal of bacteria by

Zeta plus filter

Magnetite

Celite

Gram-positive Bad!lus brevis B. ({nus B. drtulal/S B. lichL7lijimllis B. pumilils B. subtilis

11.1 92.6 48.9 57.2 63.6 65.8

0 0 0 0 0 0

17.6 86.5 73.8 13.8 63.6 0

La(/obadlills Cl/seii var. cascii L pla11tarum

86.4 0

0 0

97.0 0

JJ icrococcus roseus Micro(()ccus sp.

20.3 82.9

0 14.1

32.8 n.d.

Staphylococcus sp. Staph. aureus Staph. warnerii

72.5 75.6 92.9

76.4 74.6 70.5

82.9 96.8 94.2

Streptococcus jill'calis Strep. lactis Sirep. lactis var. maltigou's

88.1 85.4 71.8

33.0 18.3 20.5

94.1 95.7 66.0

Gram-negative Achromobacta sp.

50.0

Organism

Ad11etobclcter sp.

84.3

/!eromm/as sp.

89.4

Alcaligem's jaecalis

60.6

Chromobacterium sp.

78.7

0 0

60.3 17.5 20.0

Citrobacta FClmdii

71.3

E11terobacter agglomera11s

79.5

escherichia coli

65.4

Serratia liqucfilciL7ls Serr. marceSCL7lS

66.3 36.2

50.4

Pseudmlll!l1as fluorescL7ls Ps. Fagi Ps. lemOll11ieri Ps. putida Ps. stutzeri

47.8 78.5 67.2 60.8 70.4

()

n.d., not determined.

19.8 0 0 ()

0 0 0 ()

58.9 63.2 93.5 91.3 36.9 82.6 61.1 84.5 49.4 51.3 86.9 22.2 84.4 93.8 n.d.

22

J.

M. W. GRIFFITHS AND

D. PHILLIPS

2. Removal of bacteria from milk by electrostatic charge

TABLE

Average

% removal f\1atrix

Number of e",:periments

of bacteria from milk

Range (%)

6 6

40.0 29.6 70.9

4-93.3 0-65.6 29.4-99.3

Celite Magnetite Zeta plus filter

TABLE

22

3. Removal of bacteria from milk by immobilized leetins % Removal of bacteria from milk

Lectin Concanavalin A Helix pomatia lectin Triticum vulgaris lectin

93.0 45.0 25.0

Values shown are means of three milk samples.

.

4

5-oJ

/

~ ::J

(.)

/

0...

f-

/

/

/

/

/

/

/

0

«

. • .. • .... /,...

C

/

/

• ..

3

••

9

en

/

/

/

/

/

/'/

/

0

~/.

-oJ

//

./

•

2

3

4

5

6

7

8

Log 10 total bacterial count (cfu/mll FIG. 4. Effect of removal of bacterial cells from raw milk by adsorption on Celite before ATP assay.

BACTERIAL ATP IN MILK

23

ATP assay of entrapped cells and the TBC of the original raw milk. The relation was linear over the TBC range 3.8 x 103 cfu/rnl to 1.7 x 108 cful ml. A weaker correlation existed when magnetite was used as the adsorbing matrix (r = 0.53; n = 101) for bacterial cells in milks with counts ranging from 1.8 x 10 2 to 2.1 X 108 cfu/ml. Initial results using the NRS/L-NRB e},.1:raction system for nucleotides from bacterial cells entrapped in Zeta plus filters have not been encouraging. This was mainly due to the physical nature of the membrane which made it difficult to retrieve the small volumes of reagents added. The cost of the extractants would make the use of large volumes uneconomical for routine testing. The use of other, cheaper, methods of ATP extraction such as trichloroacetic acid (Lundin & Thore 1975) are under investigation. AT? assay of bacterial cells concentrated by affinity techniques

Following reaction of the bacterial cells in milk with immobilized Con A their numbers were assessed using luciferin-luciferase. There was a weak correlation (r = 0.59) between the bacterial ATP present in the adsorbed cells and the TBC of the original milks. However, the milks examined had bacterial counts in the range 3.3 X 103 to 1.5 X 105 cfu/ml. This was well below the level at which bacterial cells could be detected using the conventional bioluminescence assay technique (Bossuyt 1981, 1982). Thus affinity binding techniques of this kind have an application in improving the sensitivity of the bioluminescence assay for the bacterial content of milks. Shelf-lift test.for pasteurized milk

Previous work described the use of bioluminescence in combination with a pre-incubation procedure for evaluating the shelf-life of pasteurized milk from retail outlets (Phillips & Griffiths 1985). The application of the method to freshly pasteurized milk has also been studied (Griffiths & Phillips 1986). A curvilinear relation existed between the shelf-life of the freshly pasteurized milk and the bacterial ATP content of pre-incubated samples (Fig. 5). The corrc1ation coefficient was 0.64 using the derived equation: shelf-life (days)

=

10.63 - Y(41.67 x log PINe ATP count - 83.79)

The shelf-life of milk samples with an expected shelf-life ofless than 6 days at 6°C could be predicted with an accuracy of between 80 and 90% using this method (Table 4). The reliability of the method fell when used to predict milks of better keeping quality.

24

M. W. GRIFFITHS AND ]. D. PHILLIPS 5

o

5

4

o

...J

0::

c:;,

o u a..

3

I-

.3

2

00

o

@

10

I

---------+~-----+--------

!

I

2

4

I

!

I

1

6

8

10

12

~

14

Shelf-life (days)

FIG. 5. Relation between shelf-life and bacterial ATP content of fresWy pasteurized milks after pre-incubation at 21°C for 2S h in the presence of cl)'stal violet-penicillin-nisin.

Hygiene testing using in-line sampling in conjunction with pre-incubation procedure During the collection of freshly pasteurized milks from the two processing sites, in-line samples of milk were taken at various points in the production line. The milks were then subjected to the pre-incubation procedure followed by bacterial count using the bioluminescence technique as oudined for pasteurized milks. The quality of in-line samples from the two creameries is shown in Table 5. Creamery A showed no significant change in shelf-lives of in-line samples taken at the pasteurizer, at the homogenized milk tank or at a point immediately prior to the filler. The bacterial ATP counts on preincubated samples of these milks were also very similar. However, after packing, there was a significant change in shelf-lives and A TP counts. This was due to contamination at the filler which was of the piston type. These have been shown to cause contamination of the product due to air/product contact in the filler bowl and cleansing problems in the sliding areas of the filling mechanism (Schroder & Bland 1983). For creamery B, there was a slight loss in shelf-life in milk sampled from storage tanks prior to the filler compared with milks obtained from the pasteurizer. This was reflected in a slight increase in the bacterial ATP content ofpre-incubated storage tank milk. There was no appreciable difference between milks from the storage ta~ and canoned milk for this creamery

25

BACTERIAL ATP IN MILK TABLE

4. Reliability o/pre-incubation A7P count in predicting shelf: liji: o//reshlr pasleurized mill:

Expected shelf-life at 6°C (days)

4 6

8 IO

Predicted ATP count after pre-incubation*

'~o Samples

(RLU)

correctly dassified t

1096 302 132 87

89.5 80.7 68.0 69.6

* Pre-incubation ATP count predicted from regression equation for product having the expected shelf-life indicated. t Percentage of samples falling into predictive quartiles (e.g. 88.4% of samples with an ell..p ected shelf-life of <4 days or >4 days had a pre-incubation ATP count of > 1000 or < 1000 respectively). RLU, relative light units. TABLE

5. HygiC11ic quali!:y

Creamery

A Milk ex-pasteurizer Milk ex-homogenized milk tank Milk before filler Cartoned milk B Milk ex-pasteurizer Milk ex-storage tank Cartoned milk

0/ in-line samples Average shelf-life (days)

Average ATP pre-incubation count (log RLU)

7 7 7 8

11.8 12.1 11.7 6.5

2.11 2.04 2.08 2.33

10 10 10

14.7 I\.4 I\.6

2.07 2.17 2.16

No. of samples

RLU, relative light units.

either in terms of shelf-life or pre-incubated ATP count. This was due to the use of a Tetra Brik machine for filling which reduced contamination of product to a minimum. A separate e:\'])eriment to examine the usefulness of the bioluminescence technique coupled with pre-incubation to detect unclean plant gave similar results. Milk was processed at the Institute using inadequately cleaned plant and this resulted in a short shelf-life and high pre-incubated ATP count (Table 6). \\Then improvements were made to the cleaning operations there was a concomitant increase in shelf-life and lowering of pre-incubated ATP count.

26

M. W. GRIFFITHS AND TABl.E

J.

O. PHILLIPS

6. De/eelion of illadequate plant cleaming operatiollS Average shelf-life (days)

Cleansing procedure Inadequate (cold ODe, slow circulation) Adequate (hot ODe, rapid circulation)

Average pre-incubated ATP count (log RLU)

8.8

5.1

3.21

16.7

7.1

2.10

RLU, relative light units.

Discussion Raw milk testing

The estimation of bacterial numbers in milk by bioluminescent ATP assay is complicated by the presence of free ATP associated with the calciumphosphate -citrate complex of the casein micelles (Richardson et al. 1980). Free ATP has been found at levels of 0.13-0.31 ~mol/l (average 0.23 ~mol/l or c. 130 ng/ml) in milk. The total ATP present in milk containing somatic cells (4.5 x 105 /ml) and bacteria (2 x 104 /011) has been given as 210 ng/ml (Theron et al. 1986a). \Vith ATP at 0.4 fg/cfu (D'Eustachio & Johnson 1968) or 1 fgl cfu (Sharpe et al. 1970) for bacteria, the free ATP concentration in milk is equivalent to that contained in c. 1 X 108 bacterial cful ml. Poor correlations of ATP levels with bacterial numbers in milk samples with fewer than 1 x 105 cful ml may result mainly from ATP associated with casein micelles (Packard & l\1arth 1983). Casein micelles may be dissociated by the use of chelating agents (Lin et at. 1971). This results in improved access to the calcium phosphate bound ATP for the apyrase present'in the assay nlixture. Incorporation of EDTA into the NRS - somase reagent resulted in a decrease in background ATP levels (Theron et al. 1986a). The concentration of EDTA present in the reaction mixture is crucial and may account for some variation between results produced when using different batches of reagents (Neaves, personal communication). An improvement in the correlation of bacterial counts, as estimated by plate assay, with ATP determination was found when a small sample was removed after treannent with NRS - EDTA- somase reagent for reaction with L-NRB and luciferase. This was due primarily to decreased quenching of the light output by the milk present in the assay mixture. This quenching is

BACTERIAL ATP IN MILK

27

mostly caused by optical interference (Bossuyt 1978; Theron et al. 1986b) but chemical inhibition of the luciferase enzyme may also contribute (Aledort et al. 1966; Denbury & McElroy 1970; Thore 1979). Thus, using the protocols and reagents currently available for ATP assay, only bacterial levels in raw milk greater than about 5 X 105 cfu/ml can be detected. The reason does not depend solely on the sensitivity of the luminometers available. In this study, a luminometer (Lumac Biocounter M2500) was evaluated: this could detect 0.1 pg of ATP and would be expected to detect c. 5 X 103 bacterial cfu/ml by the assay procedure described. ATP can also be derived from somatic cells in milk: this ATP may approach 300 fg/cell (Bossuyt 1978). Theron et al. (l986a) have shown that the NRS-EDTA-somase treatment does not eliminate all non-bacterial ATP. The use of these extractants may also lower the ATP content of bacterial cells in milk. (Theron et al. 1986a). In addition, somatic cells can produce ATP-hydrolysing enzymes when present in large numbers (2 X 106 cells/ml) and these enzymes can hydrolyse extracted ATP (Botha et al. 1986). Pseudomonas fluorescens was also found to synthesize ATP-ase although bacterial counts of 1 X 108 cfulml were required before enzyme activity was detected (Botha et al. 1986). In order to improve the sensitivity of the bacterial ATP assay for use with milk, either the methods for destroying non-bacterial ATP must be improved or techniques to remove bacteria from milk before assay must be sought. Methods have been described for removal of bacteria from suspension. Most micro-organisms are negatively charged at pH values greater than 5.0 and will adsorb to positively charged matrices (Wood & Gibbs 1982). Many ion exchange matrices are available for use (Daniels 1972; Wood 1980) and methods for the removal of bacteria from water by electro-adsorption have been described (Oren et al. 1983). Magnetite, the magnetic oxide of iron, has been used eJl.'})erimentally to adsorb bacteria (De Latour & Kolm 1976) and has the advantage of being easily removed by magnetism. MacRae & Evans (1983) showed that more than 95% of cells of Escherichia coli, Streptococcus Iaecalis and Pseudomonas stutzeri could be adsorbed by magnetite at pH 6.0. The use of clectropositively charged filters for removal of bacteria from foods, including milk, has also been described (Kroll 1985). In the present study, magnetite, Celite (a commercially available diatomaceous silica) and Zeta plus filters were investigated for their ability to adsorb bacteria. For magnetite, 12 of the 31 strains of bacteria studied could be removed from suspension with efficiencies ranging from about 14 to 76% removal. The Gram-positive bacteria seemed to be adsorbed by magnetite more effectively than were the Gram-negative organisms studied. Streptococcus faecalis was adsorbed but none of the Bacillus spp. tested could be removed from suspension by magnetite. None of the Pseudomonas spp. tested, nor E.

28

M. W. GRIFFITHS AND

J.

D. PHILLIPS

coli, was adsorbed by magnetite and this makes its usefulness for removing bacteria from milk questionable. Such was the case, with the magnetite effectively removing bacteria from milk in only about 33 (~o of samples. This may have reflected the age of the milk as the microflora of freshly drawn raw milk consists mainly of Gram-positive bacteria whilst Gram-negative bacteria predominate in stored milks. Celite and Zeta plus filters were equally effective in removing bacterial cells from suspension in nutrient broth. There appeared to be little difference in the selectivity of the binding of Gram-positive or Gram-negative organisms. When the systems were applied to milk samples Zeta plus filters were the more effective in adsorbing bacterial cells. This was probably due to the physical nature of the filter which led to the retention of more bacteria by entrapment. The physical nature of the Zeta plus filter made it less amenable to use in conjunction with ATP methodology. Reasonable results, however, were obtained by using the bacterial ATP assay on cells removed from milk by Celite. Results indicate that the sensitivity of the method can be increased by at least one log cycle when this bacterial concentration step is incorporated in the assay system. Lecrins bind with polysaccharide residues of the cell wall of Gram-positive bacteria and the lipopolysaccharides of the cell envelope of Gram-negative bacteria. Among the bacteria that react ffith leetins are some commonly found in milk, such as streptococci (Hamada et ai. 1977) and members of the Enterobacteriaceae (Le Minor et al. 1973). Lectins with different specificities can be obtained and this present study investigated the use of Concanavalin A which is specific for (Y-D-mannose and (Y-D-glueose residues (Reeke et al. 1974), Helix pomatia lectin specific for N-acetyl-D-galactosamine (Hammarstrom el al. 1972b) and Triticum vulgaris lectin which reacts with sialic acid and N cetyl-D-glucosamine (Allen et al. 1973; Nagata & Burger 1974). Gram-positive bacteria including Bacilmus subtilis, Lactobacillus plantarum and Streptococcus faecalis were agglutinated by Concanavalin A because their cell walls contained teichoic acids substituted by (X- D-glucopyranosyl residues (Doyle & Birdsell 1972; Archibald & Coapes 1971; Bauer et al. 1974) whilst both 'Con A and llelix pomatia lectin have been shown to interact with the lipopolysaccharides of Gram-negative bacteria (Doyle et al. 1968; Hammarstrom & Kabat 1971; Hammarstrom et al. 1972a; Pistole 1981). Immobilized lecrins could, in fact, be used to remove bacteria from milk. Con A was the most effective in removing bacteria from raw milk with the Triticum vulgaris lectin the least effective. Subsequent assay of bacteria retnoved by inlmobilized Con A using the ATP technique gave a significant correlation with total bacterial counts in milks containing less than 1 x 105 cful ml.

BACTERIAL ATP IN MILK

29

Pasteurized milk testing

It is well documented that the main factor governing the shelf-life ofpasteurized products is the growth of psychrotrophic, Gram-negative bacteria introduced as the result of post-heat-treatment contamination (Griffiths et al. 1985). Although these organisms grow well at refrigeration temperatures their optimum temperature for growth is about 20- 22°C (Lawton & Nelson 1954). Indeed, a good correlation has been obtained between the growth of psychrotrophs in milk at 6°C for 14 days and at 21°C for 25 h (Oliveria & Parmelee 1976). Thus, they may be selectively grown at a temperature of 21°C for 25 h with incorporation of an inhibitor system which prevents Gram-positive bacterial growth. Of the inhibitors studied, crystal violet-penicillin-nisin had least inhibitory effect on Gram-negative growth whilst still preventing growth of Gram-positive bacteria (Phillips & Griffiths 1986). The bacteria that grew under these pre-incubation conditions could be estimated using the ATP assay. This provided a good indication of the quality of pasteurized products including milk (Phillips & Griffiths 1985; Griffiths & Phillips 1986) and cream (Griffiths et al. 1984a; Phillips & Griffiths 1985; Griffiths & Phillips 1986) within 26 h of production. A similar test for predicting the keeping quality of pasteurized milk has been described (Wacs & Bossuyt 1981, 1982). Pre-incubation conditions of 30°C for 24 h in the presence of benzalkoncrystal violet to inhibit Gram-positive bacterial growth were used prior to bacterial ATP estimation. Benzalkon was, however, more inhibitory towards Gram-negative bacteria than the crystal violet-penicillin-nisin mixture and many psychrotrophs did not grow well at 30°C (Langeveld et al. 1976). The use of the pre-incubation test in conjunction with in-line sampling at the processing site allowed the monitoring of the efficacy of plant cleaning operations. The source of contamination could be identified within a useful time frame and remedial measures taken. References ALWORT, L.M.R., WEW, R. & TROUP, S.B. 1966. Ionic effects of firelly bioluminescence assay of RBC ATl'. Analytical Biochemistry 17, 268-277. Al.LEN, A.K., NEUBERGER, A. & SHARON, N. 1973. The purification, composition and specificity of wheatgerm agglutinin. Biod1l'mimt ]ounJal131, 155~ 162. ARCHII.lALD, A.R. & COAPES, I I.E. 1971. The interaction of concanavalin A with teichoic acids and bacterial walls. Biodlemiml]ounJal 123, 665-667. EMlER, 11., E\RR, D.R. & IIORISBERGER, M. 1974. Ultrastructural localization of cell wall teichoic acids in Streptococcus jaew/z'· by mcans of concanavalin A. Archives o/Miaobiolo!..'J! 97, 17-26. BOSSUYT, R. 1978. Usefulness of an ATl' assay technique in evaluating the somatic cell content of milk. Milduvissetlsc!llIji 33, 11-13. BOSSUYT, R. 1981. Determination of bacteriological quality of raw milk by an ATP assay

30

M. W. GRIFFITHS AND

J. D. PHILLIPS

technique. Mi/cllWisSt'l1schafi 36, 257- 260. BOSSUYT, R. 1982. AS-minute ATP platform test for judging the bacteriological quality of raw milk. Netherlands Milk and Dairy Journal 36, 355 - 364. BOTHA, W.e., LUCK, H. & JOOST£. P.J. 1986. Detennination of bacterial ATP in milk - the influence of adenosine triphosphate-hydrolysing enzymes from somatic cells and Pseudomonas jiuoresce1ls. Journal of Food Protectioll 49, S22-82S. DANIELS, S.L. 1972. Adsorption of microorganisms onto solid surfaces. Review. Developments in Industrial Microbiolog)1 13, 211- 253. , DE LATOUR, C. & KOLM, H.B. 1976. High-gradient magnetic separation a water treatment alternative. Journal of the American Water Workers Association 68, 325 - 327. DENBURY, J.L. & lVlcELROY, \V.D. 1970. Anion inhibition of firefly luciferase. Archives of Biochemistry aud Bioph,ysics 141, 668-675. D'EUSTACHIO, A.]. & JOHNSON, n.R. 1968. Adenosine triphosphate content of bacteria. Federalio11

Proceedings of the Federation ofAmerican Soaeties of Expen'mC11tal Biology 1968, p. 761. DOYl.E, R]. & HIRDSE!.L, D.C. 1972. Interaction of concanavalin A with the cell wall of Bacillus

subtilis. Journal of Bacteriology 109, 652-658. DOYLE, R.j., WOODSIlJE, E.E. & FISHEL, C.\V. 1968. Protein and polyelectrolyte interactions:

The concanavalin A precipitin reaction with polyelectrolytes and polysaccharide derivatives. Biochemical}ourna/106, 35-40. GRIFFITHS, M.W. & PHILLlI'S, J.D. 1986. The application of the pre-incubation test in commercial dairies. Australian Journal of Dairy Technology 41, 71-79. GRIFFITHS, Nl.W., PHILLIPS, J.D. & Mum., D.O. 1980. Rapid plate counting techniques fc)r the enumeration of psychrotrophic bacteria in pasteurized double cream. Joumal of the Societ;y oj Dairy Tedmology 33, 8-10. GRIFFITHS, M.\V., P~IILl.IPS, J.D. & NluIR, D.O. 1984a. 1\1ethods for rapid detection of postpasteurization contamination in cream. Joumal oj tilt' Society of Dairy Teclmology 37, 22 - 26. GRIFFITHS, M."V., PHILLIPS, J.D. & lVlulR, D.O. 1984b. Rapid detection of post-pasteurization contamination. Hannah Research Institute, Bulletin No. 10. GRIFFITHS, M.\\I., PHILLIPS, J.D. & MUIR, D.O. 1985. Post-pasteurization contamination - the major cause of failure of fresh dairy products. Hannah Research 1984, 77-87. IIAMAOA, 5., GILL, K. & SL:\DE) H.D. 1977. Binding of lectins to Streptococcus mutans cells and type-specific polysaccharides and effect on adherence. Injec/ion and Immunio' 18, 708 - 716. lIAMMARSTR()M, S. & KAUtH, E.A. 1971. Studies on specificity and binding properties of the blood group A reacth;e hemagglutinin from Helix pomatia. Biochemistry 10, 1684-1692. lIAMMARSTROM, S., LINDBERG, A.A. & ROI-:lERTSSON. £.S. 1972a. Precipitation of lipopolysaccharides from rough mutants of Salmonella typhimurium by an A-hemagglutinin from Helix

pomatia. European ]oi/mal

(~r Biochemistry

25, 274- 282.

IIAJ\\MARSTROM, S., "VESTHOO, A. & BJORK, I. 1972b. Subunit structure of Hehr poma/ia A hemagglutinin. Scandinavian Journal oflmmunolof!J' I, 295-309. I lARDING, F. 1987. The impact of central testing on milk quality Dairy Industries Intematiollal 52(1), 17-19. KROLL, R.G. 1985. Elcctropositivcly charged filters for the concentration of bacteria from foods. Food A1icrobiology 2, 183-186. LA.NGEVELD, L.P.M., CUPERUS, F.) VAN BREEMEN, P. & DIjKERS, J. 1976. A rapid method for the detection of post-pasteurization contamination in HTST pasteurized milk. NetherJilfuls Milk alld Dairy .Joumal 30, 157-173. L'\ \\TON, \V.e. & NEI.SON, F.E. 1954. The effect of storage temperatures on the gro\\.1h of psychrophilic organisms in sterile and laboratory pasteurized skim milks. ]oumal of Dairy S'o'e11ce 37, 1164-1172.

BACTERIAL ATP IN MILK

31

LE MINOR, L., TOURNIER, P. & CHALON, A.M. 1973. The agglutination by concanavalin A of certain Gram negative bacilli: A study into the relationship with the somatic antigens of Salmonella serotypes. Annals ofivIicrohiology, Paris 124A, 467-476. LIN, S.l I.e., DEWAN, R.K., BLOOMFIELD, V.A. & MORR, CV. 1971. Inelastic light scattering studies of the size distribution of bovine milk casein micelles. Biochemistry 10, 4788-4793. LCNDlN, A. & THORE, A. 1975. Comparison of methods for extraction of bacterial adenine nucleotides determined by firefly assay. Applied Microhiology 30, 713 -721. MM:RAE, I.C & EVANS, S.K. 1983. Factors influencing the adsorption of bacteria to magnetite in water and wastewater. Water Research 17, 271-277. j\,IUIR, D.o. & PHILLIPS, ].D. 1984. Prediction of the shelf-life of raw milk during refrigerated storage. Mildlll'issetlsdza{i 39, 7 -11. N.AGAT.A, Y. & BURGER, lv1.M. 1974. Wheat geml agglutinin. Molecular characteristics and specificity fi.Jr sugar binding. ]oumal ofBiologiwl Chemistry 249, 3116-3122. Ol.lVERLA, j.S. & PARA1EI.EE, CE. 1976. Rapid enumeration of psychrotrophic bacteria in raw and pasteurized milk. ]oumal oj'Milk (lIId le/JOtI Tl'Chnolob.1' 39,269-272. ORFN, Y., Tom.As, II. & SOFFER, A. 1983. Removal of bacteria from water by e1ectroadsorption on porous carbon electrodes. Bioell'Ctrodle1nistry and Bioenergetics 11, 347-351. PAcKMm, R.A. & MARTH, E.II. 1983. JVlodifying the ATP bacterial test for use with raw milk. }oumal of Dairy Science 66, Supp!. I, 67. PHILLIPS, ].D. & GRIFFITHS, ,\tW. 1985. Bioluminescence and impedimetric methods for assessing shelt~life of pasteurized milk and cream. Food Microbiology 2, 39- 51. PHII.I.IPS, ].D. & GRIFFITHS, M.W. 1986. Estimation of Gram-negative bacteria in milk: A comparison of inhibitor systems for preventing Gram-positive bacterial growth. ]oumal of Applied Bal'lerioloKJ! 60, 491- 500. PHlI.I.lPS, ].D., GRIFFITHS, M.W. & MUIR, D.o. 1984. Preincubation test to rapidly identifY post-pasteurization contamination in milk and single cream. ]oumal of Food Protection 47, 391-393. PISTOLE, T.G. 1981. Interaction of bacteria and fungi with lectins and lectin-like substances. Annual Rf'vjews oj'lvlicrobiology 35, 85 -112. RI.I·:KE, G.N., BECKER, ].W., CUNNINt;IHM, B.A., GUNTHER, G.R., WANG, ].L. & EDELMAN, GAl. 1974. Relations between thc structure and activities of concanavalin A. Annals of the New York Aau/i'1I1y o{Scie1lce 234,369-382. RICILARDSON, T., McG:iNN, T.CA. & KEARNEY, R.D. 1980. Levels and locations of adenosine 5-triphosphate in bovine milk. Journal o{Dajry ResClmh 47, 91-96. SCHRODER, MJ.A. & BLAND, M.A. 1983. Post-pasteurization contamination and shelf-life of IITST-pasteurized milk when filled in Liqui-Pak conventional or Model 820A cartoning machine. ]ounwl oj'the Society o{Dairy Tedll/oloKJ! 36, 43-49. SHi\RI'E, A.N., WOODROW, M.N. & JACKSON, A.K. 1970. Adenosine triphosphate (ATP) levels in foods contaminated by bacteria. ]ourl/al o{Applied 8a(faiolo[.,'JI 33, 758-767. STi\NN.ARD, C]. & GmBs, P.A. 1986. Rapid microbiology: Applications of bioluminescence in the food industry - a review. Jouml/l of Biolu1l1i1lescfllu a1ld Che1l1ilumi1lcscetlee 1, 3-10. THERON, D.P., PRIOR, B.A. & LHEGAN, P.M. 1986a. Determination of bacterial levels in raw milk: Selecthity of non-bacterial ATP hydrolysis. Joumal of Food Protaljlm 49, 4-7. THERON, D.P., PRIOR, B.A. & L.ATEGAN, P..VI. 1986b. Sensitivity and precision of bioluminescent techniques ft)r enumeration of bacteria in skim milk. ]oul71al of Food ProtatiOlI 49, 8-11. TIIORE, A. 1979. Technical aspects of the hioluminescent fireflv luciferase assay of ATP. Scimce 71lOls 26, 30-34. ' \V·\\.s, G. & Bossun, R. 1981. :\ rapid method to detect postcontamination in pasteurized milk. Nli/chwissl'llsdwji 36, 548-552.

32

M. W. GRII"FITHS AND ]. D. PHILLIPS

G. & BOSSUYT, R. 1982. Usefulness of the Benzalkon-crystal violet-ATP method for predicting the keeping quality of pasteurized milk. Journal of Food Protection 45, 928- 931. WOOD, ].M. 1980. The imeraction of microorganisms 'With ion exchange resins. In Miaobial Adhesion to Surfaces, ed. Berkeley, R.C.W., Lynch, lM., Melling, l, Rutter, P.R. & Vincent, B. pp. 163-185. Chichester: Ellis Horwood Ltd. WOOD, J.M. & Gums, P.A. 1982. New developments in the rapid estimation of microbial populations in foods. In Developmetlts in Food Microbiology, ed. Davies, R. pp. 183-214. London: Applied Science Publishers. WAE5,

DEFT: Recent Developments for Foods and Beverages G. L. PETTIPHER 1 , R. G. KROLL 2 , L.J. FARR 3 AND R. P. BETTS 4 I Cadbury Schweppes PLC, Group Research, The Lord Zuckennan Research Centre, The University ofReading, Reading RG6 2LA, UK; 2 Department ofMicrobiology, AFRC Institute ofFood Research, Reading Laboratory, Shinjield, Reading RG2 9AT, UK; 3 Foss Electric (UK) Ltd, The Chantry, Bishopsthorpe, York Y02 lQf, UK; and 4 Campden Food 0 Drink Research Association, Chipping Campden, Gloucestershire GL55 6LD, UK General Principles The direct epifluorescent filter technique (DEFT) was originally developed for counting bacteria in raw milk (Pettipher et al. 1980; Pettipher 1983). This method, which uses membrane filtration and epifluorescence microscopy, takes less than 30 min to complete and does not suffer from many of the disadvantages of other microscopical methods. Pre-treatment of the milk with lyses somatic cells and makes fat a proteolytic enzyme and surfactant at globules sufficiently fluid to enable 2 ml of milk to be filtered easily. Filtration concentrates and distributes the bacteria in a manner that makes counting easier, and the technique is about 100 times more sensitive than the Breed smear. The use of a fluorescent stain and an epifluorescence microscope produces well-stained micro-organisms that are easily distinguishable from the small amounts of fluorescent debris. A polycarbonate membrane filter is used, the flat surface of which is better suited to microscopy than the uneven surface of cellulose acetate membranes. Micro-organisms on DEFT slides can be counted by automated methods, thereby considerably reducing operator fatigue. DEFT can be used for the rapid enumeration of micro-organisms in urines, pharmaceutical products, beverages, and, with additional sample pre-treatment, a variety of foods, in addition to milk.

sooe

Apparatus, Reagents and Methodology Details of the apparatus, reagents and methods are given in the chapter by Shaw and Farr (this volume). Cflpj'righl © 1989 It), Ih,' SO';,1J' jrJr Applied BarlennlnK)' All rights ({ rcprodlU:lion in

Rapid Microbiological \lethods for Foods, !leverages and Pharmaceuticals

an)1

fium rl'Scrved

0-632-02629-4

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34

G. L. PETTIPIIER ET AL.

In addition to the count/ ml of sample, a presumptive identification of the types of micro-organisms present on DEFT slides may be made. Generally the following types of bacteria are discernible by morphology; streptococci, micrococci/staphylococci, and some bacilli and coryneforms. Fungi, yeasts and spores can also be distinguished. A tentative identification of the types of micro-organisms present in the sample may be useful in locating their source, e.g. a high bacterial count in refrigerated farm milk, where most organisms are streptococci, is usually indicative of mastitis in the herd.

Automated counting The manual DEFT count is suitable only for 30-40 samples per day per operator as prolonged use of the microscope results in operator fatigue. Micro-organisms on DEFT slides can be counted automatically using the 40-10 image analyser (Foss UK Ltd, York). A closed circuit TV camera (scanner) attached to the microscope camera port detects the microscopic image and the resulting video signal is analysed to obtain quantitative data. With the image analyser an operator can count about 50 slides per hour. The image analyser can be controlled by a microcomputer which processes the generated data. The relevant information, e.g. sample code, count/ml and grade, is printed out and can be recorded simultaneously on to a magnetic disc for storage purposes and report writing. The semi-automated count of bacteria on DEFT slides agrees well with the corresponding visual DEFT count with a correlation of 0.94 (Pettipher & Rodrigues 1982a). The counting system could be fully automated by including a cassene loading system for slides and focusing under microcomputer control. The technology for these modifications is already available. Automation of DEFT preparations (which would involve sampling, pre-treatment, filtration, staining, and rinsing stages) combined with automated counting, would provide a system capable of testing hundreds of samples each day.

Cost of equipment and disposable items The cost of equipping a laboratory for the DEFT is approximately £5000 (in 1988). The major capital cost is the epifluoresccnce microscope which retails for about £4000. The other e},.'Pensive items are the filter manifold and water bath. The additional cost of the instrumentation for semi-automated counting is about £7800. This includes the TV image analyser system and the microcomputer which, although not essential, greatly facilitates data logging, record keeping and report writing.

DEl'T: RECENT DEVELOPMENTS

35

The current cost of disposable items used in the DEFT is about £0.55 per test, of which the Nuclepore membrane filter is the most expensive single item. Alternative membranes are currently being evaluated. Reproducibility of the DEFT count seems to be similar to that of the plate colony count. The DEFT count correlates well with the plate colony count for farm, tanker and silo milks, with an overall correlation coefficient (r) of 0.91. The DEFT count is both sufficiently rapid for monitoring tanker and silo milk and sensitive enough for grading farm milk on the basis of bacteriological quality.

Foods The DEFT method was found to be unsuitable for counting micro-organisms in suspensions of food prepared in a stomacher because of the presence of food debris. Pre-filtration of the suspension through nylon filters (mesh size 5 [tm) removed most of the food debris and only slightly reduced the recovery of micro-organisms, as determined by the plate colony count (Pettipher & Rodrigues 1982b). For milk, 0.6 [Lm pore size polycarbonate membrane filters are used in the DEFT but for some 'cleaner' liquids, such as certain pre-filtered food suspensions, beverages and water, a smaller pore size (0.4 or 0.2 [Lffi). membrane filter is recommended. Depending upon the nature of the food, 4-15 ml of food suspensions (10 g food plus 90 ml diluent) could be filtered through a single nylon prefilter and 3 - 10 ml of each pre-filtered suspension could be filtered in the DEFT apparatus. A sample size of 2 ml was used routinely in the DEFT, together with a microscope factor of 57000. For fresh meat and fish, the pre-filtered DEFT count agreed well with the plate colony count over the range 10 4 -10 10/ g, with a correlation coefficient of 0.91. For counts on frozen meat and frozen fish, the pre-filtered DEFT count also agreed well with the plate colony count over the range 5 X 104 - 5 X 1Q7/ g. Of the other foods tested, there was good agreement between the prefiltered DEFT count and plate colony count for cooked meats, cream doughnuts and spices (Pettipher & Rodrigues 1982b). Beverages Methods based on the same principles as the DEFT are used to estimate viable and non-viable yeasts and bacteria in wines and on winery equipment surfaces (Cootes & Johnson 1980). The method for wine takes only 15 min to complete and has a sensitivity of less than 1 micro-organism/m\.

36

G. L. PETTIPHER ET AL.

Selective Pre-incubation for the Detection of Low Levels of Spoilage Bacteria by the DEFT The microbiology of pasteurized milk and cream is complex. During the summer months spoilage due to the growth of psychrotrophic spore-forming bacteria can be important (McKinnon & Pettipher 1983; Phillips & Griffiths 1986a). It is now generally held that low-level post-pasteurization contamination by Gram-negative rods is the most significant factor in reducing the keeping quality (KQ) of these products (Phillips et al. 1981; Schroder et al. 1982). The counts of the initial microbial flora of pasteurized milk and cream are thus of little value for predicting their KQ (Bishop et al. 1984), because the vast majority of viable bacteria present initially after pasteurization are thermoduric non-psychrotrophs which do not contribute to the eventual spoilage of these products. The definitive method of assessing the psychrotrophic contamination is by incubating plates at 7°C for 10 days. Such retrospective results are obviously of little practical value and more rapid estimates of this contamination would be most beneficial for monitoring production standard and product quality and to identifY sources of contamination. The numbers of these Gramnegative psychrotrophic contaminants are well below the current detection limits of the truly rapid methods of estimating microbial numbers, e.g. the assay of ATP or the DEFT. One approach has been to use a pre-incubation stage to increase the concentration of these organisms to detectable levels, the

premise being that the final concentration of organisms after pre-incubation is proportional to the original number of organisms present. The DEFT and ATP methods cannot, at present, easily distinguish between the types of organisms in the sample (but see Rodrigues & Kroll 1985), so the preincubation stage needs to be selective against the Gram-positive organisms (generally non-psychrotrophic). The outer membrane of Gram-negative bacteria has a restricted permeability to many compounds, including several inhibitors, which renders these bacteria more resistant to some inhibitors. This, together with different selective modes of action on Gram-positive bacteria, has been exploited to achieve selective pre-incubation stages. The procedure, in practice, is simple. Samples for testing are incubated at a controlled temperature for a set time with a mixture of inhibitors and the normal procedures of the assay (ATP or DEFT) are then performed on the samples. Wacs & Bossuyt (1982) successfully used a mixture of benzalkonium chloride (0.03% w/v) and crystal violet (0.002% w/v) as the selective inhibitors in the pre-incubation stage before assay of ATP. Rodrigues & Pettipher (1984) also used this combination and demonstrated that the DEFT could be used successfully to predict, within 24 h, the KQ of >80% of samples of

37

DEFT: RECENT DEVELOPMENTS T,\ULE I. CllIssijimlioll o/pasleurized milk samples 1111 Ihe basis 0/ KQ al 5°C alld 1JOC kv Ihe DEFT wUIII folllllvillg pre-illcubalioll al ](J'C iiJr 18 h

Storage temperature eC)

DEFT count/ml after pre-incubation

Corresponding KQ (days)

o/.. Correctly

10 4

10.6

106

7.6

J(f

88 80 80

lOx

6.0 4.5

95 91

10' 10;

3.8 2.85

82 88

106

1.9 0.95

92 90

10;

11°

classified

)07

9.1

From Rodrit,'ues & Pettipher (1984).

pasteurized milk stored under conditions of satisfactory (5°C) or unsatisfactory (10°C) refrigeration (Table 1). Griffiths et at. (1984) used a different mixture of inhibitors (crystal violet, 2 mg/ml; nisin, 40000 units/ml; penicillin, 20000 units/ml) and compared the ability of the ATP assay and the DEFT to detect post-pasteurization growth in pasteurized cream stored at 6°C. They found that both methods correctly identified samples as unacceptable or acceptable (DEFT, 91%; ATP, 94%). Kroll & Rodrigues (1986a) also studied the ability of tlle DEFT for predicting the KQ of pasteurized cream stored at 5° or lO°e. They found that the DEFT classified >81 % of the samples correctly, but only when undiluted cream was filtered (using the 20-ml 0.1 'Yo Triton X100 modification of the Pettipher & Rodrigues (1981) method). In this work the pre-incubation stage included only benzalkonium chloride at 0.05% (w/v) as inhibitor. All the pre-incubation systems described appear to function correctly and the DEFT (and assay of ATP) can be used to quantifY the low-level contamination present in these products. The ideal selective system that inhibits all the Gram-positive bacteria without any inhibitory effect on the Gram-negative bacteria probably docs not exist. Phillips & Griffiths (1986b) have recently made a useful study of seven possible inhibition systems that could be used for this purpose. They showed that the two mixtures that have already been used for this purpose appear to be the most effective at inhibiting Grampositive organisms and not inhibiting Gram-negative organisms in skimmed milk. A variety of pre-incubation temperatures have been used. Although the

38

G. L. PETTIPHER £1' AL.

Gram-negative post-pasteurization contaminants are psychrotrophic they are not psychrophilic and in our experience will grow well at least up to 30°C. as pre-incubation Rodrigues & Pettipher (1984) used 15°, 20° and 300 temperatures for 18 h and they found that the DEFT gave the best prediction of the KQ of pasteurized milk after pre-incubation at 30o e. Griffiths et al. (1984) pre-incubated samples at 21 DC for 25 h and found that the assay of ATP and DEFT gave satisfactory results at this temperature. Kroll & Rodrigues (1986a) found that the DEFT gave a good prediction of the KQ of pasteurized cream after pre-incubation of samples at 20 0 or 25°C for 18 h. It is not yet clear which time/temperature combination is optimal. The temperature must be chosen to give the maximum final yields to bring the cell density above the detection limits of the chosen method (especially with samples with low levels of contamination) while not being so high as to inhibit the growth of the psychrotrophs. Further work is needed to clarify the position, but when Kroll & Rodrigues (1986a,b) used the cytochrome c oxidase test for this purpose they encountered many false negatives with pre-incubation tenlperatures above 20o e. It should be emphasized, however, that this was not due to lack of growth in these samples so this should not affect assay by ATP or DEFT. A cautionary note must be made about the estimates of KQ in these studies. The end of KQ has been taken to be when the cell density reaches the arbitary value of 5 x 106 bacterialml (g). In practice, some milk samples can have higher cell densities than this although the product may not have spoiled (Rodrigues & Pettipher 1984). In principle, this approach could be used to detect low numbers of other types of spoilage or pathogenic micro-organisms in foods.

e

Osmotolerant Yeasts in Confectionery Products The presence of even low numbers of osnlOtolerant yeasts in creme fondant, a confectionery ingredient, is undesirable as growth during storage and transportation may cause spoilage of the products. In view of the potential cost of spoilage it is essential that rapid, sensitive and reliable methods are used to assess the quality of this ingredient. Results obtained using naturally spoiled creme fondant showed that, in the DEFT, 2-~m pore size Nucleporc nlembrane filters retained similar numbers of yeasts as 1.O-~m or O.6-~m pore size filters. For routine usc, 5 g of fondant and pre-treatment with trypsin and Triton X-IOO was necessary for efficient filtration. The DEFT could be used to detect high numbers (>200/g) of osmotolerant yeasts immediately or low numbers after pre-incubation in a medium containing 10% (\v/v) sucrose. The detection limits for osmophilic yeasts in the pre-incubated DEFT count, as extrapolated from the initial plate count, were c. 1/g after 25 hand 1/(10 g) after 49 h (Pettipher 1987). A pre-incubated plate count takes 4-5

DEFT: RECENT DEVELOPMENTS

39

days to give a result. The DEFT may also be of considerable value in plant hygiene surveys to pinpoint areas of yeast proliferation. Selective Enumeration of Bacteria by the DEFT At present, a major limitation to the truly rapid methods of microbial detection and enumeration (i.e. the DEFT and assay of ATP) is that they cannot specifically identify and quantitY or even give an indication of the types of organisms present in a sample, whereas this can be achieved with methods relying on changes in the impedance/conductance of the medium, although the results are obtained more slowly. If such advances could be made to the truly rapid methods, this would greatly increase the application and acceptance of these methods in microbiology. An advantage of the DEFT is that in some cases tentative identifications of the types of organisms present can be made in a sample of raw milk. With bacteria, however, identification on the basis of morphology alone is not valid. One modification has been made which allows the DEFT to give a total count of the Gram-negative bacteria present (Rodrigues & Kroll 1985). This method uses the normal Gram staining procedure of bacteria on the polycarbonate membrane, except that 95% ethanol is used as tile decolorizing agent and acridine orange (125 I-tg/m!) is used as the counter-stain. In a trial of this modification the active Gram-negative bacteria, but not the Gram-positive bacteria or inactive Gram-negative bacteria, fluoresced orange and the method was shown to give a good relationship between Gram-negative DEFT count and Gram-negative plate count (crystal violet agar) with samples of raw milk (r = 0.94). Occasional problems were encountered with clumps of streptococci which tluoresced orange, but these were distinguishable from the Gramnegative organisms by transmitted visible light illumination of the sample. Although this modification introduces a degree of selective counting of bacteria in the DEFT, there are many applications where a better differentiation between the types of bacteria present needs to be made. One approach to improve this aspect of the method has been investigated recently (Rodrigues & Kroll 1988): this is to eA'})loit the selectivity of commercially available microbiological media to grow microcolonies of the selected bacterial type. In essence, samples of meat, fish and vegetables are homogenized in a stomacher in 0.1 'Yo peptone water and the samples pre-filtered through 5.0-I-tm nylon mesh to remove the large particles of food in the normal way (Pettipher & Rodrigues 1982b). The samples are not pre-treated with enzyme or detergent as in the normal DEFT, but are immediately filtered under vacuum through 0.6-~lm Nuclepore filters held in filter towers. In some cases this reduces the volume of sample that can be filtered (typically 1-5 ml for meat, 2-10 ml for vegetables) and slightly reduces the sensitivity of the method. It

40

G. L. PETTIPIIER ET AI.

is essential, however, that the detergent/enzyme pre-treatment stage of the normal DEFT is omitted, to avoid damage to the bacteria. The filters are then removed from the filter towers and placed on the surface of the required agar medium in Petri dishes. The Petri dishes are inverted and incubated at 30°C for different periods of time. In our experience 3 and 5 h gives satisfactory microcolony formation by Gram-negative and Gram-positive bacteria, respectively. After incubation, the filters are replaced in the filter towers and stained with acridine orange by the normal DEFT procedure (Rodrigues & Kroll 1985). The mcmbrancs arc then mounted on slides with non-fluorescent immersion oil and examined by epifluorescent microscopy using a x 50 or x 100 objective lens. The microcolonies which have formed stain bright orange and are easily counted. Individual cells or small clumps «4 cells) are commonly observed. These are thought to be non-viable cells or, more likely, cells which have broken off from the microcolonies; in any event, these cells are not included in the microcolony count. In initial experiments microcolony formation by pure cultures was studied on filters incubated on nutrient agar and compared with the total viable count on nutrient agar. These experiments demonstrated that the microcolony counts in DEFT were very closely related to the number of colony-forming units in the standard plate count. A total of 26 Gram-negative and 17 Gram-positive cultures was studied and it was interesting to find that the morphology of the microcolonies was sometimes distinctive to that genus, often with spectacular formations on the membrane. These cultures were then tested on eight

selective media for their ability to form microcolonies of the correct type and in accordance with the selectivity of the medium. Only four of the media appeared to exhibit the correct properties and these were selected for further study. The natural and introduced flora of a variety of fresh and frozen fish, meat and vegetables was then examined for its ability to form microcolonies on these media (Table 2). The microcolony counts related very well to the normal plate counts but the results were available in only a few hours. The selective media for coliforms and pseudomonads gave the best results; the medium for streptococci, staphylococci and micrococci was less satisfactory, having lower correlation coefficients and higher intercept values (Table 2). This medium was also less satisfactory in that the staphylococci and micrococci microcolonies could not be properly differentiated. Despite these extremely encouraging results, the method is subject to the following restrictions: 1 The maximum sensitivity of the method with real food samples is in the range 103 - 104 bacteria/g. As coliforms or pseudomonads often form only a small proportion of the total viable count the method will be able to quantifY these organisms only in badly contaminated samples. 2 Injured cells may not have sufficient time to form microcolonies. The results with frozen fish and meat (Table 2) demonstrate slightly more scatter

41

DEFT: RECENT DEVELOPMENTS TA[JLE 2. Correlation of microcolony Dl::.rJ· counts with viable cOimls offood Medium for viable count

n

m

,.

r

Lowest count (logJ() reliably detected

Raw meat NA ABA PSA FJ,SAB

24 19 22 22

0.85 0.44 0.90 0.76

0.52 2.85 0.12 1.23

0.90 0.63 0.93 0.94

4.8 3.8 3.1 3.3

Frozl'11 meal or fish NA ABA PSA ELSAB

29 16 25 26

0.85 0.52 0.82 0.76

0.25 2.9 0.55 0.86

0.88 0.64 0.92 0.83

4.3 4.5 3.6 4.1

Frozen vegetables NA ABA I'A ELSAB

19 33 6 7

0.79 0.76 0.81 0.55

0.58 0.98 0.38 2.01

0.84 0.80 0.93 0.90

4.0 3.2 2.8 3.1

Regression data

n, the number of individual samples; m and c, values of the regression lines ofy = mx + c of plots oflogJ()cfu plate count/ml (x axis) and logJ() microcolony DEFT counts/ml (y axis); r, correlation coeflicient. NA, nutrient agar (total aerobic plate count); ABA, azide-blood agar (staphylococci/streptococci/ micrococci); PSA, pseudomonad supplement agar (pseudomonads); ELSAB, lauryl sulphateaniline blue agar (coliforms). Filters placed on NA, PSA and ELSAH were incubated fin 3 h and those on ABA for 5 h at 30°C.

than with raw foods and inadequate resuscitation of injured cells is suspected. Indeed, this is to be eJl.'Pected with the highly selective nature of the media used, which contain antibiotics or inhibitors that can be injurious to sublethally damaged cells. The results demonstrate the potential of the method with foods, however, and a detailed study of the effects of sub-lethal heat treatments on the efficiency of microcolony formation is being undertaken. 3 The selectivity of the method can only be as good as the selectivity of the medium. Few media appear to be totally selective for microcolony formation as they do not inhibit all the non-selected organisms. Many rely on differential colony morphology or changes in the medium surrounding the colony, e.g. pI I, to be diagnostic of the required organism, and many do not support the growth of microcolonies of all the strains or species of their target organisms.

42

G. L. PETTIPIIER £1' AL.

These results demonstrate that the microcolony/DEFT can be used to count specific groups of bacteria. The results are available well within a working day and this should allow better management of food supplies. The modifications also demonstrate that the full potential of the nlethod(s) has yet to be realized. There is no reason why the DEFT cannot be similarly modified specifically to detect other groups of organisms and, if used in conjunction with fluorescent polyclonal or monoclonal antibodies, this differentiation could be improved still further.

Use of DEFT for Irradiated Foods There is increasing interest in the use of gamma radiation in food preservation. Reports have suggested that the irradiation of foods with a dose of 10 kGy (1 Mrad) of gamma radiation presents no toxicological hazard and introduces no special nutritional or microbiological problems (Anon. 1986). One of the major points of concern about the use of radiation as a food preservative has been the lack of a simple, reliable method for detecting when the process has been used. Some methods for detecting irradiated foods have been proposed but most of them are elaborate and require ex])ert skills. The DEFT has the potential to be a relatively simple, inexpensive way to indicate whether a food has been irradiated. In addition, it will provide an estimate of the microbiological quality of an irradiated food prior to the process (Betts et al. 1988). This method for indicating the presence of an irradiated food relies on the comparison of the aerobic plate count (APe) and the DEFT count of the same sample. The APe will enumerate only those organisms remaining viable after the irradiating process. The DEFT, however, will show not only the organisms remaining viable but also those killed by the process. The APe of an irradiated food is therefore generally 2- 3 log cycles lower than the DEFT count, the DEFT count correlating well with the APe of the sample before irradiation (Fig. 1). Irradiation of minced beef, steak, bacon, ham, gammon, ox liver, pate and milk, at the suggested maximum dose of 10 kGy, reduced the APe from 107 -1 0 9 I g to 10 4 -10 6 / g. Milk with an initial count of 107 cful ml had an APe of zero after being irradiated at a dose of 5 kGy. In all cases the DEFT count of food samples after irradiation correlated well with the APe before irradiation. Results obtained for pure cultures are shown in Fig. 2. Initial DEFT and APe counts were in dose agreement but after irradiation at 25 kGy the APC was reduced by 6-8 log cycles, to zero, whilst the DEFT count was reduced by only 0.5-3 log cycles. The following method can be used to screen raw materials which may have been previously irradiated. The DEFT is carried out as described by

43

DEFT: RECENT DEVELOPMENTS

10

-g' c:

9

:J

10

-----_L---..'-----.

0

u Q>

9

...

!!'

8 §

8

o

ro

0u

:.a

e

Q>

'"

u

7

7

6

6

0

C; 0

--'

rt::i

o

o OJ

o

--'

5

~5

Radiation dose (kGy) FIG. 1. Effect of irradiation on the aerobic plate count (APe) (0), and DEFT count (e), of minced beef samples.

o kGy 0 kGy 0 kGy 0 kGy 0 kGy 0 kGy 25kGy 25kGy 25kGy 25kGy 25kGy 25kGy Bacillus subtilis

Escherichia Salmonella Bacillus coli typhimurium stearothermophilus Staphylococcus Pseudomonas aureus pero/ens

2. Effect of irradiation on the aerobic plate count (APC) (shaded), and DEFT count (black) of bacterial cultures in nutrient broth.

FIG.

44

G. L. PETTIPHER ET AI.

Pettipher (1983). Triton X-IOO and trypsin are used in the pre-treatment usually with 3-6 ml of homogenized sample, depending on the sensitivity required. For meat samples, 10 g of meat is macerated by stomacher in 90 ml of maximum recovery diluent or 0.050/0 peptone (not Ringer solution). The resulting homogenate is pre-filtered through a 5.0-,.un pore size filter (Shaw et al. 1987). Milk can be used without pre-filtration. The pre-treatment, filtration, staining and counting stages of the DEFT are as described in the chapter by Shaw and Farr (this volume). After the DEFT count of the sample has been established, an APC can be set up. If the DEFT count and the APe are similar then the sample has not been processed. If the DEFT count is considerably greater than the APC, then this suggests that the sample could have been irradiated. Heating is the only other treatment which is known to give results similar to this when a joint DEFT/APe is carried out. Heat-processed food in most cases will have a DEFT count higher than their APC but they usually show visual changes indicative of such a treatment. A joint DEFT/APe procedure therefore has potential as a method for the indication of an irradiated food. It also allows the estimation of the viable count of an irradiated sample before irradiation.

References ANON. 1986. Report on the Safety and Who/esom1less oIIrradiated and Novel Foods. London: HMSO. R.P., FARR, L., BANKES, P. & STRINGER, Nl.F. 1988. The detection of irradiated foods using the direct epifluorescent filter technique (DEFn. Jounzal of Applied Bacteriology 64,

BETTS,

329-335. ).R., WHITE, CM. & FIRSTENBURG-EDEN, R. 1984. Rapid impedimetric method for determining the potential shelf-life of pasteurised whole milk. Jounzal of Food Protection 46 1

BISHOP,

622-624. R.L. & JOHNSON, R. 1980. A fluorescent staining technique for determination of viable and non-viable yeasts and bacteria in wineries. Food Technology in Australia 32, 522-523. GRIFFITHS, t\1.W., PHILLIPS, J.D. & MUIR 1 D.O. 1984. Methods for rapid detection of postpasteurisation contamination in cream. Journal of the Society of Dairy Technology 37, 22 - 26. KROLL, R.G. & RODRIGUES, U.M. 1986a. The direct epifluorescent filter technique, cytochrome c oxidase test and plate count method for predicting the keeping quality of pasteurised cream. Food Microbiology 3, 185-194. KROLL, R.G. & RODRIGUES, U.~1. 1986b. Prediction of tIle keeping quality of pasteurised milk by the detection of cytochrome c oxidase. Journal ofApplied Bacten'ology 60, 21-27. McKINNON, CII. & PETTIPHER, G.L. 1983. A survey of the sources of heat-resistant hacteria in

COOTES,

milk with particular reference to psychrotrophic spore forming bacteria. Journal of Dairy Research 50, 163 -170. PETTIPHER, G.L. 1983. The Direa Epifluoresccnt Filter Technique for the Rapid Enumeration oIMicroorganisms. Innovation in Microbiology Series, 1. Letchworth: Research Studies Press. PETTIPHER, G.L. 1987. Detection of low numbers of osmophilic yeasts in creme fondant within 25 h using a pre-incubated DEFT count. Letters in Applied Microbiology 4, 95-98. PETTIPHER, G.L., l\1ANSELL, R., McKINNON, C.H. & COUSINS, C.M. 1980. Rapid membrane

DEFT: RECENT DEVELOPMENTS

45

filtration-epifluorescent microscopy technique for direct enumeration of bacteria in raw milk. Applied and Ellvironme1ltal Microbiolo&'V 39, 423-429. PETTIPHER, G.L. & RODRIGUES, U.M. 1981. Rapid enumeration of bacteria in heat-treated milk and milk products using a membrane filtration-epifluorescence microscopy technique. Journal ofApplied Bacteriology 50, 157-166. PETTIPHER, G.L. & RODRIGUES, U.l\l. 1982a. Semi-automated counting of bacteria and somatic ceUs in milk using epifluorescence microscopy and television image analysis. Journal of Applied Baeteriology 53, 323-329. PETTIPHER, G.L. & RODRIGUES, U.M. 1982b. Rapid enumeration of micro-organisms in foods by the Direct Epifluorescent Filter Technique. Applied and Environmental Microbiology 44, 809-813. PHILLIPS, J.D. & GRIFFITHS, !\'l.W. 1986a. Factors contributing to the seasonal variation of Badllus spp. in pasteurised dairy products. Journal ofApplied Baeteriology 61, 275 - 286. PHILLIPS, J.D. & GRIFFITHS, M.W. I 986b. Estimation of Gram-negative bacteria in milk: A comparison of inhibitor systems for preventing Gram-positive bacterial growth. Journal of Applied Baderiology 60, 491- 500. PHILl.lPS, J.D., GRIFFITHS, lVl.W. & MUIR, D.O. 1981. Factors affecting the shelf-life of pasteurised double cream. Journal of the Sode~v of Dairy Technology 34, 109-112. RODRIGUES, U.M. & KROLL, R.G. 1985. Increased selectivity, sensitivity and rapidity in the Direct Epifluorescent Filter Technique (DEFT). Journal o/Applied Bacteriology 59, 493-499. RODRIGUES, U.M. & KROLL, R.G. 1988. Rapid selective enumeration of bacteria in foods using a microcolony epifluorescence microscopy technique. .loumal 0/Applied Baderiology 64, 65- 78. RODRIGUES, U.M. & PETTIPHER, G.L. 1984. Use of the Direct Epifluorescent Filter Technique for predicting the keeping quality of pasteurised milk within 24 hours. Journal of Applied Bactenology 57. 125-130. SCHRODER. M.J.A., COUSINS, CM. & McKINNON, CII. 1982. Effects of psychrotrophic postpasteurisation contamination on the keeping quality at I I° and 5°C of I ITST-pasteurised milk in the U.K. Journal of Dairy Research 49, 619-630. SIHW, B.G., lIARDlNG, CD., HUDSON, W.l1. & FARR, L. 1987. The estimation of microbial numbers on meat and poultry by the direct epifluorescent filter technique. Journal of Food Protection 50, 652-657. WAES, G. & BOSSUYT, R. 1982. Usefulness of the benzalkonium-crystal violet ATP method for predicting the keeping quality of pasteurised milk. JOllmal of Food Pro/atioll 10, 928-931.

The Rapid Estimation of Bacterial Counts on Meat and Poultry by the Direct Epifluorescent Filter Technique B. G. SHAWl AND L. J. FARR 2 I AFRC Institute of Food Research, Bristol Laboratory, LangfOrd, Bristol BS18 7DY; and 2 Foss Electric (UK) Ltd, The Chantry, Bishopsthorpe, York, UK

Conventional culture methods for determining total viable counts on meat and poultry take at least 1 day to produce a result. In meat factories this is a considerable drawback in ingredient and raw product testing where results are often needed within hours (or even minutes) rather than days. Direct microscopic enumeration of bacteria using the direct epifluorescent filter technique (DEFT) (Pettipher 1983) offers a solution to this problem as results may be obtained in 35-45 min. The DEFT was originally developed for the rapid enumeration of bacteria in milk (Pettipher et al. 1980) but was later modified for application to other foods including meat (Pettipher & Rodrigues 1982b). Subsequent studies (Boisen 1983; Qvist & Jakobsen 1985; Shaw et al. 1987) have established its suitability for the rapid estimation of total bacterial numbers in a variety of red meat and poultry sample types. This article describes the DEFT procedure as applied to poultry and fresh, frozen and comminuted meat. The DEFT has been evaluated by comparison with the plate count on a variety of meat and poultry sample types to demonstrate the capabilities of the technique. DEFT Methodology for Meat and Poultry The DEFT procedure described in this article is based on that developed by Pettipher & Rodrigues (1982b) for food suspensions. The technique essentially involves the counting of fluorochrome-stained bacteria on membrane filters on which they have been concentrated from the sample suspension following removal of food debris by pre-filtration and enzyme treatment. The procedure may be conveniently divided into five stages as shown in Fig. I. Cr;pyright

© 1989 by the Society for Applied Bacteriology All rights of reproduction in any for", reserved 0-632 -02629-4

Rapid Microbiological Methods for Foods, Beverages and Pharmaceuticals

47

48

B. G. SHAW AND L. J. FARR Preparation of sample suspension

! 1 1 1

Prefiltration

Enzyme and surfactant treatment

Filtration and staining

Counting FIG.

1. Outline of DEFT procedure for meat and poultry samples.

Preparation of sample suspension The sample suspension may be prepared by any of the sampling techniques in current use (e.g. scraping, swabbing, stomachering). Specific examples of sample suspension preparation techniques which we have used successfully with the DEFT are shown in Table 1. Rubber bungs or bottIe cap sealers must not be used on any sample container or equipment used in the DEFT as this will inevitably lead to orange fluorochrome-stained debris and incorrect counting. The diluent used in the preparation of the sample suspension must contain 0.1 % (w/v) peptone, otherwise cells may not take up the fluorochrome effectively at the later staining stage. A solution of 1% (wIv) peptone (pH 7.0) is recommended but peptone salt dilution fluid (0.1 % (w/v) peptone, 0.85% (w/v) NaCI; pH 7.0) has proved satisfactory. Tween 80 should be added at a concentration of 1 % (v Iv) prior to stomachering of samples with a relatively high fat content (e.g. sausages, mechanically recovered meat) - this disperses the fat, aiding pre-filtration and improving the quality of the final stained DEFT preparations by minimizing interference from fat particles. As recommended by Boisen (1983), comminuted meats should be placed in an inner nylon-lint bag (Foss Electric (UK) Ltd, York) prior to addition of diluent and stomachering. This retains a large proportion of the solids, whilst still permitting full recovery of organisms.

Pre-filtration Pre-filtration aims to remove coarse particles from the sample suspension and should be applied to all types of preparation including swab suspensions. For

49

DEFT FOR MEAT AND POULTRY TABLE

1. Meat alld poultry sample suspmsioll procedures appropriate to the DEFT

Sample type Joints of meat

Sampling method

3 x 7 cm l areas

Diluent volume (mI)

Dispersal methods

100

Stomachering for 1 min

cut from surface Carcasses and joints of meat

50 or 100 cnl area swabbed

20

Swab compressed repeatedly \\-ith pipette

Chicken carcasses or joints

10 g skin and muscle excised

90

Stomachering for I min

Chicken carcasses or joints

7 cm 2 area scraped

10

Surface scraped in presence of diluent

Frozen blocks of meat

100-200 g thawed in microwave oven (230 W, 3 mill)

Comminuted meat (e.g. sausages, mechanically recovered meat)

10 g sample examined

Equal to weight of sample

Stomachering for 2 min

90

Sample placed ill nylon-lint bag and stomachered for 1 min

prefiltration of suspensions, 25-mm-diameter 5 f!m polypropylene filter discs (Foss Electric (UK) Ltd) are mounted in Swinnex filter holders (Millipore Ltd, London) and autoclavcd before usc. After allowing large food particles in thc homogenate to settle for 2-3 min, a 10-15 ml volume of the sample suspension is drawn into a sterile disposable plastic syringe and forced through the Swinnex filter unit into a sterile McCartney or Universal bottle. Sample suspensions with an expectcd count > 10° cful ml should be decimally diluted below this level prior to pre-filtration, as should any sample which blocks the pre-filter prior to the passage of 3 ml (the normal volume used). Enzyme and surfactant treatment

Treatment with trypsin and Triton X-IOO disperses protein, lyses muscle cells and makes fat globules more fluid, aiding the final filtration stage and improving the quality of the stained DEFT preparations (Pettipher & Rodrigues 1982b). Reagents for use in this and the latcr filtration and staining stage may be prepared and dispensed using filters and pipetters obtained as individual items. Alternatively the bio-Foss Reagent System (Foss Electric (UK) Ltd) may be used. This is purpose-designed for the DEFT including all necessary filter units, containers, tubing and pipeners and greatly simplifies and speeds up procedures.

50

B. G. SHAW AND L.

J.

FARR

Trypsin is prepared by reconstituting the freeze-dried enzyme (Difco

8103 - 97) in chilled (...... 7°C) distilled water and sterilizing by passage through a 0.2-~m disposable filter (Foss Electric (UK) Ltd). The enzyme is dispensed aseptically in 0.5 ml amounts into sterile capped test-tubes, excess of requirement being stored in these tubes deep-frozen at - 20 0 (maximum 3 months). Triton X-IOO (BDII Chemicals Ltd or Difca 8106-97) is prepared as a filter-sterilized 0.5% (v/v) solution made up in warm distilled water. For treatment 3-9 ml of the pre-filtered sample suspension (the volume dependent on the count sensitivity required) is thoroughly mixed in the testtubes using a vortex mixer with the trypsin and 2 ml of added 0.5% (v/v) Triton X-IOO solution and incubated at 50°C for 10 min in a water bath.

e

Filtration and staining This stage is vital to the success of the DEFT. A good reliable, efficient membrane filtration system is essential if consistent DEFT slides are to be prepared suitable for accurate counting. This should include membrane filtration units having stainless steel screen membrane filter supports (sintered glass bases are not suitable), filter towers, and a 5-way filter manifold preferably fitted with a vacuum gauge (a vacuum of 15 - 20 mmHg is optimal excessive vacuum may distort the membranes). The manifold, which allows processing of five samples at a time, should be connected via a 3-way valve (one vent to the atmosphere) to a side-arm flask (to collect the filtrate) and then to the vacuum source for which an electric vacuum pump is preferred to a water jet. A suitable filtration system is commercially available (Foss Electric (UK) Ltd). All reagents used in flitration and staining should be pre- filtered through 0.22 (.tm membrane filters to remove bacteria and particulate matter. The fluorescent stain is acridine orange prepared from DEFT buffered acridine orange concentrate (Difco 8101-97). The staining solution should be freshly made up every 2- 3 days and stored at <7°C when not in use. Other prefiltered reagents required are Triton X-IOO (BDI-I Chemicals Ltd or Difco 8105-32) as a 0.1 % (v/v) solution, isopropanol, and citrate-NaOI-I buffer (0.1 mol/I; pH 3) which is obtainable as a concentrate (Difco 8104-97). The filtration and staining procedure is as follows: 1 1\;lount a Nuclepore polycarbonate membrane filter (0.6 ~m pore size, 25 mm diam; Sterilin Ltd Teddington, Middlesex) shiny side uppermost in the membrane filter unit connected via the manifold and trap to the vacuum source. Pre-warm the filter by filtering 5 mJ Triton X-IOO (0.1 % v/v) at SO°C. Release the vacuum by means of the 3-way valve which should be used for application and release of the vacuum at all stages of the procedure. 2 Remove the sample from the 50°C water bath, wipe the outside of the tube, and pour into the filtration tower. Apply the vacuum and filter. Release the vacuum.

DEFT FOR MEAT AND POULTRY

51

3 Add 5 ml of Triton X-100 (0.1 % v/v) at 50°C to the sample tube to remove any residual sample. Add to the filtration tower and filter. Release the vacuum. 4 \Vith the system at atmospheric pressure, overlay the membrane with 1 ml of acridine orange stain. Leave for 30 s, then re-apply vacuum to filter. Release the vacuum. S Add 2 ml of the pH 3 citrate - NaOI I buffer to the filtration tower and filter. Release the vacuum. 6 Add 1 ml isopropanol and filter. Remove the filtration tower leaving the vacuum on at a sufficient level to hold the membrane in place. Carefully remove the membrane using blunt-ended tweezers. (To prevent carry over of debris and bacteria the filtration towers should be washed thoroughly with 5% Ocean 90, and rinsed with hot water before re-use. All other parts of the filter units should be similarly washed each day.) 7 Air-dry the membrane until it becomes evenly opaque. Failure to dry properly will result in discoloration of bacteria from orange to green, with consequential lower counts. 8 Place a small drop of non-fluorescing immersion oil (e.g. Difco 8102-97) on a microscope slide, then lay the membrane on top of this, shiny side uppermost. Place another small drop of immersion oil on top of the membrane and a coverslip on top of this. Place the slide on a firtn surface and press down and outwards to eliminate air bubbles and ensure the membrane is flat. The preparation is now ready for counting.

Counting

Counts are obtained from the acridine-orange-stained DEFT preparations using an epifluoreseent microscope under which metabolically active bacteria fluoresce orange and inactive bacteria green. Clumps of orange-fluorescing bacteria and single cells separate from other groups of cells are counted in 2- 15 fields ofview according to the number of dumps ofcells/field (Pettipher et al. 1980) as follows: 15 fields for 0-10 clumps/field; 10 for 11-25; 6 for 26-50; 3 for 51-75; 2 for 76-100. The sample should be diluted and the analysis repeated when > 100 clumps/ field occur. When an automatic counting system (see below) is used, it is convenient to count a set number of fields ,of view (usually 20) whatever the count per field, with the proviso again that the sample should be diluted and repeated when > 100 clumps per field occur. Cells fluorescing green are not included in the count. The DEFT count/ml of sample suspension is obtained by multiplying the

52

B. G. SHAW AND 1.. J. FARR

average number of clumps/microscope field by the microscope factor (MF). The microscope factor is calculated as follows:

MF

=

Area of membrane through which sample is filtered (mm 2) Microscope field area (mm 2) X Sample volume (ml)

The area of the filter used is calculated from the internal radius of the filter tower. The area of the microscope field of view is calculated from the radius of the field which is measured using a stage micrometer. Manual counting of DEFT preparations is cAtremely laborious and may cause eye fatigue. Automatic counting is possible using a television scanner linked to a suitable image analysis system as described by Pettipher & Rodrigues (1982a). A commercially available automatic counting system (Foss Electric (UK) Ltd), including microprocessor controlled image analyser and automatic slide stepper, requires the operator merely to focus the microscope and operate a hand switch. Counts/ml of sample suspension are automatically calculated and may be produced as a printout or transferred to a computer to give counts/g or / cm 2 and for storage of results in a file. Comparison of DEFT and Plate Counts The DEFT as described in this article aims to produce the equivalent of the total viable count. To evaluate the technique, Shaw et ai. (1987) have compared DEFT counts with plate counts on a variety of uncooked red meat and poultry samples examined as shown in Table 1, covering common sampling techniques in current usc. The procedure used for the DEFT was as described in this article except that Tween 80 was not added at the stomachering stage. The apparatus used was the bio-Foss Automated Microbiology Systeln (Foss Electric (UK) Ltd) shown in Fig. 2 which includes the purpose-built reagent and filtration equipment and automatic counting system. Total counts in these comparisons were made by surface plating on plate count agar (PCA, Oxoid) + 1% NaCI with incubation for 5 days at 20o e. The summarized results (Table 2) show that the DEFT count correlated well (correlation coefficient (r) = 0.95 -0.96) with the plate count for frozen or chill-stored lean red meat and for red meat carcasses (beef, pork, lamb). For frozen meat and swabbed samples the DEFT gave good agreement with the plate count over a range adequate tor routine quality evaluation (5 x 103 /g or /cm 2 and above). For samples taken by surface excision with plate counts of I04/cm 2 or less, DEFT counts were >0.5 lOglO unit higher, indicating a loss of sensitivity. If required, precision at these lower count levels could be enhanced either by use of a larger sample volume (6 ml were used for red meat samples) or by increasing the number of microscope fields counted. For stored and unstored raw chicken sampled by skin scraping or

DEfT FOR "IEAT AND POULTRY

53

FIG. 2. The hio-Foss Automated .\licrobiology System equipment fiJr the DEFT.

stomachering good overall agreement (r = 0.88-0.89) was obtained between the DEFT count and the plate count in the ranges 1.1 X 103 to 1.3 X 107 1 cm 2 (scraping) and 1 X 10 4 to 9.5 x Hl'/g (stomachering) even thoug'h the DEFT overcounted by >0.5 10glO unit on samples on which little or no growth had occurred (plate count <10 5 /g or Icm 2). This overcounting cannot be explained on the basis of loss of sensitivity or interference from stained non-bacterial particles. Pettipher & Rodrigues (1981) have shown that some types of bacteria killed by heat treatment remain acridine-orangestainable and it is possible that the DEFT count from fresh chicken includes damaged or non-viable cells resulting from the 50°C hot water scalding treatment briven to the carcasses. Ilowever, since reasonably good overall linearity is obtained between DEFT and plate counts on chicken, a predetermined regression equation can be used to obtain a more accurate estimate ofthe plate count on fresh samples if this is required. This can be incorporated in software in the automatic counting system. Comminuted meats such as mechanically recovered meat (MR1Vl) or sausages contain a relatively large amount of particulate material which can block the til tel'S or cause interference at the counting stage where it may fluoresce orange. In the examination of MRM, Shaw el al. (1987) needed to dilute the sample suspension to prevent blockage of the pre-filter even though

TAIlLE

2. Relationship betwet'll log/o DEFT count/g or /cm 2 (y) and loglO plate count (x) jOr various meat and poultry sample types Log lO plate count/g or Icm 2

Material and sampling method

No. of samples

examined

Correlation coefficient (r)

Range covered

Range over which DEFT gave good agreement

= 1.28 + O.83x

0.96

3.8-8.4

4.2- 8.4

+ 0.78x

0.95

2.3-7.0

3.7- 7.0

Regression equation

70

J'

Beef, pork and lamb carcasses and aerobically stored beef joints - swab samples

50

.y = 1.26

Chicken joints - surface

49

.y

= 3.51 + OA9x

0.89

4.0-6.8

5.0- 6.8

41

.y

= 3.26 +

0.45x

0.88

3.1-7.1

5.0- 7.1

Frozen beef - stomachered

60

.Y

0.95

3.8-10.4

3.8-10.4

!v1echanically recovered

50

= 1.85 + 0.74x J' = 5.34 + 0.28x

0.49

4.2-8.3

Vacuum packed or

aerobically stored beef joints - surface excised

skin and muscle excised

Chicken joints - surface scraped

meat

55

DEFT fOR MEAT AND POULTRY

many particles were contained in the inner nylon-lint bag during stomachering. However, even with this pre-dilution, much orange-red debris was seen in the stained preparations and there was consequently a poor overall correlation (r = 0.486) between the DEFT and plate counts, tlle DEFT overcounting by between 5- and !ODD-fold. As a result of this and similar observations made in other laboratories, Tween 80 (1 % v/v) is now routinely included at the stomachering stag'e for all comminuted meats to aid the dispersal of particles of fat. This results in cleaner DEFT preparations and correspondingly much better correlations between the DEFT and plate counts. Using this procedure Sacree & Brown (Unilcver Research, personal communication) have obtained the following correlations between automated DEFT counts and plate counts: Beef including hard fat samples, gristles and MRM Pork including fat samples and MRM Chicken including skin samples and MRM

r = 0.91 r = 0.81 r = 0.90

The DEFT has not been evaluated intensively for application to cooked meats, although Qvist & Jakobsen (1985) compared DEFT counts with total counts on heat-treated Vienna- and Bologna-type sausages. The poor relationship between the DEFT and plate counts soon after heat treatment, when the total count was low (10' to 105 /g), was attributed to the inclusion in the DEFT count of cells killed during heat treatment. The DEFT count could therefore be used retrospectively as an indicator of the microbiological condition of t1le product before heat processing. Good agreement was obtained between DEFT and plate counts after storage when the total count was between 106 and lOll/g. The DEFT can therefore be applied to heat-treated meat products, but it seems essential to establish the relationship between DEFT and plate counts on each product type before using the technique routinely.

Applicability of DEFT to Meat and Poultry The DEFT performed as described in this article is applicable to the rapid estimation of t1le plate count in many types of meat and poultry samples. Given appropriate equipment there is no reason why the technique should not be used successfully in any routine quality control laboratory. Even when a proven relationship between the DEFT count and plate count has been preestablished elsewhere for specific sample types it is nevertheless recommended tIlat each laboratory initially evaluates the technique by comparison with the plate count tor every product sample type before using the DEFT as a routine metllod. This is necessary to establish tIle extent (if any) of influence of particulate material or non-viable cells on tile relationship between DEFf and plate counts on the particular material examined by that laboratory. Due regard to any such effects can thcn be taken in tile interpretation of the

56

B. G. SHAW AND L.

J.

FARR

DEFT count, and if required the DEFT count may be routinely converted using the obtained regression equation to give a better estimate of the plate count. Using a filter manifold system and automatic counting it is possible to obtain DEFT counts on five samples within 35-45 min. However, as currently developed,-the technique is demanding on a technician's time, requiring attention virtually throughout this period. Allowing for a necessary period for preparing reagents and cleaning or sterilizing equipment, this limits the daily throughput from one operator to c. 25 samples. Automating the pre-treabnent, enzyme treatment and filtration and staining stages would obviously enhance the appeal of the technique considerably. The DEFT is still a relatively new technique and developments should be possible to improve further sample preparation methodology, sensitivity, and perhaps rapidity (Pettipher 1986). Differential enumeration of Gram-negative and Gram-positive bacteria by the DEFT has already been achieved (Rodrigues & Kroll 1985), and it is in the area of improved selectivity that the technique has the greatest potential for development (see Pettipher et ai., Ch. 3, this volume).

Acknowledgefllent The valuable technical assistance of Mrs C.D. Harding and Mr W.I-I. Hudson (Institute of Food Research, Bristol Laboratory) in the evaluation of DEFT for application to meat and poultry is gratefully acknowledged.

References F. 1983. Practical experience with a microbiological rapid method by the application of the direct epifluorescent filter technique. Dansk Veterinaertidsskrifi 66.22, )) -15. PETTIPHER, G.L. 1983. The Direct E'pifluorescent Filter Technique fOr the Rapid Enumeration ofMicroorganisms. Letchworth: Research Studies Press. PETTIPHER, G.L. 1986. Review: the direct epifluorescent filter technique. Journal ofPood Technology 21, 535-546. PETTlPHER, G.L., MANSELL, R" McKINNON, C.H. & COUSINS, C..M. 1980. Rapid membrane filtration-epifluorescent microscopy technique for direct enumeration of bacteria in raw milk. Applied and Environmental Microbiology 39, 423-429. PETTIPHER, G.L. & RODR1GU£S, U.1\1. 1981. Rapid enumeration of bacteria in heat-treated milk and milk products using a membrane filtration-epifluorescent microscopy technique. Journal BoISEN,

ofApplied BtUteriology 50, 157-166. G.L. & RODR1GUES, U.M. 1982a. Semi-automated counting of bacteria and somatic cells in milk using epifluoresccnce microscopy and tele"i.sion image analysis. Journal of Applied Bacteriology 53, 323-329. PETTIPHER, G.L. & RODRIGUES, V.M. 1982b. Rapid enumeration of microorganisms in foods by the Direct Epifluorescent Filter Technique. Applied and Environmental Microbiology 44, PETTIPHER,

809-813. S.H. & JAKOBSEN, M. 1985. Application of the direct epifluorescent filter technique as a

QVIST,

DEFT FOR MEAT AND POULTRY

57

rapid method in microbiological quality assurance in the meat industry. lnternalional]oumal 139-144. RODRIGUES, U.M. & KROLl., R.G. 1985. Increased selecti\~ty, sensiti\~ty and rapidity in the. Direct Epifluoreseent Filter Technique (DEFT).]ournal ojApplied Bac/eriology 59,493-499. SHAW, B.G., HARDING, C.D., HUDSON, W.I I. & FARR, L. 1987. Rapid estimation of microbial numbers on meat and poultry by the direct epifluorescent filter technique. ]oumal oj Food

/1 Food Microbiology 2,

Proleclion 50, 652 -657.

Medical and Pharmaceutical Applications of the Direct Epifluorescent Filter Technique (DEFT) S.

P. DENYER),

R. A.

P. LYNN)

AND P.

S.

POVER 2

I Department of Pharmaceutical Sciences, Universi~y of Nottingham, Universi~y Park, Nottingham NGl 2RD, UK; and 2 Analytical Measuring s.ystems, London Road, Pampisjiml, Cambridge CB2 4EF, UK

The microbiological examination of biological or phannaceutical fluids for infecting/contaminating org'anisms is a routine procedure in hospital bacteriology, or industrial quality control laboratories, respectively. Many samples, often with widely differing bioburdens, may need to be processed daily, and urgent clinical intervention or corrective action may be indicated by the results. Traditionally, quantitative cultural techniques have been employed, but alternative 'rapid' methods have been considered in an effort to overcome the inevitable delay associated with lengthy periods of incubation and to offer labour-saving screening methods. In this context, the Direct Epifluorescent Filter Technique (DEFT) (Pettipher 1983) can be adapted for application in both medical and pharmaceutical areas, and this chapter describes protocols appropriate to the examination of urine and the microbiological quality control of intravenous infusion fluids. The principles and detailed methodology of the DEFT are described in Chapter 4 (Shaw & Farr). Urine Examination by the DEFT Urinary tract infections (UTI) are common and urines are usually the most frequent specimens processed by hospital diagnostic laboratories. Urines from healthy individuals are normally sterile whereas those from patients with UTI contain large numbers of bacteria (> 105 / ml) and/or abnonnally high levels of polymorphonuclear leucocytes (>2.5 X 104/ml). It is the detection of this 'significant bacteriuria', typically found in only 20 - 30% of urines tested, that forms a major diagnostic burden of the pathology laboratory. The most widely used method for detecting bacteriuria is semi-quantitative culture of a 2 III urine sample streaked on selective media (e.g. Columbia blood, MacConkey or cystine lactose electrolyte deficient (CLED) agars). A Copyright Rapid Microbiological :\1ethods filr Foods, Beverages and Pharmaceuticals

© 1989 ~)' the Solietl' for Applied Ba'1aio!fJKY All ni:hts of npmductifm ill allY jimn re",roed 0-632-02629-4

59

60

s. P. DENYER

E1' AL.

specimen is considered positive if 200 or more colonies are observed following overnight aerobic incubation at 37°e. Several alternative cultural techniques are available including blotting paper inoculation (Leigh & Williams 1964), spiral plating (Gilchrist et al. 1973) and multipoint inoculation (Henrichsen & Moyes 1987). Polymorphonuclear leucocyte (polymorph) levels are routinely determined by microscopy. The DEFT has been shown to be applicable to the enumeration of bacteria and somatic cells in milk (Pettipher & Rodrigues 1982) although separate methods and preparations are necessary for the two analyses. Since high bacterial and polymorph counts indicate bacteriuria, techniques were developed which would enumerate them both in a single preparation. Initial studies showed that urine specimens varied in the quantity and speed with which they could be filtered under vacuum through polycarbonate filters (Pover & Pettipher, unpublished observations). This was attributed to the varying concentration of bacteria, polymorphs, erythrocytes, epithelial cells, crystals and amorphous debris present in the samples. I~Ieavily contaminated specimens could be filtered, however, after dilution in quarter-strength Ringer solution. A simplified DEF'T protocol, drawing on the general principles and reagents of the DEFT (Shaw & Farr, this volume) but with no enzyme and surfactant pre-treatment, was then developed to facilitate routine bacteriuria screening. Urine DEFT analysis Method

1 Examine the urine for cloudiness, discoloration or debris in order to judge its filterability. If it is clear and straw-coloured it should filter with ease, otherwise dilute it IO-fold in quarter-strength Ringer solution (Oxoid). 2 Pipette 2 ml of the sample on to the O.6-llffi pore size polycarbonate membrane filter installed in the filter tower (Fig. 1A) and apply the vacuum. If it filters successfully, proceed to stage 3; otherwise, pipette off the excess and clean the filtration unit. Dilute the sample by a further factor of 10 and filter through a new membrane filter. 3 Overlay the membrane filter with 2.5 ml of acridine orange for 2 min then re-apply the vacuum. 4 Rinse the membrane by filtering through it 2.5 ml of citrate- NaOI-I buffer. S Filter 2.5 ml of propan-2-o1 through the membrane ensuring that the contact time is minimal. 6 With the vacuum still applied carefully remove the filter using pointed forceps and blow- or air-dry.

APPLICATIONS OF DEFT

61

FIG. 1. Filtration apparatlls associated with (A) conventional DEFT and (B) post-incubation DEFT.

7 l'vlount the dry membrane filter on a microscope slide as described previously (Shaw & Farr, this volume) and examine under the microscope. Bacteria tluoresce an orange-red or green colour and vary in intensity from very dull to very bright (Fig. 2). For a viable bacterial count include only the orange fluorescing bacteria. Polymorphs appear in varying condition from distinct bright cells, with dearly defined nuclei (usually bright orange) surrounded by a rounded envelope of cytoplasm (usually green), to dull disorganized debris. Count only those which appear as distinct, intact cells. Epithelial cells are clearly distinguishable, usually with yellow cytoplasm and a green or orange nucleus. Occasionally epithelial cells may have many bacteria adhering to them. Erythrocytes do not Iluoresce but may be distinguished by virtue of fluorescent particles adhering to them, thus silhouetting the cells against a slightly fluorescing background. Also present may be spcrmatazoa, yeasts

62

S. P. DENYER 1:.'1' AI..

FIt;. 2. B
(Candida spp.) and crystals as well as amorphous debris (probably remnants of lysed polymorphs or epithelial cells). 8 Exanline 15 randomly selected fields of view and count clumps of the orange bacteria and polymorphs in cacho The respective counts/ml are calculated by multiplying the average number in each microscope field by the microscope f~lCtor (see Chapter by Shaw & FaIT, this volume). Account must be taken of any sample dilution.

63

APPLICA TlONS Of DEfT TABl.E

1. Comparison oIthe DEFTexalllillillion oI514 urine salllplesjiJr signijicanl batleriuria* milh ClI/lVl7llirmal bru'lerial adlure and polYlIlorph CfJunling It'lJmiques Numher and percent of samples showing significant bacteriuria according to cell type examined

Sample category A. Distribution o/specimens True negative True positive False negative False positive

Total

Bacteria

Polymorphs

%

279 lJ3

54.28 25.88 2.14 17.70

340 138 13 23

66.15 26.85 2.53 4.47

II

91 514

514

75.41 'X, 92.36'X, 5(U8°;',

93.66% 91.39%

96.21';1"

96.32%

B. Prediflive values t Specificity Sensitivity Positive predictive value Negative predictive value

85.71 'Yo

.. 'Significant hacteriuria' indicated by bacterial and/or polymorph counts greater than 105 /ml and 2.5 x 104 / ml respectively. t The predictin: values were calculated by the method of Ransohoff & Feinstein (1979) as follows: specificity = TN/(TN+fP); sensitivity = TP/(TP+FN); positive predictive value = TP/(TP+FP); and negatiw predictive "alue = TN/(TN+FN), where TP is true positive, TN is true negative, FP is {,lise positiw, and Fl\ is false negative.

Evaluation of the technique The method described requires (. 5 min to prepare the DEFT slide and another 5 min to examine it and record the number of bacteria and cells present. The latter can be facilitated by using standard recording sheets. Application of the DEFT to urine examination has been evaluated by comparing the results obtained from 514 fresh hospital urine specimens to those obtained by conventional bacterial culture and cell counting techniques (Pover, Pettipher & Johnston, unpublished results). The results given in Table I were obtained with a positivity threshold for 'significant bacteriuria' of lOs/ml for bacteria and 2.5 x lO-l/ml for polymorphs. The DEFT offers a useful alternative to conventional culture and counting techniques and also to other direct methods (e.g. dye tests, Pfaller et al. 1983; fixed smears, Hoff el at. 1985; automated fluorescence microscopy, Manson et al. 1985) or indirect methods (e.g. flow calorimetry, Beezer et at. 1978; impedance/conductance measurements, Cady et al. 1978; nephelometry, Kelly

64

s.

P. DENYER E1' AL.

& Balfour 1981; endotoxin detection, Jorgensen & Alexander 1982; ATP bioluminescence, Nichols et al. 1982) for enumerating bacteria and polymorphs in urine. It is suggested that in its present fonn the DEFT test may be used as a rapid 10-min bacteriuria screen where a result is required for an individual patient. In such cases the lengthy preparative stages and examination would not he considered inappropriate. In addition, the DEFT may be valuable as a direct reference method to provide useful information on urine composition for researchers developing novel bacteriuria screening techniques or instruments. The DEFT can also be applied to the exaulination of other clinical specimens, e.g. cerebrospinal fluid, peritoneal fluid (Dcnyer & Sansom, unpublished), pleural fluid, bile and wound exudates, although modifications to the procedures described here may be necessary.

Analysis of Intravenous Fluids by the DEFT Microbial contamination in sterile fluids can have serious consequences (Denyer 1982). Considerable attention is paid therefore, to the microbiological quality of the manufacturing environment and raw materials to ensure a minimal bioburden at sterilization. In-process and end-product microbiological monitoring thus form an important part of the quality assurance procedures associated with the manufacture of sterile products. In addition, the potential for in-use contamination of sterile products is significant, and microbiological testing is also carried out on products suspected of being an infection source. The microbiological quality of injectable products is currently assessed using conventional methods of bacteriological culture, often combined with membrane filtration, which are either of the 'part sample' or 'total sample' type (Denyer 1982). The principal disadvantage associated with these methods is the lengthy incubation period and hence the retrospective nature of these tests when applied at intermediate stages of product manufacture. Rapid methods have been applied to the microbiological examination of infusion fluids but these have generally relied upon the detection of microbial components Oorgensen & Smith 1973; Anderson et at. 1986) or metabolites (Bapp & Wachsmith 1981; Anderson et aL 1986), and have usually been limited to instances of relatively heavy contamination with Gram-negative bacteria (c. 10 4 organisms/ml). Denyer & \Vard (1983) described the application of the DEFT to the rapid detection of bacterial contaminants in intravenous fluids. For parenteral fluids the method has a minimum workable detection level of c. 2S organisms/mL Recently, this technique has been modified to include a pre-incubation stage, resulting in at least a further SOD-fold increase in sensitivity (Denyer & Lynn 1987). Both approaches to the detection of

APPLICATIONS OF DEFT

65

microbiological contamination in infusion fluids by the DEFT are detailed below.

Conventional DEFT anazysis of contaminated fluids Method 1 Introduce the sample of contaminated fluid into the filter tower (Fig. lA) and filter under vacuum. For volumes in excess of 15 ml the vacuum should be applied and maintained during addition of the fluid. 2 Rinse the filter with 5 ml of Water for Irrigation (WFI; United States

Pharmacopoeia 1985). 3 Release the vacuum and overlay the filter with 2.5 ml of acridine orange for 3 min. 4 Re-apply the vacuum to remove the stain and follow by rinsing with two 5 ml volumes of WFI. 5 Turn off the vacuum, remove the polycarbonate filter carefully and mount on a slide as described previously. This is a 'wet mount' procedure and care should be taken to prevent the filter from drying out. 6 Store the prepared slides in the dark and examine within 1 h of preparation. 7 Examine a minimum of ten fields of view, chosen at random, for both orange-red and green fluorescing organisms (see Chapter 4, Shaw & Farr), and count at least 200 cells or the cells in a maximum of 35 fields of view. Calculate the level of contamination by reference to the microscope factor and volume filtered. Post-incubation DEFT analysis of contaminated fluids The inclusion of a short pre-incubation stage (3 - 5 h) before conventional DEFT examination allows the method to be extended to situations of lowlevel microbial contamination. Since an enrichment stage is employed, careful attention must be paid to aseptic technique and the elimination of contamination. It is imperative, therefore, that in the procedures outlined below, operatives exercise all necessary aseptic precautions and carry out all pre-DEFT manipulations under appropriate laminar air flow (LAF) conditions using sterile equipment and materials. In this post-incubation DEFT approach a modification to the general design of DEFT filtration equipment is necessary to ensure a closed sterile unit. In our experience, this can be achieved by closing the filter tower with a resealable rubber closure (Subaseal, W.H. Freemans, Barnsley) and venting with a 12-mm-diameter O.2-llm pore size cellulose nitrate membrane filter

66

S. P. DENYER ET AL.

and needlc assembly (Fig. IB). The wholc device may thcn be wrapped in Kraft paper and sterilized by autoclaving (121°C, 15 min). When required for usc, the sterile filter apparatus is re-assembled under LAF conditions to include an ethylene oxide sterilized O.6-llm pore size polycarbonate filter supported on a similarly sterilized O.2-llffi pore size cellulose nitrate menlbrane filter; this assembly is then connected to a sterile vacuum manifold.

Method

1 Introduce 5 ml \VFI into the filter tower through the 700~) alcoholswabbed resealable closure via a sterile syringe and needle. Remove "VFI by evacuation. 2 Place the manifold and tower assembly in an incubator at 37°C for 30 min to equilibrate to temperature. 3 Remove the manifold and tower assembly from the incubator and place in an LAF cabinet. Swab the resealable closure with 70% alcohol and allow to dry. Inject the desired volume of sample into the tower using a sterile, disposable syringe and needle and apply the vacuum. For volumes in excess of 15 mI, the sample should be introduced, under vacuum, directly from the container either using an appropriate delivery set or with the resealable closure removed. 4 Break the vacuum and inject 10 ml of pre-warmed (37°C), pre-sterilized (I2IoC for 15 min) Tryptone Soya Broth (Oxoid) through the alcoholswabbed resealable closure, via a syringe and needle attached to a sterile O.22-J.tm pore size cellulose nitrate membrane filter. Stages 3 and 4 should be completed within 5 min in order to minimize heat loss from the apparatus.

5 Close the air vent and vacuum line to prevent the gradual passage (under gravity) of broth through the polycarbonate filter and return the apparatus to the incubator for the required time period. 6 After incubation, unclamp the vent and vacuunl lines and remove the broth by vacuum filtration, followed by two rinses of the membrane with 5 ml of WFI. Strict asepsis need no longer be maintained and all manipulations can now be performed with the rubber resealable closure removed. 7 Release the vacuum and overlay the filter with 2.5 ml acridine orange stain for 3 min. S Re-apply the vacuum and rinse the filter twice with 5 ml \VFI. 9 Remove and prepare the polycarbonate filter for conventional 'wet mount' DEFT analysis, as described above. 10 Count as for conventional DEFT analysis as described above. Bacteria detected by this method will normally fluoresce orange-red but the total bacterial ccn count should be recorded. The average count per field of view

APPLICATIONS OF DEFT

67

can then be used to calculate the initial contamination level by reference to the following formula:

G . log (c' M) - O.30It G

log no

(1)

where no = initial number of bacteria/ml of sample; G = mean generation time (min); c = average cell count/field of view after incubation; M = microscope factor; and t incubation time (min). 11 Mean generation times can be calculated in the classical manner (see Equation 2) by conventional plate counts of pure or mixed cultures incubated in tryptone soya broth. An appreciation of the likely contaminating flora is necessary for determination of the relevant values. Some calculated mean generation times are given in Denyer & Lynn (1987). G

=

OJOIt-----log V - log Va

(2)

where ~) = initial viable count; V = final viable count; and t = incubation time (min).

Evaluation of the techniques The methods described above have been variously applied to the examination of 103 samples of deliberately contaminated 0.9°1<, (w/v) sodium chloride or 5% (w/v) dextrose infusion solutions. When examined by either conventional or post-incubation DEFT the best relationship with pour plate colony count (Denyer & Ward 1983) was given by the total membrane fluorescence count derived from the sum of both green and orange-red fluorescing bacteria. In ell.'})eriments conducted without an incubation period, an excellent correlation (r = 0.991) was shown to exist between the total membrane fluorescence count and the pour plate colony count for 41 intravenous fluid samples contaminated with either Stap/~ylococcus au reus, Escherichia coli or Pseudomonas aeruginosa (Fig. 3). Contamination levels as low as 25 organisms/ ml were detected in a SOO-ml sample. Total membrane fluorescence counts, obtained after filtration of bacteria suspended in 0.9'10 (w/v) sodium chloride and incubation for periods of up to S h, showed linear relationships (r 2: 0.94) with pour plate colony counts. This was true for a range of organisms including Bacillus pumilus spores (Fig. 4). The inclusion of a period of incubation permitted the detection of as few as six viable organisms in the volume of fluid sampled. This sensitivity was confirmed using fluid samples deliberately contaminated with a mixed industrial flora where initial contamination levels calculated from post-incubation DEFT results were in close agreement with the conventional

s.

68

P. DENYER ET AL.

5

4

~

E :J

0

0

Qi 3

~

:+: 0

ci

0

....l

2

• /

1 / 1

0

2

3

4

5

L0910 plate count/ml

FIG. 3. Correlation between logw membrane filter fluorescence count/ml and loglO pour plate colony count/ml for 41 contaminated infusion fluids. 0, Escherichia coli; 0, Pseudomonas aeruginosa; 1:1, Staph.ylococcus aureus. Closed &)'IIlbols, 5% (w/v) dextrose; open ~JII11bols, 0.9% (w/v) sodium chloride; - - , fitted regression line (y = 0.178 + 0.85lx); - - -, line of equivalence. (Reproduced with permission from Journal of Parmteral Scientc and Technology.)

TABLE

2. Microbiological examination of three nonnal saline infusion fluids contaminated with a mixed industrial flora Conventional membrane filtration sterility test Volume sampled

Sample

(ml)

A B C

480 10 390

Viable count (cfu/ml)

6 h Post-incubation DEFT Volume sampled (ml)

0.35

IO

2.00 0.05

50 100

Filter count after incubation (per ml of fluid)

Initial number of organisms (per ml)*

2.54 x 103

0.61

103

1.57 0.04

6.52

X

1.53 x 102

* Calculated using Equation (1) and a mean generation time of 30.0 min (Denyer & Lynn 1987).

69

APPLICATIONS OF DEFT 6

/:

/

C 5 :J o

I"

II

o

S

6

l

4

/

0> --.J

3

/

o

o

0/ S

4

/

3

1

a

Log 10 plate count

b

6

I

": /0 /

o

345

~!/(/

r /

0>

o

--.J

o

2

/I

5

o

/~

l

o

C :J

o

234

5

Log 1D plate count

6

C 5 :J o o

~

~4 0>

/

o

--.J

/

l

i

/

§

5

o o

~

;;=

S 4 0>

o

--.J

3·

3

o

1

c

Log 1D plate count

234

5

o

1

d

Log lO plate count

234

5

FIG. 4. Correlation hetween IOglO memhrane tilter fluorescence count (l') and IOglO initial pour plate colony count (x) for Esdlerichia wli (a), PSl'udollllJ1laS aeruKillosa (h), Badllus sublilis (c) and Bacillus pumilus spores (d). 0, no incuhation before DEFT; ., 3 h incuhation before DEFT; 6, 5 h incubation before DEFT; titted regression lines given hy .y = a + bx. (Reproduced with permission from .toun/al o/PaYl7lleral Sciellce alld Tedll1ology.)

membrane filtration sterility test Crable 2). A full statistical treatment of these results is presented in the original papers (Denyer & Ward 1983; Denyer & Lynn 1987).

Conclusion The DEFT has been successfully applied to the analysis of unne for 'significant bacteriuria' and for the detection of contaminants In

70

S. P. DENYER ET AL.

aqueous infusion fluids. In its conventional form it is capable of providing a workable detection sensitivity (Le. 1 cell/field of view) in the region of 5 X 10.1 organisms/sample, dependent upon the microscope factor employed. '''ben used following a 5 h pre-incubation stage, as few as six viable organisms can be detected, irrespective of sample volume processed. The DEFT offers the benefits of rapid enumeration, discrimination between cell and particle types, and the opportunity for tentative identification by cell morphology. In intravenous fluids, comparisons between pre- and postincubation DEFT counts afford confirmation of cell viability. At present, the technique is limited in its routine medical and pharmaceutical laboratory applications by the labour involved in sample preparation and manual Iuicroscopy. It is now feasible, however, for the staining procedure and microscopic examination to be automated (Pettipher & Rodrigues 1982), and suitable image processing hardware and computer-controlled XYZ microscope stages are available, otTering the potential for adaptation to suit Individual applications.

References ANDERSON,

R.L.,

HKiHS,," 11TH,

A.K. &

HOLl.AND,

RW. 1986. Comparison of standard pour

plate procedure and the ATP ami Limulus Amoebocyte Lysate procedures for the detection of microbial contamination in intravenous fluids. ]()umaJ of Clillim/ /\!1icrobio/01.TJ' 23, 465~468.

A.E., BETTELHEI.\I, K.A., AI.-S,\UHI, S. & SH.-\\\, E.). 1978. The enumeration of bacteria in culture media and clinical specimens of urine by microcalorimetry. Science Tools

BtTZER,

25,6-8. EOI>'" CA. &

\V.\CHS.\'IITH, I.K. 1981. Ludferase assay to detect bacterial contamination of intravenous fluids. Amer;can ]ounw! ({Hospital Plwnluuy 38, 1747-·-1749. CADY, P., DUFOUR, S.\V., L\\\LESS, P., NUNKE, B. & KRAEt,;\R, S.]. 1978. Impedimerric scn:ening {or bacteriuria. ]ounwl of Clinical iHiaobjology 7, 273~278. DENYER, S.P. ]982. In-use contamination in intravenous therapy - the scale of the problem. In Injitsiolls and Infix/ions. The Ha::Alrds of Iu-use Coutaminalion in Illlmvellous Therapv, cd. D'Arcy, P.F. pp. 1-15. Oxford: Tbe Medicine Publishing Foundation. DENYER, S.P. & LYN~, R. 1987. A SCnSillye method for the rapid detection of bacterial contaminants in intravenous fluids . .7oumal 0/ Parmlt'raI Sdnw! and Tedln%b'Y 41, 60- 66. DENYER, S.P & \V,-\RJ), K.I 1. ] 983. A rapid method for the detection of bacterial conlaminants in intrayenous fluids using membrane filtration and epitluorescence microscopy. .7ounJtll 4

Parmleral Scient!.' awl TedwoJof...'}137, ]56-158. JE., CAMPUEI.I., ].E., DONNELLY, C.B., PEELER, j.T. & DU.ANEY, ).'\1. 1973. Spiral plate method for bacterial determination. Applied Jlicrobiolo!:.1J' 25, 244- 252. IIFI\,lRICHSEN, C. & J\Io\'Es, A. 1987. A semi-automated method for the culture, identification and susceptibility testing for hacteria direct from urine specimens. iWet/fca! Labora{ory Sdow:s

GII.CHRIST,

44,50-58. NF,\\;'Vl:\.!'\, D.E. & ST:\NECk, J.L. 1985. Bacteriuria screening by use of acridine orange stained smears. Journal of Chuiwl Alitrobiolob.1J' 21, 513- 516. jOl
IIoFF, R.G.,

APPLICATIONS OF DEFT

71

jOR(;ENSEN, j.I I. & S\lITH, R.F. 1973. Rapid detection of contaminated intravenous fluids using the Limlllus ill vitro endotoxin assay. Applied Miaobilllo!{y 26, 521 -524. KI·:I.1.Y, 1\1.'1'. & BALFOUR, L.c. 1981. Evaluation and optimisation of urine screening by Autobac. ]oun/al oj' Clillical Microbiolo!,')! 13, 677 -680. LEiliH, D.A. & WU.I.IA\\S, J.D. 1964. i\lethod for the detection of significant hacteriuria in large groups of patients. ]lIlImalll[Clil1ical Pathlllo!,,)! 17, 498-503. :\l.\NSON, R., SCiIOLEFIEI.I), J., JOHNSTON, R.]. & SCOTT, R. 1985. The screening of more than 2,000 schoolgirls for bacteriuria using an automated fluorescence microscopy system. Urological Research 13, 143 -148. NICHOLS, W.W., CURTIS, GD.W. & JOHNSTON, lUI. 1982. Analysis of the disagreement between automated bioluminescence-based and culture methods for detecting significant bacteriuria, with protocols for standardising naluations ofbacteria detection methods.Jllumal II[ Clilliat! MicrobiologJ! 15, R02-R09. PETTII'IIER, G.L. 19R3. 771t' Direct Epijlmlrt:st't'l1t Filter Tcehllique jilr the Rapid Fllumaatilill o[ iHicroorgtlllisms. Letchworth: Research Studies Press. PETTII'IIER, G.L. & ROJ)RlliuEs, U.M. 1982. Semi-automated counting of bacteria and somatic cells in milk using epifluorescence microscopy and tele\ision image analysis, ]oumal I!I' /lpplied Bae/erilllll!..')! 53, 323-329, PFALI.ER, .\I.A., B,YU\I, C.A" NII.ES, A.C. & MeRR.w, P.R. 1983. Clinical laboratory evaluation of a urine screening device. ]oumal o[ Clilliml Miallbio!lIgy 18, 674-679. R.YNSOHOFF, D.F. & FLiNSTLlN, A.R. 1979. Problems of spectrum and bias in evaluating the etlicacy of dial,'llostic tests, Nap Ellglalld Jllumtllllj';Hedidlle 299, 674-679. Ullited States PllIlnllampllo'a 198.1. Twenty-first revision incorporating National Fommlary, 16th edn, Rochille, :\laryland, USA: United States Pharmacopoeial Convention, Inc.

The Use of Image Analysis for MIC Detennination and Bioassay B. J. BROOKS AND K. COLEMAN Beecham Phannaceuticals Research Division, Chemotherapeutic Research Centre, Brockham Park, Betchworth, Surrey Rill 7AJ, UK

Many microbiological assays involve large numbers of measurements, and are tedious in terms of the amount of time and effort taken to read the test, calculate the results and report the summary data. Two such tests that are widely used in antibiotic research are the determination of the Minimum Inhibitory Concentrations (MICs) of an antibiotic against a range of microorganisms, and the microbiological assay of antibiotic concentrations in biological fluids. Both have been successfully automated using a computercontrolled image analysis system, with consequent improvements in reproducibility and data processing time. What is Image Analysis? Image analysis involves the processing of video images to provide a source of numerical data. The image to be processed is obtained from a video camera focused on an object or photographic image (positive or negative), although it is also possible to mount the camera on a light or electron microscope, if the application requires this. The video camera scans the object and translates the image field into a number of lines. Within the analyser, these lines are subdivided into a series of square picture points (pixels) of fixed size, and the intensity of each of these pixels is assessed and assigned a numerical value - the grey level (or contrast) number. In most systems, this grey level number usually ranges from 0 (black) to 256 (white), although low-performance devices may operate on a scale of 0-64 or 0-100. The spatial resolution of the image - the smallest element that can be measured by the system - is governed by the total number of pixels, and this, in turn, is governed by the number of scan lines and the frequency of the Paper submitted '\ul,'Ust, 1987. if}Sf) hI' 'he ,)'miff)' /ilT A/t/llil'd /JudniologJ rights of rcprrlllUdi'nl i,~' ~rJJI Jilrrn n:){'rv(;d i

Rapid ,\:Iicrohinlogical .\:Ierhnds lor l'oods, Beverages and Pharmaceuticals

0-632-02629-4

73

74

B.

J.

BROOKS AND K. COLEMAN

system clock, which is responsible for digitizing the information along each scan line. Most video cameras transmit an image in 256-750 lines, and each line is divided into 256-1024 pixels, depending on the system. Thus, a lowperformance system will have about 65000 pixels per image (256 x 256) whilst high performance systems have in excess of 500000 pixels per image. The grey level resolution ('contrast') of the image is improved by relatively slow scanning rates, and, in practice, rates of 10 frames or less per second are generally employed. Once the image has been digitized, the operator can select a grey level range to delineate certain features within the image. Thus, if the operator is interested in a set of dark features on an image, the detector can be set to generate a bright display superimposed on the video image to identify all pixels with a grey level below, say, 25. Each image analyser has a number of measuring capabilities, allowing the user to count the number of highlighted features, or measure the area, circumference, width, height, etc. of one or more of the features within the field of view. In many devices, some or all of these functions are provided by hard-wired logic boards, whilst in other systems these functions are softwarebased and are consequently slower to perform. Image Analysis at Brockham Park Our association with image analysis began in 1978, with an OPTOlVlAX I, one of the first systems to be produced by Micromeasurements of Cambridge (now AMS of Saffron Walden). We interfaced this device to a HewlettPackard 9815A desk-top calculator, with a 20-column paper printer, a very non-standard programming language, and a 'massive' 2K program memory. With this equipment we wrote a suite of programs for diffusion assays which read a series of zones, unscrambled the latin square template used on the plate and then calculated the standard line and sample concentrations. In 1984 we invested in a System III image analyser (also from AMS) and interfaced this to a PC350 microcomputer (DEC, Reading) with SI2K main memory, lO-Mbyte hard disk and two 640K floppy disks. A suite of menudriven Fortran 77 programs was written for plate assay and MIC determination using the image analyser. A link to a VAX 785 minicomputer allowed the rapid transfer of data from the image analyser to application software, including shared biological databases and RS I (BBN Software Products, Staines), an eA1:remely powerful data analysis/graphics package. There is currently a large choice of image analystT;; on the market, ranging from the Mastascan II (Mast I,aboratories Ltd, Bootle), which is designed specifically for MIC work and cannot be used for any other type of application, to the extremely versatile Quantimet 520 (Cambridge Instruments,

IMAGE ANALYSIS FOR MIC AND BIOASSAY

75

Cambridge) with its own programming language. The System III, used in our laboratories, is no longer commercially available, but the manufacturers have adapted part of our MIC reading software to a smaller instrument in their range, the 40-10. Another manufacturer now producing a system suitable for MIC and diffusion assay applications is Seescan Ltd, of Cambridge. Application to Bioassay The methodolob'Y involved in the microbiological plate diffusion assay is reviewed in detail elsewhere (see for example Hewitt 1977) and will not be described further in this paper. Perhaps the most difficult part of this assay procedure is the measurement of the zones of inhibition (or exhibition). Most assay organisms give zones with a fuzzy edge, and reading such a variable zone boundary by eye will generally give rise to some inconsistencies. By using an image analyser, the zone boundary can be set at a particular grey level, thus increasing the reliability of the zone measurement, and the assay. Occasionally, the zone will not be a true circle, and then the average of a series of diameter measurements must be taken. With an image analyser, the total area of the zone can be rapidly measured, and reduced to an average zone diameter by the computer. Any required magnification or reduction of the image may be simply achieved by altering the height of the camera above the assay plate. Before reading a plate, the computer program requires the following data to be keyed in: 1 A calibration factor. The system is calibrated by measuring the area (in pixels) of a I-em-square hole cut out of a sheet of opaque plastic. 2 The number of standards and their concentrations. 3 Details of any check standards used. 4 The template used. Details of a series of mirror-image and latin square templates are held in disk files on the PC 350. These templates contain details of the total number of zones on a plate, the number of sample replicates used, and the order in which standards and tests will occur during reading. In this way, individual zone data can be stored in an array in the appropriate sequence. S Study details, such as laboratory notebook references, operator name, protocol reference numbers, etc. can be entered if required, and it is also possible to define a multi-plate study such that the sample results from each plate are stored as part of a summary table. The operator sits in front of the camera stand, manoeuvering each zone into place beneath the camera. Once the zone is correctly located, a foot pedal is depressed to initiate the reading procedure. A buzzer is used to signal that the reading was taken satisfactorily, and anomalies, such as a zone diameter

B.

76

(a)

(b)

J.

BROOKS AND K. COLEMAN

IMAGE ANALYSIS FOR MIC AND BIOASSAY

77

(c) FIG. 1. (a) Displayed image of inhibition zone on an agar phne. This inhibition zone consists of a bright circle of clear agar surrounded by a darker area of confluent bacterial growth, but the image displayed on the monitor is actually a negative one, with bright tCatures sho\\TI as black. The imag(> analyser has superimposed a white display on .111 bright features within the circular frame. Notice the features within the inhibition zone which are not displayed in white; these are caused by the edge of the sample well and by a colony of resistant bacteria. (b) The 'Hole Fill' option will put a white display over any dark features which are entirely surrounded by a bright feature. Using this option interference from both the sample well and the resistant colony are removed. (e) Interference from the sample well can be eliminated by generating a circle on the monitor image widl a diameter slightly larger than that of the sample well. In this example, the senmd circle is shown without the bright fCature white display.

less than or equal to the diameter of the sample well, are notified on the PC 350 monitor before the next reading is taken. The option also exists to step backwards and forwards through the plate to re-read particular zones. The System 1II has a number of feature switches which enable additional processing of the image, prior to taking the reading. Two commonly encountered problems in plate assay are, firstly, that the sample well is often of a very diHerent grey level from that of the zone being measured, and secondly, that some of the assay organisms used can give rise to resistant colonies within the zone of inhibition (Fig. la). It is possible to fill in any unhighlighted features

78

B. J. BROOKS AND K. COLEMAN

which are completely surrounded by a highlighted feature using the 'Hole Fill' option (Fig. 1b). Alternatively, one could blank out the area around a poorly contrasted sample well by generating a circle on the monitor image with a diameter slightly larger than that of the well (Fig. 1c). On completion of the reading process, the control data are plotted on the PC 350 monitor along with the line of best fit (Fig. 2a). The option is available, at this stage, to exclude certain of the control zones from the standard line calculation (Fig. 2b), or to break the line in one or more places if the standard 'line' is non-linear (Fig. 2c). Once a satisfactory standard line has been generated, the estimated sample concentrations may be calculated for each replicate, and each group of replicates averaged. Once again, individual replicates can be excluded, and the average re-calculated prior to printing out the final report.

Application to MIC Determination The lowest antibiotic concentration needed to inhibit growth is known as the Minimum Inhibitory Concentration or MIC, and this is an important parameter in the evaluation of antibiotic activity in vitro. Tests to determine MIC values are often performed by incorporating a series of dilutions of an antibacterial agent into an agar medium, just before it is poured into Petri dishes. A multipoint inoculator (Ridgway- \Vatt 1975; Denley, Billinghurst) can then be used to replicate as many as 40 different organisms on the surface of each petri dish, and, after incubation, the gro'wth of each organism is recorded on each plate.

(a)

IMAGE ANALYSIS FOR ,\lIC A!\D BIOASSAY

79

(b)

(c)

FIG. 2. (a) Screen plot of concentrations of standards vs. zone diameter with line of best fit. (b) A standard line with some control zones excluded. (c) A standard line with a break.

A typical tcst in the authors' laboratory consists of about 150-200 plates, and would take about 6 man-hours to prepare. Following incubation, the dcgree of growth of each organism on each plate is scored semi-quantitatively, resulting in some 6000 data items, which must then be reduced to about 400 MIC values prior to the preparation of summary statistics. The summary

80

B.

J.

BROOKS AND K, COLEMAN

statistics can include geometric mean MIC, MIC range (min and max), the concentration required to inhibit 50, 75, 90 and/or 95 % of the sample population, and plots of the cumulative percentage of strains inhibited vs. MIC. If such a test were to be read manually, it would take 2-3 h, with a further 1- 2 h for data reduction and anything from 2- 20 h for a summary report. In 1982, a VAX 11/750 minicomputer was installed on site, and one of the first applications up and running was RS 1, a scientific data analysis system. We wrote RSI procedures which could process lvnC data and generate the appropriate statistics and cumulative percentage graphs within seconds. While these procedures were found to be extremely useful, the MIC data had to be keyed into the computer manually; as this was generally done by the scientist responsible for the test - seldom the fastest or most accurate of typist') - delays and transcription errors were often encountered at this stage. An attractive feature of the AMS System III is the ability to set up a square frame anywhere within the field of view, under computer control. A suite of programs was written which allowed the user to read a MIC test using the System III, the aim being to speed up the data reduction stage, and to allow for the rapid transfer of MIC data to our RSI software on the site minicomputer. Once again, the program requires certain information before readings can commence. This includes: 1 Pin-pattern data. The user can pre-define a number of agar dish templates, with each template file containing data on the size of the square frame, the number of inoculating pins used on each plate, and the X and Y co-ordinates of the centre of each pin location. The software allows for a maximum of 20 such templates, although, in practice, only four are routinely used: 20-, 36- and 40-pin templates for a standard 9-cm dish, and a 25-pin template for a IO-cm-square dish. 2 Strain data. The namc of each organism used in the tcst can be entered, to improve the clarity of the final results table. The name can be up to 20 characters long, but may be left blank if not required. The option is also available to store lists of frequently used organisms in permanent data files. 3 Treatment data. Details of each of the antibiotic (or treatment) sets must be entered, to include the name of the treatment, the number of plates in the series, the highest concentration of antibiotic, and the dilution factor used for this series (usually Vz). Each treatment must begin with an antibiotic-free control plate. Once this is positioned under the camera, the computer sets up a square frame on the plate image, around each pin location in turn, and measures the total area of highlighted feature(s) representing microbial growth (Fig. 3). Each of the treatment plates is then read in the same way, from the lowest antibiotic

L\lAGE ANALYSIS FOR l\llC AND .BIOASSAY

81

FIG. 3. A System III monitor display sh,ming a MIC plate on which 40 organisms were inoculated. The anlibiotic concentration in the agar was sufficient to inhibit growth of some of these organisms. The square reading fram,' can he seen as the degree of growth of one organism is measured.

concentration up to the highest. When the whole series has been read, a print-out is produced (Table 1), expressing the growth of each organism as a percentage of the growth seen on the control plate. According to NCCLS guidelines (NCCLS 1985), the ,\:lIC is defined as the lowest concentration that completely inhibits growth, disregarding a single colony or faint haze. In this system, the MIC for most org'anisms is assumed to be reached when the area of the colony falls below 5% of the area of growth on the control plate, although some strains may occasionally produce a single colony with area > 501<). In such cases, the operator can intervene during the reading stage to modify the end-point. During the initial trials with this system, it was found that the inoculator pin pattern was not in a consistent position on each plate, but tended to wander; this movement was due to the plates not being located in exactly the same place on the Denley inoculator. When plates were located carefuHy, pin movement was little more than 1 mm, but could be far greater if the plate

82

B. J. BROOKS AND K. COLEMAN TABLE

1. Raw AUC (/,ala

oulpul

file

Area* of growth with antibiotic at dilution Organism

1 2 3 4 5 6 7

8 9 10 11 12

13 14

15 16 17 18 19 20

Control area 433 394 365 486 445 444 411 325 434 397

350 378 323 389 407

382 362

406 418 467

124 122 79 93 86 82 87 75 81 86 67 86

75 75 79 75 79 36 81 75

2

3

4

5

143

100

97

115

99

0

70 25 76 79 86 88 83

0

0

2 69 88 85 82 84 89 82 98 83 85 88

2 7 93 95

85

86

99 0 91

88 98 75 99 95 83 92 15 103 0 72

74

73

52

88 76

85 72 77 75 77 84 30

83

0 0 0 1 0 82 89 63 84 87 16 92 77

7

8

9

66 0

0

0

0

0 0

0 0

0

0 I

0 0

0 0

0

0 0 95 100 76 92 101 6 99 86 0

0

0

0

82

93 94 44 83

94 79 38 89

59

6

0

81 45 79 85

10

0 0

52 I)

59

88

87

82

0

0

8 58 0

0

0 0

39

0 0 0 0

0 0

81

5

0

0

0

0

0

0

0

102

0 70

0

0

0

0

0

0

0

0 0

21 0

2

0 0

0 0

0 0 0 0

43 73 0 83

0

0

* For the twenty organisms in this test (column 1), the area of growth on the control plate is recorded in pixels (column 2), followed by the area of growth on each of the ten antibiotic concentrations recorded 3,.<; a percentage of the control area.

were poorly located before inoculation. To correct for this, it was found necessary to introduce a set of alignment frames into the program. Up to four of these frames can be defined in the pin pattern template file, and they arc displayed over the plate image just prior to the reading of each plate (Fig. 4). A footswitch allows the operator to step through a series of alignment frames as many times as necessary before the reading cycle commences. This may sound a time-consuming process, but in practice an operator familiar with the system can align a plate in 2 - 3 s. A second problem was the occasional poor reading, caused either by badly contrasted growth, an off-centre inoculum which fell across (or outside) the square frame, or a single colony with an area in excess of 5 % of the control plate. None of these occurred very often, but it was clear, from the early development stage, that the square frame had to be moved around the screen at a speed slow enough to allow the operator to check each reading, and that

I.\lAGE ANALYSIS fOR .\He AND I3IOASSA Y

83

FIG. 4. !\ System III monitor display showing one of the large rectangular frames used to align the MIC plate prior to measuring the degree of growth of e'lch of the 40 organisms.

manual intervention should be possible once a plate was completely read. The manual reading stage allows a trame to be enlarged, reduced or moved from its origin, a reading to be retaken after adjusting the grey level, or a reading to he keyed in manually. /\. further problem was that, occasionally, an organism would exhibit an interrupted growth pattern, showing no growth on a particular plate, although growth was seen on plates containing higher antibiotic concentrations (sec, for example, organism 1 in Table 1). The program alerts the operator to this situation and waits for the series to be inspected. The program will only continue to run after the best estimate of the NIle has been keyed in. The organisms used in lVllC tests in the authors' laboratory are predominantly enterobacteria and pseudomonads. Both groups grow well on clear media, so sub-stage dark-f,'Tound illumination is ideal. Wilen opaque media (such as blood agar) are used, overhead illumination is needed, and this has been found adequate for most Gram-positive organisms, except Streptococcus pllewnolliae and Strcp. P'VOgCllCS, which often contrast very poorly with the

84

B. J. BROOKS AND K. COLEMAN TABLE

2. Final MIC table lor

seVe1l

antibiotics

Antibiotic tested Organism used

Eschen'chia coli E 96 coli £124 coli P 91 coli SA 15 coli SA 22 Klebsiella pneumoniae pneum011iae pneumoniae pneumoniae pneumoniae Proteus mirabilis mirabilis mirabiJis mirabilis mirabilis Proteus vulgaris vulgaris vulgaris vulgaris vulgaris

>512 64 >512 16 32 A E 70 Ba 95 A 112 I 77

C889

Il12 SAI7 P 97

Q 8 SA 6 Ba 3 N 11 Q136 D

2

3

16 16 4.0 4.0 8.0

16 8.0 16 4.0 64

256 128 32 32 32

>512 >512 512 >512 >512

128 32 32 32 32

64 128 256 64

2.0 2.0 2.0 2.0 2.0

16 256 32 32 64

16 128 4.0 16 16

2.0 2.0 2.0 2.0 4.0

>64 16 16 16 16

32

4

512 64 256 4.0 8.0 128 64 32 16

32

5

6

0.50 1.0 1.0 2.0 8.0

2.0 0.50 2.0 4.0 8.0

>64 8.0 4.0 1.0 1.0

>64 8.0 4.0 1.0 1.0

7

512 128

512 16 32 128 64 32 16 64

16 16 16 8.0 16

0.50 0.25 0.25 0.25 <0.13

0.25 0.25 0.25 0.50 0.25

32 32 16 16 16

8.0

<0.13 0.50

0.25

8.0 256 32 32 32

256

8.0 8.0 16

2.00 0.25 <0.13 0.50

<0.13 <0.13 <0.13

The calculated MICs of the seven antibiotics are given for each of the 20 organisms used in the test.

TABLE

3. Estimated process timitlgs and operator errors Read manually Time (h)

Reading

2-3

Transcription Summary statistics Secretarial

1 1-12 2~4

Read by image analyser

Errors

Time (h)

Errors

++ + +++ ++

2-3

+/-

Inunediate Immediate Immediate

+/-

l'v1AGE ANALYSIS FOR 1\11C AND BIOASSAY

85

growth medium. Since these two organisms are not used extensively in our laboratory, little work has been done on this problem, but we feel that it could be overcome by a change of camera. Our system uses a I-in Chalnicon camera, which is a fairly robust unit. A Plumbieon camera has about 10 times the light sensitivity of the Chalnieon and would therefore be a better choice for low-light or poor-contrast applications, although it must be used with care, as excessive light can very easily destroy the photosensitive layer of the Plumbicon. There is a new generation of solid-state cameras emerging at present which do not show the same degree oflight sensitivity as the Plumbicon, and it is possible that one of these may prove a better choice for use with poorly contrasted organisms. Finally, when all treatments have been read satisfactorily, the final MIC tahle is printed out (Table 2), and can be transferred to an RS 1 file on the site computer for summary statistics or graphs to be generated. Reading an MIC test in this way is no quicker or slower than reading the same test by eye, but tremendous savings are made in both time and accuracy during transcription, calculation of MICs, transfer of data to computer files, and generation of reports (Table 3). The reading stage could be speeded up, but we feel that this would give rise to a conveyor-belt mentality, where no thought is given to the readings being taken, widl consequent loss of reliability. References Miaobiological AsslI.Y. All In/mdl/dimz /0 QUlIli/i/ative Pril/tiples {md Evaluatiol/. New York: Academic Press. Nccl.s, 1985. Approved Stal/dard /VIeth ods jilr Dilutioll AntilllimJbiai Sum:ptibili!y Tests jor Bacten'a that GmwAerobim/lv. Approved Standard M7-A. Villanova, PA, USA: National Committee filr Clinical Laboratory Standards. RIDGWAy-WATT, P. 1975. An automatic multipoint inoculator for petri dishes. Labora/ory Prattic<' 26, 350-351. IIU\T1"1', W. 1977.

Optimization of Automated Electrometric Methods D. M. GIBSON

Ministry ofAgriculture, Fisheries and Food, Torry Research Station, 135 Abbq Road, Aberdeen AB9 8DG, Scotland, UK

Over the past decade automated instruments for measuring the impedance, conductance and capacitance changes of growing microbial cultures have become generally available and are now found in many routine quality control laboratories. Their main use is for estimating numbers of micro-organisms in terms of detection time (DT), i.e. the time from inoculation to a pre-selected change in the electrical parameter. The DT can be calibrated against the results from conventional counting methods, and is available in 1O-30'Yo of the usual incubation time. Higher counts give shorter detection times. The instruments are also used for detecting specific pathogens such as salmonellas. They have been described by Firstenberg-Eden & Eden (1984) and Gibson & Clark (1987). Conductance is an extrinsic parameter, an electrical property of solutions. When an alternating current of suitable frequency (usually 2-10 kHz) is passed through culture media, it encounters impedance to its flow. Impedance is a complex quantity and in this case consists of two components, resistance (reciprocal of conductance and capacitative reactance. The conductance depends on the number and nature of charge carriers in the solution and the metabolism of any microbes present generally changes it by converting poor charge carriers such as carbohydrates, proteins and lipids to more effective charge carriers such as acids, amines, etc. Thus, there occurs a change in conductance of the medium which can be measured and used in microbiological analyses. It has been shown that the change in conductance is proportional to the change in number of viable micro-organisms and this is the basis for calibration of conductance assays as a measure of growth rates (Richards et al. 1978). The change in impedance and capacitance of the growth media can also be measured and used in a similar way (Firstenberg-Eden & Eden 1984).

Copynghl Rapid Microbiological Methods for Foods, Beverages and Pharmaceuticals

©

1989 b), Ihe SO£;e(y jur Applied Batteriolol!J' A II riKh Is of reprod,u,tion in any form reserved

0-632-02629-4

87

88

D. M. GIBSON

They are influenced more by electrode effects than solution effects (Harris et ai. 1987). Electrometric assay is thus an empirical measure of the activity of a culture. The D"I's have to be calibrated against conventional microbiologkal assays. There are large errors in the aerobic plate count method, ±37 to ± 11 for 30-300 colonies/plate, respectively (Hall 1982). How can electrometric assays be optimized? As assays arc used to yield data for many different purposes, there cannot be a unique answer. For example, in the food industry, the assay may be used as a measure of product quality and stability, and hygienic state of products and production processes. A single assay can usually give the answer to one question. Therefore, the users of cIcctrometric instruments must know clearly which question they are trying to answer. Growth conditions can be made to relate more to those in the sample than in a conventional bacteriological assay. Thus, a total viable count (TVC) may, after the usual dilution for conventional methods, give the number of microbes able to gTow on a particular medium. But in an electrometric assay where little or no dilution is needed, more of the sample is present and the assay measures the ability of the inoculum to grow in the test material supplemented with media. This may be advantageous for stability studies as natural antimicrobials are not diluted out. \Vhen electrometric assays are done on the same samples using many media fonnulations, along with other measurements of quality, interesting differences between data may arise. For example, Gibson (1985) attempted to predict the quality and remaining shelf-life of chilled packaged fish by conductance assays from a data set which included conventional bacteriological assays and sensory taste panel data. lIe showed that the shortest detection times, i.e. those relating to quality, were found with assays in seawater broth, but the highest correlation with TVC was given by the data from assays in Wood & Baird's trimethylamine oxide broth, which also correlated highest with the shelf-life as found by the taste panel. This latter medium mimicked the food more closely and it selected the part of the bacterial flora known to be active in fish spoilage. Assays for the presence or absence of specific organisms such as Escherichia coli or Salmonella spp. present a different set of considerations. The taxonomic criteria which define the species, or some other property ,-,:hich correlates highly with them, may be incorporated into the assay, or it may suffice to obtain an indication of high probability that any change in the assay is due to the organism sought, this being confirmed in subsequent testing. Outgrowth in the media is paramount in this case and the ecological conditions need not resemble those of the host sanlple but should be optimal for the organism's recovery and growth. Enumeration may be done in conductance assays but when pre-enrichment procedures are incorporated, as for salmonellas, this is not possible.

AUTOMATED ELECTROMETRIC METHODS

89

It must be stressed that electrometric assays only reflect the microbiological conditions prevailing. What can be achieved by such assays can often be achieved by conventional assays but with much longer incubation times, greater physical, and perhaps greater intellectual, etIort. Electrometric assays arc a means to an end and not the end in themselves. The appropriate microbiological question must be posed and attempts made to provide the best answer in the most usable form rather than simply converting a conventional assay to an automated form. General Hints

It is axiomatic that the instruments should be operated according to the manufacturers' instructions and suitable controls run to ensure that the system is fully functional. The design of control assay is important. It has been found that after repeated sub-culture and growth some salmonellas are unable to grow characteristically in selective media. Sub-cultures taken from selective media are not advised. For best reproducibility, media should be left tor at least 4 h after autoclaving. For highest accuracy, media should be dispensed after autoclaving. One particularly useful technique with Malthus electrodes is to autoclave them in glass tubes containing 1 ml water and then transfer them to the assay medium held in either glass ampoules or sterile disposable plastic tubes. If the instrument permits, the initial resistance of the assay assembly can be checked to see if it is in the normal range. Small changes in the assay temperature produce changes in conductance and impedance, similar to those produced by microbial growth. Thus, conductance assays are done in precisely controlled water baths, as with the Malthus instrument. If possible the assays should be set up at or close to their subsequent incubation temperature perhaps by pre-heating the medium but without exposing the microbes to temperature shock. This reduces the equilibration time so that rapid changes caused by high inoculum levels (> 107 /ml) are not masked.

Curve Quality As microbes grow, the conductance, impedance and capacitance of the culture medium change. Plots of these changes with the time since inoculation resemble conventional growth curves. For data equivalent to conventional counts, the detection time (DT) is sought. It is defined in different ways depending upon the algorithm used in the instruments. Its determination depends on there being a clear change in slope of the conductance change with time and this requires a short equilibration time, minimal drift due to non-microbial effects and a distinct microbial signal. The equilibration time

90

D. M. GIBSON

depends on the difference between the temperature of the assay and the incubator, and particles settling out or on the electrodes. In conventional plate and turbidity assays, only the liquid phase of homogenates, etc. is taken {to avoid confusion between particles and colonies on the surfaces). In conductance assays particulate matter can also be taken. Settling out depends on the size and density of the material, and the viscosity of the medium which can be raised with dextran (Curtis et al. 1985), agar or similar materials. For samples containing lipids the assay volume can be increased to ensure that the lipid rises clear of the electrodes. Simple coarse filtration through a tea strainer has been found to be very effective in improving the electrical stability of assays on homogenates of hamburgers (Gibson, unpublished). Electrometric assays are usuaHy done on undiluted homogenates; if high levels of contamination sufficient to give an immediate electrical change are suspected then the sample should be diluted to avoid missing the D1'. To obtain a distinct change in the electrical parameter the medium must permit good growth, or more correctly good metabolism (catabolism and anabolism), leading to the formation of more and better charge carriers. Knowledge of the metabolism of the microbes involved can sometimes be usefully applied. Operational experience indicates that aldose metabolism in non-selective nledium under the micro-aerophilic conditions of the assay does not generally yield conductance change, and that phosphate buffer lengthens DTs. Metabolic products from amino acids, peptides and peptooes of <20000 daltons yield many charge carriers, especially with Gram-negative bacteria. Amino acids that have zwitterion form at the assay plI are particularly useful substrates. "Vhen a good distinctive change in conductance, impedance or capacitance has been obtained it is desirable to know which constituent of the medium (or of the inoculum) is responsible. Kell (1987) suggested that it is easier to progress by omitting constituents or substrates from the medium rather than by adding them or increasing their concentration.

Comments on some Conventional Assays Most instrument users attempt to correlate the results obtained with those from conventional assays, such as TVC by aerobic plate count methods. Some of the pitfalls of conventional assays have already been mentioned and Postgatc (1969) has oudined their errors. In constructing calibration curves relating DTs to TVC, the usual procedure is to make a homogenate and assay similar samples by both systems. A common procedure (used at this laboratory) is to store the homogenized material in a refrigerator for 30 min to allow the homogenate to settle. The liquid is then pipetted with narrow-bore pipettes to make appropriate dilutions from which equal volumes are dispensed on the surface of agar plates using a modification of the Miles & Misra technique

AUTOivlATED ELECTROMETRIC METHODS

91

(Bousfield ct al. 1973) and the plates arc incubated aerobically. Then a sample is taken for conductance assay. But when conductance assays are being done alone, a standard volume is taken immediately after homogenization by widebore pipene and placed in the assay cell. \Vhat differences may arise? It is known from microscopic examination of the homogenate that bacteria may exist in large elongated forms. At low growth rates many organisms appear as multicellular filaments (Postgate et al. 1961; Pirt 1987). They subsequently divide rapidly to adopt their more usual forms (so giving an apparent increase in number) and this can happen during the storage period. The errors in preparing dilution series, especially if the diluent is dispensed before autoclaving, are large and known. The conditions of aerobiosis in the assays are not the same, being microaerophilic in the c!ectrometric assays, perhaps more similar to t1lOse in the sample. From these considerations, it can be argued t11at data from electrometric assays, being a measure of the metabolic activity of the culture, may relate more to overall quality of the product being assayed, however measured, than simply to TVC results. Thus, high corrc!ations between conventional and electrometric assays are not necessarily desirable. Temperature The choice of incubation temperature in conventional methods has usually been a compromise. Assays on chilled products take a long time at O-IO°C and are t11erefore incubated at 20, 25 or even 30°e. While the numerical values of the counts at these temperatures are usually similar, they may result from different components of the flora outgrowing in the assay, which may give different electrical changes. In assays for enteric organisms, incubation temperatures of 35 -44°C are used. Microbes show an optimum temperature or range of temperature; above and below this the growth rate declines. The metabolic pattern and efficiency arc temperature-dependent, efficiency tending to increase at sub-optimal temperatures. Bishop et at. (1984) showed that for milk products, most types of organisms had similar growth rates at 18°e. They recommend this temperature to maximize correlations with conventional TVC assays. It may be better to optimize at the temperature most favoured by the active portion of the spoilage /lora especially if conductance assays are being done to estimate spoilage rate or shelf-life. Calibration Curves

It has been the practice for users to construct a plot correlating the results of conventional testing, for example TVC, with detection times derived from conductance assays. A typical plot is shown in Fig. 1 (Gibson & Ogden 1987).

92

D. M. GIBSON

10

8

z

6

o

Ol

.2

o 4

0

~ft 0

0 00 0

o

0 00

'tB 0

'0

00

2

0

0

5

10

15

20

25

DT (hl L A typical calibration curve relating logarithm of numbers to detection time. r 0.6 (taken from Gibson & Ogden 1987).

FIG. =:

=:

0.9, s.d.

After calibration many users rely on DTs only. How can the quality of the calibration be assessed? There are many reports showing correlation coefficients (r) assuming a linear or quadratic relationship between DT and TVC and some of these, in view of the errors in detennining TVe, are surprisingly high. But a high r value alone is insufficient; the amount of scatter about the line of correlation, expressed as a standard deviation, is important. Some of the scatter can be explained. Points above the line, i.e. DT longer than expected from the TVC, may arise from spores remaining dormant during the short conductance assay time but germinating during conventional incubation periods, from injured cells not recovering fast enough (although liquid media usually encourage recovery better than do solid media), from antimicrobial substances active in the conductance assay but being diluted out in conventional testing, and from antagonism between components of the flora. Points below the line, i.e. shorter than expected DTs for the TVC, may be due to colonies derived from dumps or chains of cells rather than single cells, as each cell in the aggregate contributes to the electrometric change, and synergism between components of the tIora, the degree of aerobiosis favouring the outgrowth of different components of the flora in each assay. For example conductance assays (in the Malthus) of 10 ml volume arc done under microaerophilic conditions and some plate assays under highly aerobic conditions. Another factor may be the distribution of microbes in the inoculum. As

i\UTOMi\'fED EI.ECTRO:YIETRIC METHODS

93

mentioned, extracts are allowed to settle before being dispensed, diluted, etc. for conventional assay but are taken immediately, as a slurry, for conductance assay. It has been assumed that the microbes are uniformly distributed between the solid and liquid phases of the extract but in view of modern work on the adhesion of microbes to nearby solids, this may not be true for all foods. Thus caution must be exercised in interpreting calibration curves. Practical Experiments on Fish A series of experiments was carried out using cod of various qualities to study the effects of parameters which might be ex.'Pected to influence the assay. From the mass of data only a summary of the salient features can be given here but it is hoped that it will assist uscrs in devising protocols for their own use. The prototype JVlalthus instrument was used for this study.

Extraction The extracts were prepared in a stomacher. The ratio of fish to extractant was tested in the range 1: 1 to 1:4, higher dilutions being thought to reduce the concentration of microbes too much. Three extractants were tested, peptonephosphate (as used in conventional tests), 0.9% saline, and 1.5°/<, saline, which is osmo-equivalent to fish tissue. The most concentrated extract was difficult to dispense and as all the other dilutions were suitable, a ratio of 1:2 was taken. The extractants had little effect and the cheapest can be used. With concentrated slurries there can be instability of the conductance signal while the solids are setting. This can be overcome by coating the electrodes with 4% agar, which does not dissolve in the medium, or by a short, lowspeed centrifugation of the total sample. Such effects are shown in Fig. 2. Because of the improved curve quality some pre-treatment may be worthwhile despite the extra eflort.

A1cdia The media used in conventional assays have been chosen to promote the rapid appearance of colonies. While they may be suitable, in liquid form, for electrical assays, other recipes may be worth testing. In this work three media were used, seawater broth (SWB), Wood & Baird's TMAO broth, used by Gibson (1985) and brain-heart infusion broth (BHI) used by Ogden (1986). All were suitable. The shortest DTs were obtained with BHI (Fig. 3) for all qualities of fish. The TMAO broth and SWB assays generally gave later results.

94

D. M. GIBSON

250

200

en

3~

150

C-

m

~

u

-g:l

100

o

u

50

6

3

9 Time (h)

12

15

18

FIG. 2. Conductance curves for fish extracts. 0 - 0, untreated extract; 6 - 6, electrodes coated with 4°!c) agar; 0 - 0, extract briefly centrifuged in assay tube.

70

en

50

..:;. Q)

0

...c

1'0

30

U ::J

"0

C

0

U

10

-1 0 L_-.L-=~=:::r:::::::::::!::::::L..._ _.....L-_----J

o

2

4

6

8

10

Time lh)

F1G.3. Conductance broth.

curn~s

for fish extracts.

[1-0,

\Vood & Baird;

0-0,

BIll; 6-6, seawater

AUTOMATED ELECTROMETRIC METHODS

95

12

10

8 £ t-

o

6 4

2

00'-----...... 2-----'4'----...... 6-----'8'-------'10 Inoculum (mil

FIG. 4. Effect of ratio of inoculum to medium on D1'. All assays contained 10 m!. Typical result f(lr top-quality fish.

Inoculum For all qualities of fish tested there was an optimum volume of extract as judged by a shorter DT (Fig. 4). Thus, increasing the volume of the inoculum, and so increasing the number of microbes inoculated into the sample being assayed, initially decreased the detection time to a minimum but then yielded an increase in DT, as the amount of medium became limiting. At low inoculum volumes, the nutritional contribution of the medium is high, thus reflecting the ability of the microbes to grow in the medium. At high inoculum volumes, there is little medium and the microbes are being cultured with the nutrients in the extract. This may be a desirable situation for assays relating to the remaining shelf-life of the fish, i.e. the ability of the microbes to grow in fish rather than in medium. Source of conductance change

It is known that in the spoilage of most marine fish, TMAO is reduced to TMA by the active spoilage flora and the TMAO/TMA reaction contributes to conductance change (Easter el al. 1983). There is ample TMAO in fresh marine fish but possibly not in stale fish. When TMAO was added to the BBI assay medium (10 mmolll, approximately the level in fresh fish) it led to reductions of 10% in DT. The total conductance change was increased but there was little effect on generation times as calculated from conductance data by the formula of Jason (1983). 'I'1vlAO was also added to the extractant to

96

D. M. GIBSON

induce enzymes involved in its metabolism but it had no effect on these shorttime assays.

Conclusions From such considerations it was found that the shortest OTs were given through the following protocol: homogenize 100 g fish with 200 ml 0.9 % saline, take 5 ml extract and add to 5 ml BIn supplemented with 0.1
Pathogen Detection One of the major uses ofconductance instruments is the detection of Salmonella spp. in foods for which Easter & Gibson (1985) published a protocol. Their assay system consisted of pre-enrichment in buffered peptone water (BP\V), supplemented with dulcitol and T.MAO, for recovery, resuscitation and growth of the bacteria and induction of appropriate enzymes, followed by a conductance assay in selenite- dulcitol- T.MAO nledium. Results are available in 24-48 h. There were some criticisms and Gibson (1987) has slightly modified the assay. This entails pre-enrichment in BP\V supplemented with mannitol and dimethylsulphoxide (DMSO) and then conductance assays in selenitcmannitol-TMAO to allow for any dulcitol-negative strains, as well as in the original selective medium. DMSO is an inducer of TMAO reductase, possibly better than TMAO itself, and mannitol induces dehydrogenases for both mannitol and dulcitol. Some dulcitol-negative strains gave a positive reaction in the selenite-dulcitol-TMAO after pre-enrichment in BP\V supplemented with mannitol and DMSO. Salmonellas can ferment mannitol. Thus, if assays take more than a few hours, induction of enzymes may be worthwhile and inducers such as DMSO for TMAO may not only be as effective as the true substrate but much cheaper.

Inorganic Constituents In conventional bacteriology little attention is paid to the inorganic ions present in media. Many contain added salt (NaCl) and phosphates but the contributions of inorganic ions from peptones, yeast and beef extracts is not

AUTOMATED ELECTROMETRIC METHODS

97

110

90

~ 70 OJ IJ C

ro 1:)

50

::J

-0

§ 30

u

10 -10'------'-------'-----'-------' o 10 30 40

FIG. 5. Effect of ion limitation on the conductance curve for SllelvUllcliu putrefiuiens NCMB 400 growing in Wood & Baird's medium. 0-0, complete medium; 6-11, -K+ -P0.1- 4; 0-0, -Mg++; .-e, -K+ l'Vlg++

-P0.1- 4 ;

"'-"',

-K+ Mg>+

known and disregarded. Media handbooks sometimes list 'typical analyses' but do not indicate the variability of their products. Easter et al. (1983) described a conductance assay for trimethylamine oxide (TMAO) reductase and showed that there was a large and consistent conductance change proportional to reduction of TMAO and to the accumulation of trimethylamine (TMA). Owens et al. (1985) challenged this finding and purported to show by manipulations of conductivity physico-chemical data that the conductance change was due solely to the changing ionic form of phosphate in the media. b:periments were done to resolve this (Gibson, unpublished) omitting the phosphate from the assay of Easter et al. (1983). I lad Owens et al. (1985) been correct, then there should be little conductance change in the absence of added phosphate. If Easter et ai, were correct, then tlle same conductance change should be found in the assays with and without phosphate, It was found that the total conductance change was the same thus supporting Easter et ai, The DT in the absence of K 2 HP0 4 was shorter and it was thought that this may be due to the phenomenon of ion-limited growth, well known in continuous culture studies (see Tempest & Neijssel 1984; Hellingwerf & Konings 1985). Furtller eA'Periments were done limiting the concentration of K+, Mg++ and phosphate ions; the results arc shown in Fig. 5. It is apparent that limiting the concentration of certain ions will reduce detection times, principally because under such conditions metabolism is less efficient and the

98

D. M. GIBSON

organism has to gain and dlen expend more energy for homeostasis. If fue main source of energy gives a conductance change, in this case TMAO reduction, then it can be incorporated into the assay_ Can this approach be exploited in assays on foods, for example, which may provide a large input of phosphate ions in the inoculum? The most obvious way is to reduce the overall phosphate content by removal of phosphates as precipitates in the assay. Many phosphates such as barium and calcium are virtually insoluble at physiological pIf values. Such ions can be added in their soluble chloride forms and rapid precipitation occurs within the time taken for temperature equilibration of the assay. Chelation of ions such as potassium and various divalent ions luay be possible but many chelators have direct effects on cell membranes and may be bacteriostatic. Of course, to be useful, the type of metabolism favoured by ion-limited growth conditions ought to produce a good conductance change.

Conclusions Direct transfer of conventional to conductance assays may give good results and useful information but a fundamental assessment of what really is required from the assays is worthwhile. The I'Ve is a measure of the ability of microorganisms to grow in the selected medium whereas a more pertinent question for food quality control might be the ability of the microbes to grow, albeit at an elevated temperature, in the sample (e.g. the food) to predict shelf-life or stability. The statistical aspects of the assays and their relationships to any calibrating system should be examined as should the implications of assaying relatively large amounts in the conductance assay, in the case described 2 g, compared with the small amount actually assayed in a TVC, perhaps only 0.002 g.

References & FIRSTENHERG-EDEN, R. 1984. A rapid impedimetric method fOJ determining the potential shelf-life of pasteurized whole milk. JOlin/a! ofPood Protection 47,

BISHOP, J.R., \VHrn:, CH.

471-475.

I.J., S.\t1TH, G.L. & TRUbvIAN, R.\V. 1973. The use of semi-automatic pipettes in the viable counting of bacteria. Joumal ofAppJied Batteriology 36, 297-299. CURTIS, GJ).W., THOMAS, CD. & JOHNSTON, II.H. 1985. A note on the use of dc:x1ran in blood cultures monitored by conductance methods. ]oumal ofApplied Baclcrio!o&'l' 56, 571-

BOUSFIEI.D,

576. GmsON, D.1\1. 1985. Rapid and automated detection of salmonella by electrical measurements. ]oumal of Hygiene, Cambridge 94, 245 - 262. EASTER, M.C., GlI.lSON, D.M. & WARD, F.B. 1983. A conductance method for the assay and study of trimethylamine oxide reduction. ]ounUllqf.4pplied Bacteriology 52, 365-375.. E'\STER, l\1.C. &

AUTO"'I ATED J:LECTROMETRI C '\1 ETIIOOS

99

FIRSTI:NllLRto-Em:N, R. & EDLI\, G. 1984.lmpedmlce .lIierobio!o!,.'Y. Letchworth: Research Studies Press. GillSON, D.M. 1985. Predicting the shelf-life of packaged fish from conductance measurements. JOlin/a! Of Applied Baeterio!ogy 58, 465-470. GIlISOI\, 0.1\ l. 1987. Some modification to the media f(lr rapid automated detection of salmonellas. ]ollnlll! oIApp!ied Bi/rterio!ogy 63, 299-304. GillSON, O.j\[. & O ..o\RK, A. 1987. Lightning conductance. Laboratory Praclite 36,47-51. GIllSON, D.;\!. & OGDEN, LO. 1987. Estimating the shelf~life of packaged fish. In Seajf)()d QlIali~y DetmllillalilJ1l, eds Kramer, D.E. & Liston, J pp. 437 -445. Amsterdam: Elsevier. I [AI.I., [ .. 1'. 1982. A ;Halllla! of;Het/lOds jilr Ihe Haclen'o!ogim! Emmillalioll ofFro::en Foods, 3rd edn. Chipping Campden: Campden Food Presenation Research Association. IIARRIS, c.:\I., TODD, R.W., BUNG'IRD, S.]., LOITlT, R.W., MORRIS, JG. & KEt.I., O.B. 1987. Dielectric pcrmitthity of microhial suspensions at radio frequencies: a novel method for the real time esrimation of microhial biomass. EII::yme alld Microbia! Tahllolof!Y 9, 181- 186. III:I.I.INti\Il:lU", K.]. & KOI';INtiS, W.N. 1985. The energy How in bacteria: the main free energy intermediates and their reg'ulatory role. Adm/itt's ill /vIicrobia! Physio!og)' 26, 125-153. ]ISON, A.e. 1983. A deterministic model for monophasic growth of batch cultures of bacteria. Alliollie vall LCCIllV<7dlOd' 49, 513 - 536. KI:L1., O.B. 1987. Forces, Huxes and the control of microbial growth and metabolism. Joun/a! of Gmera! /vIicrobio!og)' 133, 1651-1665. O{iDEN, ID. 1986. Use of conductance methods to predict bacterial counts in fish. Journa! 0/ Applied Haclerio!olD' 61,263-268. O\IENS, JO., MISKIN, O.R., W.'ICHER-VIIEROS, .\l.e. & BENtiE, LCA. 1985. Sources of comluctance changes during hacterial reduction of trimethylamine oxide to trimcthylammonium in phosphate buffer. JOlin/a! of Genera! :Hicrobio!o/.:JI I3 I, 1357 -136 I. PIRT, S.]. 1987. The energetics of microbes at slow growth rates; maintenance energies and dormant organisms. JOllnlll!o( Fenlll'liialioll Tcdlllo!o/!,JI 65, 173 -177. I'OST(i.'lTI:, JR. 1969. Viable counts and viability. In Melhods ill Mitrobio!ob,'Y, Vo!' I, eds Norris, JR. & Ribbons, D.W., Pl'· 611-628. London: Academic Press. POSTG'lTL, J.R., CRliMPTON, J .E. & IluNTLR, J.R. 1961. The measurement of bacterial yiabilities by slide culture.]olln/a! o(Cmera! Microbio!o.!.,'Y 24,15-24. RICH.'IRDS, .J.C.S., j'lSON, A.C., [IOIIIIS, G., GillSON, D.M. & CHRISTIE, R.l1. 1978. Electronic measurement of bacterial growth. ]olln/a!o/Ph)'si,:" E: Scimlijie lllslrllmmis 1I, 560-568. TE\IPEST, D.W. & NLI.lSSEI., O.M. 1984. The status of 1'.HI' and maintenance energy as biologically interpretable phenomena. Allllua! Revil'll' o/MiLTobio!o/!,)' 38, 459-486.

Conductance Techniques for the Detection of Contaminants in Beer A. L. KYRHKIDES* .c\ND P. A. TIlURSTON Grand ;Hetropolitan Brcming Ltd, Stag Bremery, 91, Britk Lane, iHort/ake, LOlldoll

SWJ4 lET, UK

Several groups of bacteria and yeasts are generally associated with breweries and some of these organisms can cause spoilage of beer (Ilough ct al. 1982). Current microbiological monitoring within the brewery relics heavily on growthdependent techniques such as plate counts and forcing tests. Incubation times for plate counts are 2 - 7 days. Forcing tests involve incubation of beer samples at temperatures in the range 25 - 35°C. The samples are then examined visually after 7 - 21 days for haze development and microscopically for the presence of micro-organisms (Ilough et al. 1(82). Therefore these tests can brive only a retrospective view of product contamination since most of the beer will have been processed by the time results are available. In addition, the methods are I~lirly labour-intensive and skilled assessment of results is often required. There is clearly a need therefore to develop rapid, automated techniques (Shaw 1987). Improved detection times would allow more rapid control over the process, and enhance the investigative capability of the laboratory. Automation would save time in processing samples, reading results and reporting information. The critical control points of the brewing process that require monitoring tall into two broad groups: (1) where brewing yeast is present and selective media arc required to detect contaminants (these samples include brewing yeast slurry, heel' fermentations, and beer in cold-storage prior to filtration); (2) where brewing yeasts and any other organisms should be absent or in very low numbers (e.g. in raw materials, bright and pasteurized beer). Electrometric techniques are suited to this type of monitoring because they are growth-dependent and therefore both selective and total viable count * Present address: Grand i\lctropolitan Foods Europe, -UO Victoria Road, South Ruislip, i\ litltllesex litH olIr ClipyriXIJ/ Rapid .\licrohiological \ 1t:thods fl)r Foods, BnTragcs Jnd Pharmaceuticals

©

1989 hi' llie SlJiidl'ji" Applied !J1/(/triO!r(,1' ,,.-JII n).!,hl.\ (~f' rr!lmtlut"/ioll in ({ll.I' j;,ym rt'so7..'cd

0-(,32-0262'1-4

101

102

A. L. KYRIAKIDES AND P. A. THURSTON

media can be used. In addition, multi-channel systems that lend themselves to automation are commercially available. These techniques have been investigated by the brewing industry (Evans 1982; Kilgour & Day 1983). They are based on the observation that actively metabolizing cells in a nutrient medium cause a change in the electrical conductivity by converting complex electrically inert substrates into smaller charged products (or vice versa). It should be possible, therefore, to detect growing organisms by measuring the change in conductance of the growth medium (Ur & Brown 1974). We describe here our experiences with the development of conductance techniques for detecting contaminants in beer.

General Procedures Conductivity changes caused by microbial growth are very small and instruments currently available can detcct a significant change in conductivity only when the microbial population increases to greater than c. 106 cells/ml (Richards et al. 1978). Therefore there is a period known as the detection time (DT) between sample inoculation and instrument detection. The DT value has previously been shown to be inversely proportional to the initial inoculum size (Kilgour & Day 1983).

Conductance monitoring

To achieve accurate and sensitive measurement of conductance it is necessary to minimize external influences on the sample responsible for noise and drift. These include temperature variation, electrical interference (i.e. mains power surges) and non-microbial associated reactions in the medium. Conductance monitoring in our e::\.rperiments was therefore done with the Malthus Model 128H Growth Analyser System (Malthus Instruments, Crawley, Sussex).

Microbial cultures Micro-organisms used for media development were sub-cultured from a collection of bacteria and yeasts in 9 ml of \V.L. Nutrient Broth (Oxoid CJ\1 501) and incubated at 28°C for 48 h before use. Ten strains of yeast were used. Three of thesc werc obtained from the National Collection of Yeast Cultures (NCYC), Food Research Institutc,

Norwich, UK: SaccharomJlccs diastaJicus (NCYC 447); Pichia membranaljilciens (Neye 326) and flansenula anumala (NCYC 682). The remaining seven yeasts were strains of Saccharol1~)Jces cerez)isiae, three of which were brewing strains and four, spoilage strains. Nine strains of bacteria isolated from brewery samples were used. Two

CONDUCTANCE TECHNHlUES FOR BEER

103

were identified to species level as Lactobacillus brevis (designated L1 and L2) and one as Enterobacter doacae; the others were unidentified strains of the following genera: Lactobacillus (designated L3 and L4),Acetobacter, Gluconobacter, Zymomonas, Pediococms.

Plate count techniques Total count medium Total microbial counts were achieved using Raka-Ray Medium (Oxoid, CNI777) supplemented with 1 mill bromocresol green (BDH 2.2% w/v) and 20 mlll ergosterol- Tween SO prepared from a stock solution of 125 mg ergosterol (Sigma) in 50 ml Tween SO (Sigma) and 50 ml industrial methylated spirit (IMS, BDH, 90°/., v/v). Membrane-filtered samples were incubated at 28°C for 2 days anaerobically followed by 2 days aerobically and spread-plate samples were incubated aerobically for 3 days.

Selective media Non-SaccharomJ'ccs wild yeasts were isolated on Lysine Medium (Oxoid, Cl\i119l) aerobically at 2SoC for 3 days. Wild strains of SaccharomyfCs were isolated on YM agar (Difco) supplemented with 19.5 mlll copper sulphate solution and 10 mlll ergosterol- Tween 80 solution. Copper sulphate was added aseptically, after autodaving the medium, from a stock solution (10 mg/ml CUS04) prepared by dissolving 1.57 g of CuS04·5HzO in 50 ml of distilled water, acidifying with 0.5 ml of I mol/l sulphuric acid, making the volume up to 100 ml and then autoclaving the solution at 121°C for 15 min. Ergosterol-Tween 80 was prepared by dissolving 250 mg ergosterol in 50 ml of Tween SO and 50 ml ofiMS. Cultures were incubated at 2SoC anaerobically for 4 days. Lactic acid bacteria were isolated on Raka-Ray medium incubated anaerobically at 28°C for 7 days. Acetic acid bacteria were isolated on Actidione Agar (Oxoid) aerobically at 28°C for 3 days. Screening of Non-Selective Media The requirements for developing a total count medium for use with the Malthus instrument were compatability with the system (i.e. electrically stable) and suitability for growth of brewery micro-organisms. Media for evaluation were chosen from defined broths commonly used for microbial culture. The four media initially investigated for yeast isolation were: yeast extractpeptone-glucose (5%, w/v) broth (YEPG) (Quain & Haslam 1979); synthetic wort medium (as for Bacto Wort Agar, Difco - excluding agar); YM

104

A. L. KYRIAKIDES AND P. A. THURSTON

Broth (Difco) and WLN Broth (Oxoid). In addition to these four media, bacterial isolation was evaluated in Brain - Heart Infusion broth (Oxoid), Tryptone Soya Broth (Oxoid), de Man Ragosa and Sharpe Broth (Oxoid), and pasteurized Manns Brown Ale (MBA). Broth (9 m!) was dispensed into each sample cell and autoclaved with the electrode in place, except for MBA which was transferred aseptically to sterile cells. Sample cells were placed in the water bath, linked to the growth analyser and stabilized for 24 h before use. Conductivity of each medium was checked for compatibility with the Malthus instrument and those found to be out of range were corrected with appropriate volumes of a sterile solution of 1% (w/v) NaCI. Dilutions of the microbial cultures were prepared in 9 ml of quarter-strength Ringer solution and 1 nll volumes were used to inoculate the stabilized Inedia to obtain concentrations ranging from 102 - 10 7 organisms/ ml. Samples were monitored for 50 h and curves were analysed subjectively for stability, reproducibility and growth response. Of the media tested YEPG and synthetic wort medium (SYWM) were found to be the most suitable for use with the Malthus instrument. They gave excellent growth responses and stable growth curves. Gram-positive spoilage bacteria (Lactobacillus and Pediococcus) gave good curves with detection times relating to the initial inoculum in SYVV~l but very poor, if any, responses in YEPG. Gram-negative spoilage bacteria (Acetobacter, Gluconobacter and Zymomonas) gave excellent curves in YEPG. Zymomonas would not grow in SYWM but Acetobacter and Gluconobacter did give some weak responses. All of the yeast strains gave good responses in SYM\V and slightly weaker responses in YEPG. YM broth was the only other medium where good growth curves were observed, although bacteria appeared to grow less favourably than yeasts. We found that the conductance changes in SYWM could be improved by adding NaCI to the medium (final concentration, 0.5% w/v) and adjusting the pI-I to 6. Such adjustments had no beneficial effect on responses in YEPG. Overall, the screening experiments showed that YEPG and modified SYWM were acceptable for use as non-selective total count media with the Malthus.

Detection Routine Evaluation Results obtained during the subjective screening of media were used to determine a practicable detection routine that would enable computer analysis to be used for indicating microbial growth more accurately. It was necessary to optimize the parameters to ensure that false detection times would be minimized without cOlnpromising the sensitivity. With the lVlalthus Foodchek routine which allowed data to be analysed with variable detection parameters the following detection routine was chosen: base level, 0.4 micro-Siemens (~S); 1st difference threshold, 0.8 ~S; 2nd difference threshold, 1.0 j.lS; start scan) 10 (i.e. 120 min); scan interval, 12 min.

CONDUCTANCE TECIINIQUES FOR BEER

105

Development of Selective Media Selectivity is primarily required f(Jr brewery samples containing brewing yeasts which may be present at exceptionally high levels in relation to the contaminant being sought (e.g. 107 yeast to one bacterium). Brewery contaminants are broadly divided into three groups; Gram-positive bacteria, Gram-negative bacteria and wild yeasts. Our aim was to develop selective media for the isolation of each of these groups using some of the media already evaluated.

Wild ycast mcdium S1'WM was chosen as one basal medium for wild yeast isolation as most yeasts had previously given good responses in it. In addition, 1'M broth was included for evaluation due to its known ability for supporting growth of a wide spectrum of yeasts (Taylor & Marsh 1984). Two selective agents which permitted the growth of wild yeasts, while effectively suppressing growth of brewing yeasts were chosen for evaluation. These were crystal violet (CV) and copper sulphate (CUS04), both of which have previously been used for similar selective requirements (Longley ct al. 1978; Lin 1981; Taylor & Marsh 1984). Minimum inhibitory concentrations (MIC) ofCV and CUS04 were determined in 1'M and SYWM for a selection of brewing and wild yeasts. Malthus cells containing 9 ml of 1'M or SYWM were prepared with CV concentrations ranging from 6-34 ~!g/ml in increments of 2 ~g/ml and copper sulphate concentrations ranging from 180-270 ~g/ml in 10 ~g/ml increments. Crystal violet was added before autoclaving (1'M at 121°C for IS min; SYWM at 115°C for 15 min) from a stock solution of 2 mg/ml prepared by dissolving I g of crystal violet (Sigma) in 100 ml IMS, filtering this solution through \Vhatman No. 1 filter paper and diluting 1 in 5 with distilled water. Copper sulphate \vas added aseptically after autoclaving from a stock solution of 10 mg/ml CUS04 prepared as previously described (sec plate count techniques). Chloramphenicol (Sigma) prepared by sterile filtration of a stock solution was added to each vial after autoclaving' to obtain a concentration of ISO ~g/ml to inhibit bacterial growth. One ml of a selection of cultures of brewing and wild yeasts was inoculated into 9 ml of incubation medium to obtain approximate concentrations of 10'-101 wild yeast/ml or 105 -107 brewing yeast/m!. Samples were monitored for c. 50 h with the Malthus instrument and the MIC of copper sulphate (as CUS04) and crystal violet for the yeast were calculated. The MIC of crystal violet {()r brewing' yeast ranged from 14- 26 ~g/ml in 1'M and 14-1() ~g/ml in S1'WM. Corresponding MICs of crystal violet for wild yeasts ranged from 6-14 ~g/ml and 6-12 ~g/ml in 1'M and S1'WM, respectively. It is therefore evident that the concentrations of crystal violet required to inhibit brewing yeast would not allow the growth of wild yeasts in

106

A. L. KYRIAKIDES AND P. A. TlIURSTON

both YM and SYWl\1, rendering it unsuitable for use as a selective agent for wild yeast detection. The NlICs of copper sulphate (Cusa.. .) were difficult to determine because of the unstable nature of the conductance curves, probably as a result of the interference of free copper ions on the electrodes. Brewing yeasts were inhibited in YM at euso.. . concentrations ranging fronl 210-220 ~g/ml and wild yeasts by concentrations of 230- 250 !lg/ml. In Sy\VM brewing yeasts 4, whilst wild yeasts were inhibited at levels as low as 180- 21 0 !lg/ml were inhibited at 200 ~g/ml, making the use of euso.. . in SYWM unsuitable. Despite some reservations regarding stability of the conductance curves, YM + eusa.. . at a concentration of 220 ~tg/ml supplemented with chloramphenicol (150 ~g/ml) was chosen as the lllOSt appropriate medium for selective isolation of wild yeast in the presence of pitching yeast and bacteria.

eusa

i\1edium jilr Gram-negative bacteria Gram-negative brewery bacteria grew preferentially in YEPG, and this was therefore chosen as the basal medium. Gram-positive spoilage bacteria did not grow in YEPG so it was not thought necessary to develop a Gram-positive inhibitor. Cycloheximide was studied as a yeast inhibitor and the NlIC for brewing yeasts was in the range 0-100 ~!g/ml. The effect of cycloheximide on the growth responses of Gram-negative brewery bacteria in YEPG was assessed at the same time. Cycloheximide was prepared as a 10 mg/ml stock solution by dissolving 1 g in 100 ml distilled water and autoclaving at 121°C for 15 min. l\lalthus cells containing 9 ml of YEPG supplemented with the appropriate cycloheximide concentrations were autoclaved at 121°C for 15 min. One ml of each organism was inoculated into 9 ml of YEPG plus cycloheximide to obtain concentrations of c. 10 1 - 103 bacteria or c. lOs - 10 7 brewing yeast/ml. Samples were monitored for t. 50 h using the Malthus instrument and NlIC of cycloheximide was calculated for the yeasts tested. The MIC of cyclohexinlide for brewing yeast in YEPG ranged from 15 to 25 !lg/ml. In the majority of samples containing brewing yeast, a long period of constant base-line drift was noted on the conductance curves. This was probably associated with the gradual sedimentation of yeast and limited growth before inhibition. Cycloheximide at a concentration of 100 ~g/ml appeared to give more stable curves than lower concentrations. Therefore it was decided to usc YEPG plus cycloheximide (100 ~g/ml) for the selective isolation of Gram-negative bacteria and there was little effect on detection times of Gram-negative bacteria at this concentration of cycloheximide (Table 1).

CONDUCTANCE TECIlNIQ,UES FOR BEER ·L\HI.I. I. Till' 1'(1;'1'1 o(l'j'doileximitll'

1111

107

lile deln/ill1l liml's o/Gram-lll'ga/ivl' bat/tria ill Ytas/ ex/raclpep/olle-'glu/1Jse brolh therage difference from control detection time* (h) at cycloheximide concentration (~lg/l1ll)

Organism or genus

15

25

40

100

Ail'/obl/cll'r Glu({)/lIJbaclcr EII/erobaclcr doawe

1.7 1.I

0

2.5

2.1 1.7

1.0 0.9

L2 1.0

0.8 0.0

0.0 L2 0.0 0.0

7}II}IOmmlliS

* Calculated hy suhtracting the DT for samples containing cycloheximide from the DT of the corresponding control (i.e. where no cycloheximide was added).

Medium jilr Gram-positive bacteria Gram-positive brewery bacteria were shown to grow preferentially in SYWM and so this was used as the basal medium. Cycloheximide was again studied as a yeast inhibitor. The MIC of cycloheximide for brewing yeasts and the effect on Gram-positive brewery bacteria was determined as previously detailed for the medium for Gram-negative bacteria, SYWM also allowed the growth, in some cases, of Gram-negative bacteria such that the development of a Gram-negative inhibitor was additionally required. Three inhibitors were initially tested in SYWM: 2-phenylethanol (I mg/ml), streptomycin (10 mg/ ml) and thallous acetate (1 mg/ml), Streptomycin and thallous acetate had destabilizing inlluences on conductance curves, causing false detection times but 2-phenylcthanol had no adverse effect. 2-phenylethanol was therefore studied for use as a Gram-negative inhibitor by determining the MIC for a selection of Gram-negative brewery bacteria in SY\VM in concentrations of 1.0, 1.5, 2.0 and 3.0 mg/ml. The effect of the inhibitor on the growth responses of Gram-positive brewery bacteria was also assessed. 2-phenylethanol (100 mg/ml in distilled water) was added to the medium before autoclaving at 12l °C for 15 min. One ml of the appropriate culture was added to 9 ml of medium to obtain levels of c. 10 1 _IO J Gram-positive bacteria/ml or c. 102 IO-l Gram-negative bacteria/m!. The MIC of cycloheximide for brewing yeast in SYWM ranged from 25-40 rtg/ml. The detection times of Gram-positive spoilage bacteria were not significantly affected by levels of cycloheximide up to 100 rtg/ml Crable 2). As for the medium for Gram-negative bacteria, conductance curves were more stable at higher concentrations of cycloheximide.

108

A. L. KYRIAKIDES AND P. A. THURSTON

TABLE

2. The ejfict of tJ(loheximide 011 the detection times oj Gram-positive bacteria in

~yntheli(

wort medium Average difference from control detection time (h) at cycloheximide concentration (!lgl mt) Organism or genus

Lat"tobaciJ/us (L3) Lactobacillus (L4) Lactobacillus brevis (Ll) Pediococcus

TABLE

15

25

40

100

2.0

2.1 2.0

4.0 2.0

0.0

2.0 2.1 0.0

0.0

0.8

2.4

1.2

0.0 0.4

1.7

3. The ejfict of2-phmylethal101 on the tktection times of Gram-positive bacteria il1 wort medium

~)!nthetic

Average difference from control detection time (h) at 2-phenylethanol concentration (j.Ag/ml) Organism or genus

Ltutobacillus Lactobacillus Lactobacillus Lactobacillus Pediococcus

brevis (L2) sp. (L3) sp. (L4) brevis (L 1)

1.0

1.5

2.0

3.0

4,4 1.6

8.2 1.4

16,4 14.7

No grO\\1h No gro\\1h

4.0

4.6

2.6

12.8

3.9

4.6

7.S

25.8

0.0

1.0

2.2

2.4

2-phenylethanol inhibited Gram-negative spoilage bacteria (Acetobacter, GluconobaaerandZymomonas) at 1 mg/ml but Enterobaaercloacae was unaffected at levels as high as 3 mg/m!. Gram-positive brewery bacteria were mostly inhibited at 3 mg/ml of 2-phenylethanol) and concentrations of 2 and 1.5 mg/ml also caused unacceptable lengthening of detection times (Table 3). Although 1 mg/ml also increased the detection times of Gram-positive bacteria (Table 3)) the increase was deemed acceptable by us. Selective isolation of Gram-positive brewery bacteria was therefore based on the use of SYVVl\tl supplemented with cycloheximide (l 00 ~g/ ml) and 2-phenylethanol (1 mg/ ml). The possible growth of Ellterobacter cloacae and other related organisms in the Gram-positive selective medium was not thought a serious problem as such organisms are infrequently encountered in brewery samples. As a result of these development studies, five media were chosen for selective and non-selective isolation of brewery micro-organisms (Table 4).

109

CONDUCTANCE TECilNIQUES FOR BEER T\BI.L

of. Scll'di,,£' IIl1d

/11111/ Wlilit

Basal mediulll

lI/l'dili dl1'doped .IiJr limper)' .Iidd Iria/

Inhibitor(s)

Application

Synthctic wort medium

None

Total count medium (in conjunction with YEPG)

Yeast extract·,· pcprone--glucosc broth

None

Total count medium (in conjunction with 5\"V1\1)

Synthetic wort medium

Cycloheximide (100 pg/ml) + 2-plH'nylethanol (1 mg/ml)

Gram-positi\'e bacterial selectiye medium

Yeast extract,·· pq)((lI1e-glucose broth

Cycloheximide (100 llg/ml)

Gram-negatiye bacterial selective medium

1'\\ broth (Difco)

Copper sulphate (220 llg/ml) + chloramphenicol (ISO !Ig/ml)

Wild yeast selective medium

Field Trial The media developed for the isolation of brewery micro-organisms Crable 4) was studied in detail with the Malthus instrument in a brewery quality control laboratory to determine relationships be!\Veen plate counts and detection times. Methods adopted for the examination of the samples are shown in Table 5. All experiments were carried out with negative controls of Malthus media inoculated with filter-sterilized beer to ensure that conductance curves were true growth and not merely electrical drift. Malthus detection times were determined both by the growth analyser based on the detection routine and by visual assessment of the growth curves. There appeared to be a tendency for the former to be slightly less reliable than the latter due to the Malthus assessment being based on a rigid detection routine. It is evident that tile detection routine could be improved by modifying the detection parameters and it was therefore thought more representative if results were expressed based on the visual assessment. Percentage agreement tables were produced for each category of Malthus medium to determine the reliability of the technique by comparing the !\Vo metllods solely on the basis of detection/non-detection ofcontaminants (Tables 6 and 7). Percentage detection of contaminated samples by each technique was calculated on the assumption that Malthus-positive/plate-count-negative results arose due to the increased sensitivity of the Malthus instrument whilst Malthus-negative/plate-count-positive results arose because of the greater reliability of the plate count technique. Scatter diagrams were also drawn and correlation coefficients calculated for results from the individual categories of media that were Malthus-positive/plate-count-positive in order to assess

110

A. L. KYRIAKIDES AND P. A. THURSTON TAI3LE

5. Techniques uSt'd for the comparative field trial

Conventional plate count method

Sample (a) Selective isolation Beer ex-fermenter Pitching yeast slurry Cask beer Rough beer

Malthus method

I ml inoculated into 9 ml selective media

0.1 ml spread plate on selective media

I

(b) Total COUlll isolatiou Filtered beer pasteurized

Membrane filtrationincubation on TeM

Membrane filtration filter inserted into 10 ml total count medium

Filtered beer unpasteurized

Membrane filtration or 0.1 ml spread plate on TeM

Membrane filtration or I ml inoculated into 9 ml total count medium

Sterile re-processed beer

Membrane filtration or 0.1 ml spread plate on TeM

Membrane filtration or I ml inoculated into 9 ml total count medium

TABLE

]Halthus growth medium

6. Perctmtage agreement be/ween the ,:vIalthus mid plate t'Ounts for total viable counts it] brewery samples

No. of samples analysed

YEPG

49

S\Wi\l

54

1\-1althu5 positive Plate count positive (%)

1\hlthus negative Plate count negative (%)

lVlalthus positive Plate count negative (01<,)

I\lalthus negative Plate count positive (%)

52.9 51.9

30.6 33.3

4.1 5.6

6.1 9.2

Percentage agreement (+ + and - -) was 89.8% and 85.2% for YEPG and s\r'VM respectively. Percentage detection of contaminated samples using !\lalthus was 91.2% and 91.7% for YEPG and SYW1'v! respectively, whilst the corresponding percentage detection using plate counts were 94.1% and 91.7%. Malthus-positive results were determined by visual assessment of data rather than l\1althus detection times, for reasons given in the te:\1.

accurately the potential of using Malthus detection times to estimate microbial contamination (Figs 1-4). Total count medium

There was good agreement between conventional plate count methods and the Malthus method when the total count media were used (YEPG plus

'f.·\BLE

7. Penentar,e agreement between the Afalthus results and plate counts jin the detcaion of organisms in brewery samples No. of samples analysed

Malthus positive Plate count positive

Gram-negative organisms" YEPG + cycloheximide Gram-positive organismst SYWM + cycloheximide + 2-phenylethanol

Malthus growth medium

Wild yeasts! MYGP + CUS04 + chloramphenicol

Malthus positive Plate count negative

(%)

;Vlalthus negative Plate count negative ('Yo)

(%)

l\lalthus negative Plate count positive (o/., )

102

24.5

48.0

21.6

5.9

81

12.3

70.4

13.6

3.7

63

12.7

49.2

33.3

4.8

,. Percentage agreement (+ + and - -) 72.5 %; percentage detection of contaminated samples using Malthus = 88.7%; percentage detection of infected 58.5%. samples using plate counts t Percentage agreement (+ + and - -) = 82.7%; percentage detection of contaminated samples using Malthus = 87.5%; percentage detection of infected samples using plate counts = 54.2%. ! Percentage agreement (+ + and - -) = 61.9%. Percentage detection of contaminated samples was not calculated due to dle negative controls showing detection times on the Malthus indicating that Malthus-positive/plate-count-negative results were false detections rather than increased sensitivity of the Malthus technique.

112

A. L. KYRIAKIDES AND P. A. THURSTON

6 :=-5

E

~

~4 +-'

c:

:::)

8 3 ~ ctl

0. 2 o

0)

o

1

-J

i l__.. ~.~~ 10

5

15

20

25

30

35

40

45

50

No Malthus detection time Malthus detection time (h) FIG. 1. The relationship between Malthus (01') and plate counts for brc\very samples, using YEPG as the Malthus medium. Correlation coefficient (r) = -0.41.

61 I

E

-

5

:J ~

4

+-'

C ::J

0

u

3

~

ctl

0. 2 ;?

C)

0

-J

1j

I

L___, 5

10

15

20

25

30

35

40

45

50

No Malthus detection time Malthus detection time lh)

2. The relationship between l\lalthus (D1') and plate counts for brewery samples, using SYWM as the Malthus medium. Correlation coefficient (r) -0.39.

FlU.

CONDUCTANCE TECIINIQUES fOR BEER

113

6

E

5

:J

:§ 4

....C :J

0

()

3

Q)

ro a. 0

2

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114

A. L. KYRIAKIDES AND P. A. THURSTON

SYWM) (Table 6). In addition, the percentage detection of contaminated samples using the two Malthus media was only slightly lower than that of plate counts, illustrating the reliability and rapidity of the Malthus method as the latter gave results within 48 h as opposed to 3 - 4 days. Scatter diagrams of infected samples for YEPG and SnVM (Figs I and 2 respectively) revealed very poor correlation between detection times and plate counts. Such results give little hope of using detection times to determine initial contamination levels or even categorizing contamination into acceptable, borderline and unacceptable bands. The wide degree of variation in detection times relative to plate counts could probably be attributed to the presence of a mixed microflora in the brewery sanlples. The total count media have the capacity to support the growth of a wide range of yeasts and bacteria. Consequently, a variety of growth rates and tnodes of metabolism in anyone conductance cell will lead to an overall detection time which cannot be accurately correlated with the initial level of contamination or the type of contamination in the sample.

Medium ftr Gram-negative bactfn'a The selective Gram-negative medium (YEPG plus cycloheximide) had a relatively low percentage agreement with plate counts because of the high number of samples registered as contaminated by the former and not the latter method (Table 7). These may have arisen due to the increased sensitivity of the Malthus technique (using 1 ml as opposed to 0.1 ml samples by the spread plate method) rather than false detection times, as negative controls revealed stable conductance traces. The percentage detection by the Malthus medium was therefore much greater than conventional methods, although correlation between the two sets of results for contaminated samples was again poor, removing the possibility of categorizing levels of infection based on detection times (Fig. 3). In this instance it seems likely that poor correlation resulted from the presence of brewing yeast together with variable growth rates of the bacteria isolated. Although brewing yeast in the samples is inhibited by cycloheximide, it is possible that the physical presence of such high levels of yeast (105-1 06; mt) may have suppressed bacterial gTowth. Cycloheximide exerts its effect quickly but not instantaneously, therefore allowing the yeast in the selective category of samples Crable 5) to continue to metabolize for a short period with consequent oxygen uptake and carbon

dioxide .release. The resultant micro-aerophilic/anaerobic conditions could suppress the growth of sonIC Gram-negative bacteria as many of those occurring in brewery samples are strict aerobes. Therefore, with several factors (i.e. CO 2 release, O 2 uptake, differential growth rates) in existence which have a

CONDUCTANCE TECHNIQUES FOR BEER

115

bearing on conductance it is perhaps not surprising that the correlation between detection time and plate count is poor.

Medium fOr Gram-positive bacteria The effIciency of selective Gram-positive medium (SYWM plus cycloheximide plus 2-phenylethanol) was difficult to assess accurately as the high percentage agreements (Table 7) merely retIect the high numbers of samples that were not contaminated and were registered negative by both the Malthus and plate count methods. The few positive results were statistically analysed but were not sufficiently representative for useful conclusions. It is evident however, that samples containing less than c. 50 bacteria/ml are, in general, not detected within 50 h by the Malthus method but levels greater than this are detected in 30 h or less (Fig. 4). Further investigations with more brewery samples are required to confirm such relationships.

Wild yeast medium Detection of wild yeast with the Malthus technique was highly unreliable Cfable 7) as negative controls containing no micro-organisms appeared to give numerous false growth curves. Results that were recorded as Malthus positives may therefore have been false positives, thus precluding any statistical analysis of the results. We presume that the false positive results arose because of interference of copper ions on the electrical stability of the medium, as previously noted. Stability could not be improved by the addition of 0.1 % (w/v) agar to dle medium in subsequent experiments. Discussion At present the Malthus technique cannot be recommended for use with the media developed in place of plate count methods for the estimation of microbiological contamination of brewery samples because of the poor relationships existing between plate counts and Malthus detection times. The Malthus method, together with the total count media developed for the system (yEPG and SYWM) does allow reliable and fast detection «48 h) of microbial contamination at levels ranging from as low as 2 organisms/m!. It can therefore be recommended for use with brewery samples where results indicating the presence or absence of contaminants rather than actual counts are required (e.g. pasteurized beer, sterile re-processed beer, where microbiological specifications are for zero organisms per sample). In addition, the Malthus method with YEPG and SYWM would be useful for activities such

116

A. L. KYRIAKIDES AND P. A. THURSTON

as monitoring post-pasteurizer samples after a suspected pasteurizer breakdown or during pasteurizer commissioning. The application of the Malthus method with selective media, for the isolation of brewery spoilage micro-organisms was less successful. Gramnegative and Gram-positive bacteria could be detected only in their respective media (yEPG + cycloheximide and SYVVM + cycloheximide + 2phenylethanol) within 48 h at levels greater than 1 x 102/ ml and 5 X 10 I ; ml, respectively. As quantification of contamination based on detection times is not possible, such methods would be useful only ifmicrobiological specifications for samples requiring selective bacterial isolation were in the range of 10-10 2 organisms/ml. Any detection time less than 48 h would represent sample failure and the sample would be rejected but no detection would represent an acceptable sample. Since one invariably requires some degree of quantification for selectively detecting contamination, such a procedure is not fully acceptable. One point in favour of the Malthus method, however, is that as it is a growth-dependent technique, some preliminary identification (by microscopy/ Gram stain) can be made on the contaminants. Wild yeast detection with the Malthus using MYGP + CUS04 + chloramphenicol was unreliable and therefore unsuitable for use as a selective medium because of the unstable nature of the conductance traces. If the Malthus method is to become a working system to replace plate counts as a method for microbiological analysis of brewery samples it is evident that quantification is of utmost importance. The results and the experiments detailed here suggest that such a requirement would appear to be extremely difficult to achieve because of mixed populations of micro-organisms. Quantifying contamination could theoretically be achieved by developing highly selective media for species of known sinlilar growth rates and metabolism although the occurrence of shocked organisms with retarded growth rates may present an additional problem. Ideally, defined media for a microbial contaminant should be developed with knowledge of the appropriate metabolic pathways so that the metabolism of a simple substrate will give rise to a known conductance change (Owens 1985). Such conductance changes could then be maximized by optimizing the buffering system of the medium (Owens & Wacher-Viveros 1986) therefore improving sensitivity and selectivity. It is apparent, however, that such an approach to brewery samples, especially those where brewing yeast may be present at up to 10 7/ ml, would incur great difficulties and may result in a requirement for a large number of selective media due to the range of contaminants being sought. Certainly the Malthus tnethod is an accurate conductance monitoring system and we have achieved partial success in applying it to a wide range of brewery samples. Ultimately, the success of this system in the brewing industry will depend on overcoming, through media design, the problems of mixed

CONDUCTANCE TECHNIQUES FOR BEER

117

populations of often slow-growing organisms and 'interference' from a large population of brewing yeast.

References EI.\I\S, II.A.Y. 1982. A note on two uses for impedimetry in brewing microbiology. Joumlll of Applied Hlilieriolo!-.'Y 53, 423 --426.

I lOUGH, J.5., BRIGGS, D.E., STEIENS, R. & YOUI\(;, T.W. 1982. l\licrobial contamination in breweries. NIlillillg [;) Brewillg Seimil', Vol. 2, Hopped IVllri IIl1d Beer, PI'. 741-775. London: Chapman and [[all. KII.t;OUR, W.J. & D.n, A. 1983. The application of new techniques t
0/ IiiI' fllslilllll' o/Hrl'JI'illg 87,

151-154.

LONGI.EY, R.P., DENNIS, R.R, IIEYER, l\IS & WREI\, J.J. 1975. Selective SlIa"hllrolllya!s mcdia containing ergosterol & Tween SO. Jillimal IIlllie IlIslilule ofBrflvillg 84, 341-345. O\\EI\S, J.D. 1985. Formulation of culture mcdia f
RICHARDS, j.C.S., JASON, :\.C., [IOIIIlS, G., GillSON, D.l\!. & CHRISTIE, R.I!. 1975. Electronic measurement of bacterial growth. Jillimal III' Plijlsi{s E, Sdnlfi{ic IIISlrllllll'1llS II, 560- 568. SH,\\\", S., 1987. Microbiologicallllonitoring in the brewery. Brfll'ers' GlIllrdil/ll 116, 10-13. T'\YI.OR, G.T. & M,\KSH, A.S. 1984. 1\I1'GI' + copper, a medium that detects both SlIa"hlirOlIlYcl's and non-Saec!llIrtJlII,l'(CS wild yeasts in the presence of culture yeast. JlllImlll II/Ilie IllSlilllll' III BrelPillg 90, 134-145.

UR, A. & BRO\\"N, D.F.]. 1974. Rapid detection of bacterial activity using impedance measurements. Hiollll'diw! EIIgilll'Crillg 9, IS- 20.

Electrical Methods for Water Quality Testing T. E. IRVING,1 G. STANFIELD l AND B. W. T. HEPBURN 2 * J Water Research Centre, Henley Road, Medmenham, PO Box 16, Marlow, Buckinghamshire SL7 2HD, UK; and 2 Wessex Water, Regional Scientific Centre, Mead Lane, Saltford, Bristol BS18 3ER, UK

Electrometric growth analysers are now used in the food and dairy industries for a wide range of tests, but at present they have not found similar use in the water industry. This is partly because the instruments have not yet been approved for water testing by regulatory authorities and also because of a lack of information about comparability with existing standard methods. Nevertheless, interest has been shown in tllese instruments within the water industry, and the potential to handle large numbers of routine samples may be of particular use in complying with increased levels of monitoring required by various EEC Directives (e.g. Anon. 1976). In this chapter, metllOds are outlined which have been successfully used fiJr testing various types of water with both the Bactometer and Malthus instruments. There are three considerations; the first two concern methods which have been used with the Bactometer system at the Water Research Centre (WRC), and the third deals with the development by Wessex Water of a test for drinking water with the Malthus system. It is emphasized that the methods described have no standing as approved procedures, although they will be considered for inclusion in future revisions of The Bacteriological Examination of Drinking Water Supplies (Anon. 1983). The history, theory, practice and available electrometric instrumentation have been covered comprehensively by Richards et al. (1978) and by Firstenberg-Eden & Eden (1984). The advantages to water authorities of electrometric testing can be briefly summarized as follows:

* Present address: Aurora Scientific, 6 Wilmington, Newton St Loe, Bath, Avon BA2 9JB. Co/,yriKht Rapid .\Iicriobiological Methods li,r Foods, Beverages and Pharmaceuticals

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120

T. E. IRVING ET AL.

1 A more rapid availability of results allows a quicker operational response to water supply failures. 2 A simpler and more economic method of handling routine samples permits a greater throughput of work. 3 Complete integration with laboratory data handling systems allows for quicker handling and transfer of data.

Conventional Water Tests Most bacteriological tests carried out on water samples are intended to enumerate or show the presence of faecal indicator bacteria. Tube dilution (Most Probable Number) assays have been largely (although not completely) superseded by membrane filtration procedures, and they give a presumptive count of coliform bacteria in 18 h. Further tests arc then needed to confirm the identity of the presumptive colonies, or to check specifically for the presence of Escherichia coli. The membrane test selects bacteria on the basis of their tolerance of bile salts or other synthetic surface-active compounds, and their ability to ferment lactose to produce an acid reaction. After a 4-h resuscitation period at 30°C, these tests are carried out at 37°C for total coliforms or at 44°C for thermotolerant colifonns. Confirmatory tests require the production of acid and gas from lactose within 48 h at 37°C (total coliforms) or 24 h at 44°C (thermotolerant coliforms and Escherichia coli), and the production of indole from tryptophan within 24 h at 44°C (E. coli). The procedures are described in detail in Anon. (1983). Different techniques for coliform organisms in water result in different definitions of coliform bacteria. The current definition in Report 71 (Anon. 1983) relies on the fermentation of lactose and the production of gas. Many workers now accept that this type of definition is restrictive in practice, and excludes the weakly fermenting and anaerogenic strains which are also likely to be associated with chlorine damage.

Use of the Bactometer MI23 for Environmental and Recreational Water Samples Where numbers of coliform organisms are high enough for 1- ml sample volumes to be sufficient, the Bactometer disposable culture modules provide a convenient means of setting up large numbers of tests in a short time. At \VRc's laboratories, in extensive tests of growth media selective for coliform organisms, two have consistently given reliable results, good curve quality and rapid detections. These arc 0.1 % sodium dodecyl (lauryl) sulphate (SDS) medium, and the C1\1 medium developed for the Bactometer in the USA

ELECfRICAI. 'vlETI-lODS FOR WATER

121

(Firstenberg-Eden & Klein 1983). Both are best used at double strength, 1 ml of sample being added to an equal volume of medium. The 0.1 'Yo SDS medium is the same as membrane lauryl sulphate medium (Anon. 1983), and the double strength formula is (gil): Peptone (Oxoid L37), 80; lactose, 60; Yeast Extract Powder (Oxoid), 12; sodium dodecyl sulphate (BOH 44244), 2; phenol red, 0.4. The medium is adjusted to pH 7.7 - 7.8 and sterilized at 121°C for 15 min. The reaction after sterilization should be c. pH 7.4. Membrane Lauryl Sulphate Broth (Oxoid MM 615) may be used instead. Double strength eM medium contains (gil): Proteose Peptone No. 3 (Di1Co), 20; Yeast Extract Powder (Oxoid), 12; lactose, 40; sodium dodecyl sulphate, 2; sodium deoxycholate, 0.2; bile salts No.3 (DifCo), 2; bromocresol purple, 0.07. The medium is adjusted to pH 6.8 and sterilized for 15 min at 121°C. The complete medium is available in dehydrated form from Vitek Systems Ltd, I-Ienlcy-on-Thames. The Bactometer M123 ofTers the choice of measuring changes in the conductance, capacitance or total impedance of each test culture. Of these, the conductance signal is of least use for detection of coliform organisms, as it shows small overall signal changes, and later detections than the other two signals. The capacitance signal shows the greatest overall changes, but the curves exhibit a certain amount of 'noise' and instability. Therefore the impedance signal has been found the most reliable for usc with the growth media described above. The instrument's standard detection algorithm is satisfactory without modification. Incubation at 37°C has proved most satisfactory. Tests carried out at 44°C may give more rapid detections with samples containing large numbers of thermotolerant coliform organisms or E. coli, but the additional selective pressures of the elevated incubation temperature can lead to erratic results, particularly in samples containing less than 10- 20 coliform organisms/m\. A resuscitation period at a lower temperature docs not appear necessary, as it is usually less important for liquid cultures than for colonies growing on membrane filters. If a resuscitation step is required it would have to be done before the cultures were placed in the Bactometer, as any change in incubation temperature during the test would afTect the impcdance of the culture and might give a false detection. When the test is complete a visual check on the culture modules will give an additional reassurance that any positive detections have been caused by lactose-fermenting organisms. With both the media described ahove, a colour change to yellow indicates acid production.

Derivation of quantitative results jrom impedance detection times If samples are tested by both a standard technique and an electrometric test, then a calibration curve can be constructed which allows prediction of bacterial

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numbers from a detection time. This exercise should be carried out with caution, as a technique which measures metabolic activity need not reflect physical numbers of bacteria with any accuracy. Nevertheless, detection times do vary inversely with the logarithm of the initial inoculum, and Fig. 1 shows a calibration plot for 258 samples of various types of water. Here 0.1 % SDS medium was used in the Bactometer, and the impedance detection times are plotted against the presumptive total coliform count by membrane filtration using membrane lauryl sulphate broth. Extrapolation of the line suggests that one colifonn organism would be detected in about 10 h, but in practice detection times are often longer than expected at these low levels. This is possibly because the bacteria in such samples 'Ire exposed to environmental stressing factors which render them less metabolically competent, but sampling errors will also contribute to the uncertainty. Thus, quantitative results are unreliable at levels below about 10 organisms per test volume. It is also possible to perform a quadratic rCgTession analysis using the Bactometer instrument software, that is, to fit a curve to the points, rather than a straight line. This frequendy fits the data points better at the lower levels, but extrapolation to long detection times is then even more hazardous owing to the parabolic nature of the curve.

ELECTRICAL METHODS FOR WATER

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Use of the Bactometer M123 for Potable Water Samples With potable water samples it is necessary to detect a single coliform organism in 100 ml of sample, and the most obvious way to extend the sensitivity of electrometric instruments is to incubate correspondingly larger volumes of sample. Other strategies are possible, such as non-selective pre-enrichment or concentration procedures, but these usually diminish the practicality or costeffectiveness of the test. The Bactometer system is capable of using large-volume re-usable culture bottles, which are incubated remotely from the instrument in purpose-made bottle baskets. These baskets have suitable electrical connectors for the stainless steel electrodes fitted to each bottle cap, and are themselves connected to the Bactometer processing unit by ribbon cables. The largest bottles have a capacity of 250 ml, allowing 100 ml of sample to be added to an equal volume of medium. This permits a similar test mixture to be made as with the disposable culture modules, but of 100 times greater volume, and the theoretical limit of sensitivity is thus one organismllOO m\. The bottle electrodes have been found to give excellent results, but unfortunately they are no longer being supplied in the UK. No advantage has been found with double strength medium with the Bactometer bottle electrodes and the medium eventually chosen was formulated to give a final incubation mixture containing sodium dodecyl sulphate at a concentration of 0.1 % (wIv), and all other ingredients at half the strength of normal membrane lauryl sulphate medium. The medium added to the bottles therefore contains (gil): Peptone (Oxoid L37), 40; lactose, 30; yeast extract powder, 6; phenol red, 0.2; sodium dodecyl sulphate, 2. The medium is adjusted to pH 7.7 - 7.8 and sterilized for 15 min at 121°e, and the final reaction should be about pH 7.4. Membrane Lauryl Sulphate Broth (Oxoid MM 615) may be used, made up at single strength with an extra gram of sodium dodecyl sulphate per litre added before sterilization. An equal volume of sample is added to 100 ml of medium, the bottles are incubated at 37°C, and again the impedance signal is recommended. Few tests have been carried out with CM medium with the bottle electrodes, but it appears to be quite satisfactory. However, 0.1 % SDS medium has been used in preference throughout the trials as it is similar in composition to the medium used in the membrane filter test for coliform bacteria in the UK (Anon. 1983). It is felt that the use of an existing standard medium would improve the acceptability of the new technique to the water industry. Specifici~y

of large-volume tests

With the very low levels of coliform bacteria that are likely to be encountered in watcrs of good quality, calibration of thc tcst is not feasible, and the

124

T. E. IRVING ET AL.

electrometric instrument is best used merely to indicate presence or absence of detectable growth. In this case, however, it is important to know the specificity of the test for the particular organisms sought. Therefore, in a series of tests the contents of bottles giving positive detections were subcultured to solid medium for purification. In many cases a single type of organism predominated, but in some, two or more components were isolated. The conventional confirmatory tests used in water analysis, as described above, were used. The production of acid and gas from lactosc, and indole from tryptophan, both within 24 h at 44°C, were taken to con finn the presence of E. coli. Cultures not confirmed by these tests were identified with the API 20E system, as wcre any cultures that gave ambiguous results. From the results of a limited number of tests it was observed that the presence of E. coli in a water sample nearly always corresponded with a detection in the Bactometer occurring within 12 h of inoculation. In a series of SO samples, from various stages of a water treatment works and from a number of raw water sources, 33 gave a detection in the Bactometer instrument. Of these, 16 were shown to contain E. coli, and all but one were detected before 12 h. Of the remaining 17 where organisms other than E. coli had caused the detection, only one was detected before 12 h. Thus, for this limited number of samples, the presence of E. coli could be inferred from the detection time with reasonable accuracy, although a similar cut-off time to distinguish detections from coliform and non-coliform organisms could not be seen. As the presence of E. coli in a potable water sample is a highly significant finding, this arbitrary cut-off time of 12 h was used to assess the agreement between Bactometer results and those from tests for thermotolerant coliform organisms and E. coli by the membrane filter method. Table 1 shows the results from 48 samples from a variety of raw water sources. IIere a positive result with the Bactometer means that a detection occurred at or before 12 h, and a negative result means that either there was no detection, or that it occurred after 12 h. For the membrane tests, a confirmed positive means that E. coli was present, while a presumptive positive indicates that acid-producing colonies were present on membranes incubated at 44°C, but that none of these subsequendy proved to be E. coli. A negative n~sult means that no acidproducing colonies were obtained. If rows 1, 3, 4 and 5 from Table 1 are combined, 50% of the samples gave at least presumptive positive colonies in the membrane test, and the Bactometer also gavc 50% positive results (rows 1+4+6). There was not complete agreement, however, with 6 (Yo of samples showing at least a presumptive positive membrane result, but being missed by the Bactomcter (rows 3 + 5). Also 6 % gave a positive electrometric test but a negative membrane result (row 6). Such discrepancies are inevitable with samples which are at the

125

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limit of detection by both methods, and while it may be feasible to use ekctromctric instruments for testing drinking water, it is clear that CA1ensive comparisons with conventional techniques under operational conditions would be needed before a standardized method could be devised. Use of the Malthus Microbial Growth Analyser for Potable Water Analysis

Equipment The Wessex Water Authority system is a Malthus 112L capable of handling up to 224x lOO-ml samples, spread over eight thermostatically controlled water-filled incubators. It is interfaced with the laboratory's Series 1 IBM laboratory data handling system, allowing full sample data transfer. It is a standard production instrument with no modifications for water analysis. Software has been developed to suit the Authority's needs in the transfer of sample data, the program being identified as 'A(~UATICS'.

Detection medium The most important factor influencing the detection of bacterial growth using conductance measurement is the choice of culture medium in which the detection is to take place. Conventional culture media do not necessarily give the best response. Figures 2 and 3 show the gro\\>1h curves or conductivity response of E. coli NCTC 9001 in a MacConkey broth base prepared with different nutrients. They clearly demonstrate the importance of medium formula with this technique. Good conductivity response simplifies the choice of detection routine parameters, which in turn improves the accuracy of the system.

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ELECTRICAL METHODS FOR WATER

127

The final formula of the M20 medium developed for the detection of coliform organisms in potable waters is (gil): lactalbumin hydrolysate, 5; yeast extract, 2.5; sodium chloride,S; sodium dodecyl sulphate, 0.4. The medium is prepared as a 10 times concentrate, and is adjusted to pH 6.9 before distribution in lO-ml volumes to IOO-ml Malthus cells, which are then sterilized at 121°C for 5 min. When it is cooled to ambient temperature and below, the sodium dodecyl sulphate (SOS) precipitates, and the cells must be warmed to redissolve this ingredient before use. It is the author's experience that batches of ingredients vary. This is particularly relevant with respect to the clarity in solution of lactalbumin hydrolysate, where an adverse effect on growth detection has been experienced. The use of bile salts No.3 (Oxoid) as an inhibitor was tried in early versions of the medium, but was replaced by SOS when interference effects were experienced with electrodes. SOS is also a more consistent chemically defined ingredient. The medium contains no lactose or other fermentable carbohydrate, since detection is not dependent on the fermentation properties of the organism. Trials showed no enhancement of detection times when carbohydrates were incorporated in the culture medium. Figure 4 shows typical responses for E. coli, Enterobacter cloacae and Aeromonas hydrophila in M20 medium. Using the conductance measurement technique, a presumptive coliform is defined as an organism capable of a conductance detection response in M20 medium. This would be expected to occur within 12 h for a contamination 200

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128

T. E. IRVINGET AI.

level of one coliform organism per 100 ml, but 18 h should be used as the cut-off point as outlined below in the guidelines for interpretation of detection times.

Method The test is performed by adding 100 ml of sample to a 100-ml Nlalthus cell containing 10 ml of 10 times concentrated M20 coliform medium. The cell is transferred to a Malthus incubator and the cell position is reset. The test temperature is 37°C. On the detection of growth by the previously defined detection routine, the detection time is displayed on the screen. The data can be examined graphically, and close scrutiny of the curve may aid in identification. This is particularly so when dealing with 'doubtful' results showing a detection time in the range 12- 18 h which are usually due to Aeromonas, IIafnia or Serratia species, and occasionally to some strains of Citrobaaer freundii.

Detection times - interpretation of results Comparative studies have been performed on several thousands of samples. On the data produced it is possible to interpret detection times as follows:

0- 6 h: Indicates a very high level of coliform and E. coli contamination, equivalent to > 10000 per 100 mI. This would not normally be found in potable water, and data should be examined for the possibility of erroneous detection due to fluctuating data signals.

6-8 h: High level of coliform and E. coli contamination in the original sample, equivalent to a combined membrane count of > 100 per 100 m!. Immediate action required.

8-10 h: Indicates a medium level of contamination in the original sample, with coliforms and E. coli present, and equivalent to a combined nlembrane count of 10-100 per 100 ml. Immediate action required.

10-12 h: Indicates low-level contamination of the original sample with E. coli unlikely to be present. Equivalent to a total coliform count of 1- 10 per 100 ml. Possibility of positive result due to Aeromollas spp. Growth curves and 1st differential of conductance data should be examined for possible indications of Aeromonas sp.-type growth profile.

12-15 h: Indicates bacterial growth which is unlikely to be due to coliform bacteria. However occasionally coliforms, especially Citrobacter spp., may be

129

ELECTRICAL \lETHODS FOR WATER

present, and this would be equivalent to a total colifonn count of 1-2 per 100 m!' Growth curves and data should be examined for possible indications of coliform response.

15 -18 h: Indicates bacterial growth normally due to Aeromonas spp. and other aquatic bacteria. Very very occasionally a late coliform response may be detected. Growth curves and data should be examined for confinnation.

> 18 h: No coliform bacteria present in 100 ml of sample. The conductance measurement technique relies on a single 100-ml sample at a single incubation temperature, instead of two volumes of 100 ml at dual temperatures as in the membrane filtration technique. The detection time can be used as a confirmation of the presence of E. coli as outlined in the interpretation guidelines. All positive Malthus cells can be confirmed by conventional methods by transferring portions of the medium in the positive cell to confirmation media, and to MacConkey and yeast extract agars for identification with the API ZOE system.

TABLE

2. Summary Of results oj'wmparalive tests using the Malthus 112L systnl1 alld t1mvmtio/1{i! Ilwnbrtlllc filtratioll

Number

'X,

Ncgative on both systcms Positive on both systcms Positive membrane only Positive Malthus only Presumptive Malthus only Prcsumptive membrane only Presumptive both systcms Confirmed Malthus, presumptivc mcmbranc Confirmed membrane, presumptive l\lalthus Electrical problems interfering with, or giving, a dctcction

662

91.4

Total samples analysed

724

Group I 2 3 4 5 6 7 8

9 10

24 7 4 4

3.3

(,

0.95 0.6 0.6 0.85

I

No. of raw samples

No. of treated samples

23 5 2

2 2

1

0

4

3

3

0.1

1

0

0

0

0

0

4

0.6

4

0

12

1.6 56

668

100

Groups I, 2, 5, 6 and 7 show true comparability (%.25'X,). All membranc results arc based on a total sample volume of 200 m!' All \lalthus results are based on a total sample volume of 100 ml.

130

T. E. IRVING ET AL.

Comparabili~y

Table 2 shows a summary of the results of 724 samples which were tested by both methods. The membrane technique used was that outlined in Report 71 (Anon. 1983), and the culture medium was membrane lauryl sulphate broth. All presumptive positive membranes and all Malthus cells giving a detection time were checked for the presence of coliform bacteria, and all organisms isolated were identified with the API 20E system. It is eA'Pected that full details of the comparative studies will be published in the Journal ofApplied Bacteriology at a future date. Good correlation has been demonstrated, and in the author's opinion this technique would be a suitable method for the analysis of E. coli and colifonn bacteria in treated potable waters. The author accepts that the data presented in Table 2 are insufficient in terms of the number of acmal contaminated samples that were analysed. The conclusion is based, however, on a much larger sample comparison, of which the data sho\\'l1 are a typical example.

Acknowledgement The work carried out at WRc was undertaken for and funded by the Department of the Environment, whose permission to publish has been received. References ANON.

1976. Council directive of 8 December 1975 concerning the quality of bathing water.

Official Joumal of the European Communities, 19, L31/1- L31/7. ANON. 1983. Tile Bacteriological Examination o/Drinking Water Supplies 1982. Reports on Public Health and Medical Subjects No. 71. Department of the Environment, Department of Health and Social Security, and Public Health Laboratory Service. London: HMSO. FIRSTENBERG-EDEN, R. & EDEN, G. 1984. Imped4tw: Jificrobiolob.'Y. Letchworth: Research Studies Press Ltd. FIRSTENBERG-EDEN, R. & KLEIN. C.S. 1983. Evaluation of a rapid impedimetric procedure for the quantitative estimation of coliforms. Joumal of Food Science 48, 1307 -1311. RICHARDS, j.C.S., JASON, A.C., IloHns, G., GIBSON, D.M. & CHRISTIE, R.B. 1978. Electronic measurement of bacterial growth. Joumal of Physics E: Scientific InsJrumenls 11, 560- 568.

A Conductance Screen for Enterobacteriaceae in Foods S. B. PETITT UB. (Ross Youngs) Ltd, Wickham Road, Grimsby, South Humberside DN31 3SW, UK

The aims of this paper are threefold: 1 To demonstrate some possible problems in using conventional and 'special' media for the detection of coliforms in conductance systems. 2 To describe a modified conductance screen for Enterobacteriaceae, rather than coliforms, in foods. 3 To suggest a simple metllOd for reducing test time by increasing the concentration of the micro-organisms in the test cell. For heat-processed foods, a test for members of the Enterobacteriaceae or coliforms may be of value to indicate post-process contamination, or underprocessing. Such a test may also help identify poor plant cleaning, or for example as a hygiene indicator in dairy products (Cooke et al. 1985). The usefulness of tests for Escherichia coli, faecal coli, 'coliforms', 'coliaerogenes' or Enterobacteriaceae in foods generally has been the subject of much debate (see, for example, the exchanges between Howie (1981) Waite (1981) and Mossel (1981». In water, the role of 'coliform' indicator organisms and E. coli is relatively clear. In foods, for example, raw meat, raw vegetables, and fermented foods, considerable care is needed in interpreting the results of tests for these organisms. The rationale for establishing microbiological criteria for indicator groups in these foods is complex. Often, a microbiological survey of products manufactured under good conditions is the only way of establishing a specification or standard. This takes little account of the spoilage potential, or the 'safe' limit for Enterobacteriaceae in various foods. A further problem is that the test used will define the organisms detected, rather than their taxonomic relationship, indicator status, or spoilage potential. The ICMSF (1978) defined coliforms as 'the micro-organisms that are detected by coliform tests'. Clearly, a test method must be specified if microbiological

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S. B. PETITT

criteria are to be effective; obviously this is vital where values for coliforms are included. Cooke et al. (1985) suggested that when liquid media are used in the presumptive coliform test for dairy products, incubation time and inoculum size are more important than the test medium. Overall, there is a general move towards detecting Enterobacteriaceae in foods rather than coliforms (Mossel 1985). The author supports this view.

Rapid Tests for the Enterobacteriaceae If it is to be widely accepted and used, a rapid microbiological method should produce results similar to those of existing standard methods. The alternative is to use an indirect test, which may not directly correlate with conventional methods, hut does, for example, with shelf-life. Conventional broth tests for the detection ofEnterobacteriaceae or colifonns in foods take typically 24-48 h. The most probable number (MPN) test for E. coli enumeration can take up to 96 h (ICMSF 1978). Plating methods are generally faster, 20- 24 h for E. coli (I-Iolbrook et al. 1980) and a similar time for coliforms using violet red bile medium. Thus, to be worthwhile, an alternative rapid method should produce a result in substantially less than 18 h, for levels detected by the normal plating methods, i.e. > 10 cfu/g. To be of real value to a routine laboratory working office hours, 8 h or less to detect a problem sample is a realistic target. In a production unit, where samples may be collected and tested throughout the day, 4 h is a useful target test time. Rowley et ai. (1979) used radiometric methods (Bactec 301) with a 14C_ labelled substrate in EC broth at 42-45°C to detect the presence of faecal coliforms in 18 h. The same workers also developed an impedance (Z) MPN method using the Bactometer 32. Both techniques detected 100 faecal coliforms per gram of food in 10 h. A 3-h resuscitation was needed for these methods to prevent false-negative results. Petitt (1983) developed a conductance screen for Enterobacteriaceae, rather than coliforms) after the discovery that lactose fermentation was not responsible for the initial change in conductance. The medium, Enterobacteriaceae selective broth (ESBBlI) was based upon brain-heart infusion broth. Triton XIOO, antibiotics, tenlperature) minimal headspace, and the detection routine conferred selectivity. Medium details are described in 'Materials and Methods). Firstenberg-Eden & Klein (1983) developed a medium for the impcdirnctric estimation of colifonns. In meat samples 1000 coliforms/g were detected in 6.5 h with a correlation coefficient of 0.90 against a confirmed violet red bile count. The correlation fell to only 0.77 when compared to an unconfirmed

EN1'EROBACTERIACEAE SCREEN

133

count. In routine work presumptive counts may not be confirmed and thus this method could lead to a lower numerical count. Stannard (1984) used this method for the estimation of coliforms in raw lamb but used the conductance (G) and capacitance (C) signals. Correlations were poor, 0.50 for C and 0.78 for G signals. Firstenberg-Eden et al. (1984) used their earlier coliform method and the G signal for dairy cream, ice cream mix, raw and pasteurized milk. Correlations were good: 0.88-0.95. Thus G and Z were capable of giving satisfactory results although the findings of Stannard (1984) may indicate a food-related problem. lIepburn (see Irving et al., this volume) developed a medium for the detection of coliform bacteria in water using the Malthus conductance instrument. A feature of this method is that some indication of the type of coliforms present may be gained from the shape of the conductance curves. Media for the estimation of coliforms or Enterobacteriaceae in foods using the G or Z signal are now commercially available from Bactomatic (Henley) and Malthus Instruments (Crawley). ExpcrimClltal rationale Initial experiments indicated that Malthus Columbia broth (MC) produced a superior conductance signal to brain-heart infusion broth (BHI), used in the original medium of Petitt (1983). In addition, MC contains Tris buffer which may reduce drift (Firstenberg-Eden et al., 1984). Consequently, the BHI in ESBBH was replaced by MC to give modified Enterobacteriaceae selective brotll (MESH). ESBBH has been used for egg, flour confectionery, and dairy cream (Petitt, unpublished results), consequently it was decided to usc the modified medium (MESB) for a wider range of foods, particularly meat and salad vegetables. The original medium was thought to be rather inhibitory; in an attempt to determine if this were true, conductance detection times and, using a solidified form of the new medium, plate counts were compared with other media. Materials and Methods

Modified Enterobacteriaceae selective broth (MESB) Single-strength base: Malthus Columbia broth (Malthus Instruments) 32 g; agar No. 1 (optional) 1 g; Triton X-IOO (Sigma 6878) 2 g; distilled water 1000 m!. Heat and stir to dissolve, dispense into 250-ml or SOO-mt volumes

s.

134

B. PETITT

and then autoclave at 121°C for 15 min. Cool to 45°C. Add 0.5 ml of antibiotics supplement to each 100 ml of single-strength base. Antibiotics supplement: vancomycin hydrochloride (Sigma) 0.002 g; novobiocin sodium salt (Sigma) 0.001 g; distilled water 5 ml. Dissolve in water and sterilize by filtration. Add aseptically to the sterile base above. Double-strength base: Malthus Columbia Broth (Malthus Instruments) 32 g; agar No. I (optional) 1 g; Triton XIOO (Sigma 6878) 2 g; distilled water 500 ml. I~Ieat and stir to dissolve, dispense into 2SD-ml or SOO-m! volumes and autoclave at 121°C for 15 min. Cool to 45°C. Add 1.0 ml of antibiotics supplement to each 100 ml of double-strength base.

Enterobaaeriaceae selective broth (ESBBH) As above with the Malthus Columbia Broth replaced by: Brain - I-Ieart Infusion Broth (Oxoid) 37 g; plI 7.4. Other conditions as above.

Note. 1.0 gil of agar No. 1 may be added to all the broths to reduce drift caused by some foods.

Other media Broths. Brain- Heart Infusion Broth (BHI) Oxoid, Bactomatic Coliform Broth eM (BeB), Bactomatic Enterobacteriaceae Broth: BeB + 20 gil glucose (BEB), Brilliant Green Bile Broth Oxoid (BGB), Lauryl Sulphate Tryptose Broth (LTB) Oxoid, MacConkey Broth (l\1AC) Oxoid, Malthus Coliform Broth (MCB), Malthus Enterobacteriaceae Medium (MEB), Minerals Modified Glutamate Medium (MMG) Oxoid, all prepared according to the suppliers instructions, autoclaved in volumes of 250 or 500 ml and dispensed aseptically. Solid media. Violet Red Bile Agar (VRBA) Oxoid, Glucose- Violet- Red Bile Agar (GVRBA) Oxoid, plate count agar (peA) Oxoid, solid versions of MESB, BeB, BEB, MeB, and MEB were prepared by the addition of 15 gil of agar.

Conductance instrument The l\1althus commercial 128-channel version of the instrument originally described by Richards et al. (1978), with a single water bath set to 37°C, was used in all experiments. Other instruments such as the Bactometer may be suitable, but the method was designed specifically for the Malthus instrument. The method has not been tested with impedance or capacitance signals, and the use of specific detection routines is an important feature of the method.

ENTEROBACTERIACEAE SCREEN

135

Detection parameters .for 10-ml electrodes 1st difference threshold 2nd difference threshold Base level Detection starts Scan interval

3.0 fJ,S 2.0 fJ,S

0.5 f!S 5th scan 12 min

The base level figure establishes the base-line against which the differences between subsequent measurements can be compared. The first difference is that between two consecutive measurements before detection occurs. The sccond differencc is that bctwccn two consecutive first differences before· detection occurs.

Notes 1 This detection method is designed to detect organisms (Enterobacteriaceae) producing a strong Ist difference signal in stable systems. It should ignore weak signals from other organisms, but may not detect Enterobacteriaceae in samplcs that do not produce a good base-line. Therefore, it is important to examine all conductance curves. 2 For Malthus 2-ml electrodes use 1O-ml tubes and increase the 1st difference to 4.5 ~lS.

Cells Tubes. lO-ml glass blood tubes (e.g. Labco cs 1O/w/2/c) or polystyrene 16 100 mm round base (Sterilin or Lab- M).

X

Electrodes. 10 ml or 2 ml in lO-ml tubes or 2-ml electrodes in the standard 7-ml tube for the concentration method. Test volumes. 5-ml double-strength broth and 5 ml of sample suspension or 9-ml single-strength broth and 1 ml of sample suspension. Cultures Thesc included frceze-dried cultures from the National Collection of Type Cultures, American Type Culture Collection (ATCC) and laboratory strains maintained on nutricnt agar slopes and stored under refrigeration. For most routine work, however, dried discs of ATCC organisms (Difco) were used. These were rehydrated overnight in peptone watcr at 3rC and diluted in sterile distilled water.

136

S. B. PE'I'ITT

Food samples To obtain a range of counts, samples were purchased from a number of retail outlets, and tested within two or three days of purchase. Samples were neither artificially contaminated nor abused. Foods included raw salad vegetables, raw beef products and a small number of cheese, pasta, and cream cake samples.

Filtration media After sterilization by autoclaving the following are suitable for the removal of food particles from a stomached sample before centrifugation: Nylon mesh 0.1 mm (Cadisch, Finchley, London). Filter paper No. 1 (Whatmans). Stainless mesh c. 0.5 mm (tea strainers). Sterile stomacher bags incorporating mesh sieves (Seward, London) are also suitable.

Concentration method This method was developed to shorten the total test time for the enumeration of Enterobacteriaceae in chilled cream cakes. It may be suitable for other foods. \Vhen compared with a method using equal volumes of broth and a 1 in 5 dilution of food, the theoretical increase in concentration of bacteria is 40fold. This is equivalent to approximately five generations and should reduce the test time by 2-4 h for a given level of Enterobacteriaceae in the food. Centrifuge: Labofuge 600 (Heraeus Christ, Gennany) with enclosed head taking four swing-out blocks. Tubes: 55-rol sterile plastic screw-capped with a conical base (Falcon, USA). I Make a 1 in 5 dilution of the sample in single-strength MESB (without agar) warmed to 40°C. 2 Mix in a stomacher for 30 s and heat the sample in the stomacher bag back to 40°C using a water bath. 3 Sieve the sample through a sterile tea strainer. 4 Centrifuge 2 x 50 rol of the sieved sample for 30 s only. 5 Remove 50 ml of supernatant fluid to a third 55-ml tube (marked at 2.5 mI) and centrifuge at 2000 g for 10 min. 6 By suction, remove liquid above the line to leave 2.5 m!. 7 Resuspend the pellet using a vortex mixer. S Transfer 2 rol to a standard 2-ml Malthus electrode and overlay with 2 em of sterile mineral oil.

ENTEROBACTERIACEAE SCREEN

137

Sample preparatioll Food samples are diluted I in 5 or I in 10 using maximal recovery diluent (Lab-M) or MESB, and stomached for 30 s. Cultures are diluted in sterile distilled water and mixed using a vortex mixing device, and 0.1 ml added to 10 ml of broth in the cell. After inoculation, with food suspension or pure cultures, all Malthus cells are vortex-mixed.

COllvl'1ltional counts For Enterobacteriaceae I ml of decimal dilutions of the sample are plated out with GVRBA using the pour plate method. Plates are incubated at 37°C for 18- 24 h and all red colonies counted, irrespective of size. Colonies are not confirmed. A similar method is used for solidified versions of broths usually used for c1ectrometric measurements, except that all colonies are counted. Results

Condutlance signals from Enterobacteriaceae in conventional broths The curves in Fig. I demonstrate that a non-Iactose-fermenter can produce a strong change in conductance in the normal coliform media. Similar results are found for other Enterobacteriaceae and some Gram-positive organisms, e.g. Streptococcus jaemlis ATCC 19433.

Conductance signals from Enterobacten'aceae ill 'special' broths The curves in Fig. 2a and b, for two lactose-positive organisms, show that MESB tends to produce a greater change in conductance than the other broths tested, but more importantly, the initial rate of change is greater. This

0.0-13.0 Time (hi

FIG. 1. Conductance curves produced by Sailluillclfa Iyphimunulfl ATCC 14028 in conventional media using 10-011 electrodes.•, BI II; ., J'vlAC; +, LTB; 0, 13GB; 0, 1\1MG. For abbreviations see text.

138

S. B. PETITT

(J)

.3Q)

(,)0

o

~
<0

CO U

o

I

I

..go

o

c

o

U

a

0.0-17.0

b

0.0 -- 17.0 Time (h)

Time (hI

FIG. 2. Conductance curves produced by lactose-positive organisms in special media using 2-m1 electrodes. (a) E. coli ATCC 25922; (b) Enlerobacler aerogelles ATCC 13048. •, MESB; 0, BBI; .) BCB; 0, MCB. For abbreviations see tcx1.

o

o(0 I

o

a

0.0- 17.0 Time (hl

b

0.0-17.0 Time lh)

FIG. 3. Conductance curves in media designed for the detection of Enterobacteriaceae or coliforms using a Bactometer or Malthus. Signal~ produced in a Malthus system with 2-ml electrodes. (a) Citrobacter freul1dii ATCC 8090; (b) Salmonella ~yphimur;um ATCC 14028. •, MESB; 0, BEB; ., BeB; 0) .MCB; x) IviEB. For abbreviations see text.

may mean that a detection for a given number of organisms is produced in a shorter time and the detection parameters used may be made more selective and reliable. The curves in Fig. 3a and b show broadly similar signals for Citrobacter freundii ATeC 8090, and Salmonella ~yphimurium ATCe 14028. The two colifonn media produce a relatively small change in conductance, but the signal should be enough to cause a non-selective computer detection routine to function.

139

ENTEROBACTERIACEAE SCREEN

7.0

OJ

2u

r 2 = 0.88

r 2 = 0.74

25.0 OJ

.8.

o

~ CD

000

c:

>

~

o

5.0

0 0

3.0

(1J

o

'" .;: '" (1J

3.0

u

~

~ 1.0

.0

o

Q;

C

UJ

o a

4

8 Detection time

12

b

(h)

4

8 Detection time

12 (h)

FIG. 4. Scattergrams of GVRBA counts vs. conductance detection times for samples growing in i\tESB at 3rc. (a) 30 samples of salad vegetables (sample weight 0.1 g/eelI); (b) 46 samples of raw beef (sample weight 1.0 g/cdl). For abbreviations see text.

Results with .foods Figure 4a shows the combined results from a number of foods including beansprouts, watercress, onions, celery, fennel, cress, chicory, cucumber, red pepper, and lettuce. The inoculum is 1 ml of a 1 in 10 dilution and the incubation temperature 3JOC. The correlation is poor at only 0.74 and the mean population generation time, estimated from the slope of the line, is 84 min. Figure 4b shows results obtained from an assortment of raw beef products including steak, steak and kidney, mince, lean mince and similar products. The inoculum is 5 ml of a 1 in 5 dilution of the meat, to 5 ml of doublestrength MESB. The correlation is good at 0.88 against an unconfirmed GVRBA count, and the population generation time is 32 min. Discussion These results indicate that conventional media may not be suitable for the enumeration ofcoliforms by conductance in mixed populations because lactosenegative organisms also produce a signal in these media. The new medium, MESB, is an alternative to media from Bactomatic and Malthus for the enumeration of Enterobacteriaceae (by conductance), and uses the hurdle concept. Preliminary results for other foods (not shown) indicate that MESB may produce a detection slightly faster than MEB or BEB, in the Malthus system.

140

S. H. IlETITT

The problem remains that the reported generation times ofsome members of the Enterobacteriaceae in broth at 37°e can range from 16 min to 57 min (Anon. 1956). The present study confirms a wide difference in population generation times between meat and salad vegetables. Thus the method cannot be used to enumerate Enterobacteriaceae without calibration for each type of food. The poor results for salad vegetables could be improved by producing data from each food in isolation. Reducing the temperature to 35° or 300 e might also help. Increasing the quantity of vegetable homogenate in the test cell should shorten test times. Overall, the method is still rather slow, and further work on separation and concentration of micro-organisms fronl foods, medium engineering, or enhanced instrument sensitivity is required.

Acknowledgements Thanks are expressed to VB Frozen Foods for permission to publish this work and to )acqui Hall and Simon I-linton for technical help.

References 1956. Cell division frequency: J\1icroorganisms. In Halldboook of Biological Data ed. Spector; W.S. p. 97. Philadelphia: W.B. Saunders Company. COOKE, B.C.;JORGENSEN, M.A. & l\1I\CDoNALD; A.B. 1985. Effect of four presumptive coliform test media, incubation time and product inoculum size on recovery of coliforms from dairy products. Journal of Food Proiection 48, 388-392. FIRSTENBERG-EDEN, R. & KLEIN; C.S. 1983. Evaluation of a rapid impedimetric procedure for the quantitative estimation of coliforms. Journal of Food Science 48, 1307 ~ 1311. FIRSTENBERG-EDEN; R.; VAN SISE, M.L., ZINDUUS;J. & K'\HN; P. 1984. Impedirnetric estimation of coliforms in dairy products. Jouma! oj1ood Scimce 49, 1449-1452. HOLBROOK; R., ANDERSON, J.M. & BAIRD-PARKER; A.C. 1980. Modified direct plate method for counting Escherichia coli in foods. Food Technology il1 Australia 32; 78-83. 110\\,1£; J. 1981. Significance of Eschen'chia coli in cheese. Lancet 2, 939. IClYISF 1978. Microorganisms in Foods 1. Their Significance and A1ethods of Enumeraiio1l; 2nd ed; p. 8. University of Toronto Press. i\tOSSEL; D.A.A. 1981. Colifonn tests for cheese and other foods. Lancet 2, 1425. MOSSEI.; D.A.A. 1985. Media for Enterobacteriaceae. Iniernational }oumal ofFood Microbiology 2; ANON.

27-32. PETrn', S.B. 1983. Development of a rapid Enterohacteriaceae screen. In Rapid it1icrobi%gical h1ethods in the Food Industry. Leatherhead Food Research Association Tee/mimI Nole No.4, ed. \Vood, J.M. & Gibbs; P.A. Lcatherhead, UK. j.C.S., j:\SON; A.C., HOBBS, G., GIBSON, O ..M. & CHRISTIE, R.I1. 1978. Electronic measurement of bacterial growth.]ouma/ oIP1o'sics, E: Sa'eniijl'c 11lstrummts 11, 560- 568ROWLEY, D.B., VANDEMARK, P., JOHNSON, D. & SHATTUCK, E. 1979. Resuscitation of stressed fecal coliforms and their subsequent detection by radiometric and impedance techniques. Journal of Food Prolection 42, 335 - 341. RICHARDS;

ENTEROBACTERIACEAE SCREEN

141

C.]. 1984. Estimation of total colony count and coliform count of raw lamb using the Bactometer 1\ 1120. Lmtl11'rlwul Food Research Associatioll. Rescanh Report No. 478. l,eatherhead,

STANN.\I{J),

UK. W.\ITE, W.l\1. 1981. Colif,Jrtll test for cheese. Lall(/'I 2,1174-1175.

Electrical Screening of Powdered Dairy Products SHEILA

M.

FRYER I AND KATE FORDE

2

*

/ St lvel Technical Centre, Abbey House, Church Street, BradfOrd-on-Avon, Wiltshire BA1S IDH, UK; and 2 Unigate Foods Ltd, Station Road, Wincanton, Somerset BA9 9ED, UK

Quality assurance of powdered dairy products involves the application of Hazard Analysis and Critical Control Point Principles (HACCP) supported by laboratory analysis of in-line samples and finished products. The required routine microbiological testing may include a total viable count and a yeast and mould count as well as tests for specific organisms/ groups such as coliforms, staphylococci, spores, streptococci and salmonellas. The number and variety of tests required depend on factors such as final use of the product, customer specification and the nature of the product and process. Laboratory results are often required prior to despatch of product and this may result in costly holding of product before release. Electrometric methods otTer the possibility of reducing the holding time due to microbiological screening (Hardy et al. 1977; Wood et al. 1978; Firstenberg-Eden 1983). It has been possible to adapt available methods for routine use in screening powdered products. E:~q)erience has been gained with tests for salmonellas (with a Malthus instrument) and for coliforms and total counts (with a Bactometer MI23). This paper sets out the methods as used on a routine basis with an assessment of the problems and successes encountered.

" Present address: PDP Services, Mcndip House, 114 Station Road, Westbury, Wiltshire

BAI3 4TW, UK.

Copyright

© 1989 by the Society for AfrPlied Bacteriology A /I riKhts of reproduction in a,,)' jimn menxd

Rapid :Vlicrobiological Metbods for Foods, Beverages and Pharmaceuticals

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143

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S. M. FRYER AND K. FORDE

Materials and Methods

Total viable count Impedance method The impedance method for determining total viable counts of powdered products was based on the 'Bactomatic' method (Bactomatic Ltd, l-lenley-onThanles) for total counts in raw milk. Ten grams of sample were suspendcd in 90 ml of Maximum Recovery Diluent (MRD, Oxoid CM 733). After thorough mixing, 0.1 ml was inoculated into a Bactometer module well containing 0.5 ml of modified plate count agar (MPCA) containing (gil): yeast extract, 20; tryptone, 20; dextrose, 4; agar, 10, made with distilled water, boiled to dissolve and autoclaved at 121°C for 15 min. The inoculated Bactometer modules were connected to the Bactometcr MI23 instrument in an incubator at 35°C. Preliminary e:\.1Jcrience had demonstrated that 35°C incubation yielded shorter detection times and a better correlation of impedance with viable count than did incubation at 30°C.

Standard plate count The impedance method was used in conjunction with a standard plate count (i.e. serial dilutions in milk agar (Oxoid CM21) for 3 days at 30°C). Calibration curves of impedance detection time vs. total plate count, were plotted for cornflour, skimmed milk powder and fat-filled powder.

Preparation of high -count samples In order to obtain high-count samples of skimmed milk powder and fat-filled powder for calibration it was necessary to subject each product to an abuse regime using a mini spray-drier (Aeromatic AG, Model STREA 1). The samples were first dampened in the spray-drier, followed by storage at ambient temperature for up to 5 days. Treated samples were re-dried on the mini spray-drier before being testcd for total viable counts as described above.

ColijOrms Impedance method The impcdance method for coliform detcction in powder samples was based on the Bactomatic method for detection of colifonns in milk. A lO-g sample

ELECTRICAL SCREENING OF MILK POWDERS

145

was suspended in 90 ml of MRD. After thorough mixing, 10 ml of this I in 10 dilution was inoculated into 10 ml of double-strength coliform medium (double-strength Bactometer coliform medium, containing (gil): proteose peptone, 20; yeast extract, 12; lactose, 40; sodium dem.''Ycholate, 0.2; sodium lauryl sulphate, 2; bile salts, 2; 0.35% bromocresol purple; 10 ml made with distilled water, adjusted to pH. 6.8 and autoclaved at 121°C for IS min). A further I ml of the I in 10 dilution was inoculated into 9 ml of single-strength coliform medium. Inoculated samples were pre-incubated in a water bath at 35°C for 3 h and then 1 ml of each added to Bactometer module wells. Inoculated Bactometer modules were connected to the Bactometer MI23 and incubated at 35°C.

.s'tandard colijimn method The impedance method described was compared with a standard coliform detection method using MacConkey Broth (Oxoid CM5) at 37°C.

Coniirmation a/positive samples Confirmation of positive samples from either the standard or impedance method was carried out. Presumptive positive samples were streaked on 1\lacConkey Agar plates (Oxoid CM7). After incubation at 37°C for 24 h, typical coliform colonies were picked ofT into fresh single-strength MacConkey broth and Nutrient Broth (Oxoid CMI) and incubated at 37°C for 24 h. Confirmed positive coliform samples were interpreted as those giving acid and gas in MacConkey broth with a pure culture of Gram-negative rods in nutrient broth.

Salmonella detectioll Conductance method The method used to detect salmonellas by conductance was based on the method of Easter & Gibson (1985). A 1 in 10 dilution of sample was preenriched in buffered peptone water (BPW Oxoid CM509) supplemented with 0.1 °lr) trimethylamine-N-oxide (TMA.O) and 0.5% dulcitol at pH 7.2. A 0.25-ml volume of this pre-enrichment culture was inoculated into 10 ml of sclenite-cystine-TMAO-dulcitol medium (SCTO - see below) in a Malthus cell. The cells were connected to a Malthus instrument and incubated at 37°C. Samples giving a presumptive positive response were streaked on selective agars (xylose-lysine-deoxycholate medium) (XLO, Oxoid CM469), and brilliant

146

S. M. FRYER AND K. FORDE

STANDARD

CONDUCTANCE

259 sample in 225 ml BPW

25 9 sample in 225 mJ BPW/T/D

Incubate @ 3 7°C for 16-20 h

Inoculate 10 ml into 90 ml MKTT

Inoculate 10 ml into 90 ml SC

Inoculate 0.25 ml into 10 ml SC/T/O in Malthus cell

Incubate

24 h

&

48 h

Connect to Malthus instrument Incubate @ 37°C

Streak on XLD & BGA

Streak suspect samples on BGA & XLD

Confirm suspect colonies biochemically and serologically

Confirm suspect colonies biochemically and serologically

FIG. 1. Salmonella detection methods.

green agar (BGA, Oxoid CM329)) for confirmation. The method is set out diagramatically in Fig. 1. Selenite-cystine-TMAO-dulcitol medium contained (gil): bacteriological peptone (Oxoid L37), 5; Na zlIPO,f2H zO, 10; dulcitol, 5; TMAO·HCl, 5.6; sodium biselenite (Oxoid LIZI), 4. The pH was adjusted to 7.2 and the medium steamed for 10 min. Immediately before use, 1 ml L-cystine stock solution was added to each 100 ml of broth. (L-cystine stock solution: 0.1 g L-cystine dissolved in 15 rol N NaOH added to 100 ml distilled water and filter-sterilized.)

ELECTRICAL SCREENING OF MILK POWDERS

147

Standard method The standard cultural method for salmonella detection in powdered products was based on the IDF Provisional Standard 93: 1980 method (Anon. 1980). Samples were pre-enriched in BPW followed by selective enrichment in selenite-cystine medium (SC Oxoid CM395) and Muller Kauffman tetrathionate broth (MKT Oxoid CM343). Selective cultures were plated at 24 and 48 h on XLD and BGA for presumptive identification of Salmonella spp. The samples tested were mainly those submitted routinely to the laboratory for pathogen testing. They included final packed products, such as skimmed milk powder, fat-filled milk powder, and glucose plus a variety of ingredients such as whey powder, caseinate and cornflour. In addition, a large number of environmental samples from production sites have been examined. In most cases product samples were bulked together, individual 25-g portions being weighed and then combined in batches of eight or less with a corresponding volume of pre-enrichment medium. Environmental samples were tested individually with up to 50 g per sample being used, depending on the amount available. It was not possible to test all samples by both the standard and conductance methods. In order to be able to have some comparison of the methods, all environmental samples and some repeat product samples were subjected to both methods. The remaining samples were tested by the conductance method only.

Interpretation of conductance results Easter & Gibson considered a 'positive' result on the conductance instrument to be represented by a large response of 600 IlS with a rate of change of> 100 [lS/h (see Fig. 2a). In view of our lack of experience with the method we have interpreted the conductance results more broadly and treated as suspect any samples showing a response of > 300 [lS with a reasonably rapid rate of change (see Fig. 2b). Results

Total viable count Cornflour Figure 3 shows the calibration curve obtained for total viable counts on cornflour using the Bactometer. An acceptable correlation coefficient of 0.89 was obtained. The specification for total viable counts in this product was 5 x

148

S. M. FRYER AND K. FORDE

8.0

24.0

16.0

32.0

16.0

Incubation time (h)

FIG. 2. Examples of salmonella-positive curves from the Malthus instrument using the conductance detection method of Easter & Gibson (1985). (a) Typical curves; (b) atypical curves.

7

~:J

E

.... ..

Product code: CFTVC Samples: 102 Specified cfu/ml: O. 50E + 05 Mult. corr. = 0.89

..

6

Log cfu =::: 0.0353T2-1.10T+10.99 • Cutoff time 5.95 Caution time 9.25

c o

'.j;j .o.o

~

C Q.l o c: o o

5

: ..

J ..

--------~-~----~ t ,f

co

'.j;j

:~

Ol

o

4

. ..

..

.o.o

.... I

, .-..-

..J

..

.. ~....

,t" 6

8

.." . "

".o

10

.o.o

.o.o

12

14

Impedance detection time (h) FIG. 3. Calibration curve for impedance detection time at 35°C on the Bactometer MI23 versus

total colony count on milk agar at 30°C for 3 days for cornflour.

104 cful g. Using this as a basis, a cut-off (i.e. failure) time for detection of growth of 6.0 h was set. A caution time of 9.5 h allowed samples with a 'marginal' result to be identified for further investigation. The impedance method was adopted as the routine quality control monitor for total viable counts in cornflour. The system was checked monthly against the standard plate count using samples known to contain unacceptable, marginal or satisfactory numbers of micro-organisms.

ELEC:TRICAL SC:REENING OF \lILK POWDERS

149

Skimmed milk pomder (SMP) and jat-filled pOlvder (FFP) With these products it was not possible to obtain samples with naturally high total counts. Such samples are necessary for calibrating the impedance method against the standard plate count. Samples with artificially high counts were prepared as described under 'Methods'. However, after comparison between the two methods the correlation coefficient obtained for both types of powder was unacceptable (0.58 for SMP and 0.56 for FFP).

Colijimns Coliform detection by the impedance method was tested on SMP, cornflour and PlOX (a starch product). No attempt was made to correlate impedance detection with numbers of coliforms in the samples since expected levels were always low and a presence or absence test in 1 g or 0.1 g of product was therefore more appropriate. Figure 4 shows examples of typical growth curves obtained by using the impedance method for coliforms with the Bactometer M123. From the results (Table 1) it can be seen that in most cases (except cornflour at 1 g level), the impedance method showed more positives than the standard method. A X2 test showed no significant diHerence between the results obtained from the two methods (test at P = 0.05). From the results obtained it was possible to establish a 'cut-off time for positive samples of 12 h (i.e. samples giving detection of growth in 12 h or less are considered positive). A 'caution time' of 16 h was also set which ensured that samples containing very low numbers would not be falsely classified as negative. In most cases positive samples were found to detect within 12 h giving a marked saving in time over the standard method which requires up to 48 h incubation.

Salmonellas Salmonella detection in powder is a presence or absence test and therefore it is not possible to relate detection times to plate counts. Initial trials with known positive samples in our laboratory confirmed that low numbers of salmonellas in powdered products could be detected using the conductance method of Easter & Gibson (1985). Subsequently the conductance method alone was used for the majority of samples. The standard detection method was used in conjunction with conductance for environmental samples and for some other samples for c. 17 months before being phased out. Tables 2 and 3 summarize the results obtained and the total numbers of samples tested up to March 1987.

s.

150

FRYER AND K. FORDE

M.

170 ,f

'50

(i)

/ I

(ij

I

(,,)

I

: 130

{

(

(ij

/

J

.; 110 .0

~

f f 1

I I

90

Q) 0)

I 1

C

{

)

.c;

70

(,,)

I

J

I

Q)

,,

)

U

c

50

en

I

~

)

Q)

Co

.".

.-- .".

E

30 10

2

4

6

8

10

'2

14 16 18

20 22

24 26

28

Incubation time at 35 0 C (h) FIG. 4. Examples of typical coliform growth curves obtained from the Bactometer M123 using

Bactometcr colifonn medium for powdered product').

TABLE

1. Comparison of colifOrm detection in powder products by impedance (Bac/orneter) and statuklrd methods r.t1acConkey broth)

Bactometer result for samples of

MacConkey result for samples of

1g

0.1 g

1g

0.1 g

+ + + + +

+

+ + + +

+

+

+

+

SMP

Cornflour

PIOX

Total

89 47 17 7 1 12 13 J 3

71 30 9 4 0 II

180 80 32 11 1 31 22

+

20 3 6 0 0 8 2 J 0

0

5 3

Total

40

190

135

365

+

+

+

No. of samples

7 3

ELECTRICAL SCREENING TABLE

or

MILK POWDERS

151

2. Total numbers ofsamples tt~lt'd jor the prest'1lee ofsalmonellas using the eonduetana method of Easter f5 Gibson (1985) and the results obtained

Sample type Product Environmental

No. tested

Presumptive positives

Confirmed positives

1954*1 710

96* (4.9%) 134 (18.9%)

21* (1.1%) 13 (3.1 %)

* These figures include bulked and individual samples where appropriate. 1 Bulked from 14154.

TABLE

3. Compariso/I ofresults ji;r eonduetana-positive samples with the standard cultllral method (where this was done)

Number of conductance-positive samples tested by standard method

Number positive by standard method

5

5 product I environmental

o Discussion Total viable count

The potential for the use of an impedance method for estimating the total viable counts of food is already fairly well established (e.g. Hardy et al. 1977; Wood et al. 1978; Bell 1983; Stannard 1984). The results obtained for cornflour confirmed the feasibility of this method for dried products. In view of this the results for SMP and FFP were disappointing, poor correlations between detection time and total colony count being achieved. The poor correlation was attributed to the use of adulterated samples, which were used to obtain sufficient samples with high counts. A good correlation depends on the use of a sufficient total number of samples with a range of counts (i.e. covering at least 4 log cycles; Firstenberg-Eden & Eden 1984). These authors also highlight the necessity to ensure that artificially obtained 'high-count' samples have a similar microbiological flora to 'normal' out-ofspecification samples. The method used to adulterate SMP and FFP was designed with this in mind. The poor correlation obtained with these samples suggests, however, that an atypical flora may have developed during adulteration. It was not possible to investigate the specific groups of organisms present in the unadulterated and adulterated samples because of time constraints. However, further work will be necessary to allow an acceptable correlation to be obtained so that the impedance method can be incorporated into routine usc.

152

S. M. FRYER AND K. FORDE

Coliforms As previously mentioned, the low numbers of coliforms normally present in dried dairy products made the construction of a calibration curve for impedance vs. standard place count impracticable. A presence or absence test using MacConkey broth was the standard method in use and impedance detection was therefore compared with this. The results (Table 1) showed very good agreement between the two methods, with a XZ test showing no significant difference between the two methods. As might be e::\:pected, most disagreement occurred when the total numbers of coliforms present in the sample were low (i.e. positive in 1 g, negative in 0.1 g). At these marginal levels sample variation might be expected to cause variable results even with duplicate samples tested by the same method. Overall, the impedance method gave more positives in these marginal samples than the standard method (31 compared with 22). Since all positives were confirmed these cannot be regarded as 'false' positives and the results, therefore, suggest that low numbers ofcoliforms are more likely to be detected by the impedance method than by the standard method. On the basis of these results the inlpedance method has been adopted for use both for routine monitoring of product and for investigational purposes. Salmonella detection

During routine testing of powdered dairy products positive Salmonella detection is rare. Agreement between different test methods on negative samples is very inconclusive. In view of the very low numbers of positive samples found, the work reported here cannot be used to directly compare salmonella detection by the conductance method of Easter & Gibson with the standard method (see paper by Prentice et al. this volume). Our work has shown that positive samples can be detected by the conductance method. In the few such cases where the samples were tested by both conductance and standard tnethod there was good agreement (see Table 3). The conductance method was easily incorporated into the routine work of the department. Using this method considerable savings were made in both material use and technician time. In addition, the time required to dear negative samples is considerably reduced (2 - 3 days instead of up to 5 or 7 days). Since only 'suspect' samples need to be streaked on to selective agars, the number of such plates to be examined is drastically reduced: this may well improve the detection of low numbers of suspect colonies. In conclusion, we consider that electrometric methods continue to offer increasing potential for use in routine quality assurance in the dairy industry.

ELECTRICAL SCREENING Of

~1ILK

POWDERS

153

Acknowledgement The work reported here was carried out on behalf of St Ivel Ltd and Unigate Foods Ltd.

References ANON. 1980. klilk and Milk Products-Detectiul/ uj'Salmul/el/a. International Dairy Federation. Provisional Standard 93: 1980. BEI.I., e. 1983. Impedimetric estimation of micro-organisms in meat & meat products. In Rapit! iHiaubiulugiml ,tIe/hods ill the Fout! II/dustry, LeatherJuad Fuod R4 Tedl11iml Nutes Nu. 4, ed. Wood, J,IVl. & Gibbs, P.A. pp. 18-20. E.\STER, M.e. & GIBSON, D.M. 1985. Rapid and automated detection of salmonella by electrical measurements. .Journal oIHj!gime, Cambridge 94, 245-262. FIRSTENBERG-EmN, R. 1983. Rapid estimation of the numher of micro-organisms in raw meat by impedance measurement. Fuod Techl/ology 37, 64-70. FIRSTEN[JERG-EDEN, R. & EDEN, G. 1984. Impet!
An Inter-Laboratory Evaluation of an Electrometric Method for Detection of Salmonellas in Milk Powders G. A. PRENTICE,I P. NEAVES,I D. I. ]ERVIS Z AND M. C. EASTER 3 1 Milk Marketing Board, Thames Ditton, Surrey KT7 OEL, UK; 2 St Ivel Technical Centre, Abbey House, Church Street, BradfOrd-on-Avon, Wiltshire BAIS IDH, UK; and 3 Express Foods Group Ltd, 430 Victoria Road, South Ruislip, Middlesex HA40HF, UK

As the awareness of food poisoning due to Salmonella spp. has increased, there has been a corresponding increase in the frequency of examination of foods for the presence of these organisms. In some circumstances, therefore, the product is not released until it has been shown to be free from salmonellas. This requires the use of rapid tests so that the product may be released as early as possible and storage time and space are reduced to a minimum. In addition to the requirements for a rapid test, modem statistical sampling schemes demand the testing of large numbers of samples from each batch if the necessary degree of security is to be achieved. These schemes permit the bulking of samples but the extent of this is limited by the volumes of preenrichment media involved. The current standard method for testing for the presence of Salmonella spp. (Anon. 1986) involves several stages: 1 Pre-enrichment in either media containing brilliant green for spray-dried milk powders or in buffered peptone water for others (16- 20 h). 2 Selective enrichment in both selenite-cystine and Miiller- Kauffmann tetrathionate broths (24-48 h). 3 Isolation of colonies on at least two selective agars (24-48 h). 4 Identification of suspect colonies biochemically and serologically (24-48 h). This process is very labour-intensive and expensive and the minimum time required to obtain a negative result is 72 h. Positive results may take longer depending on the ease of identification of the organism isolated. Copyright

© 1989 by the Society for Applied BIJt1eriololO' All rights of reprod.wion in allY form resmxd 0-632 -02629-4

Rapid Microbiological l\lethods for F OOd5, Beverages and Pharmaceuticals

155

156

G. A. PRENTICE ET AL.

Easter & Gibson (1985) have developed a conductance method of testing for salmonellas. This involves: 1 Pre-enrichment in a special pre-enrichment broth (16 - 20 h). 2 Measurement of conductance changes during incubation of selective broth. 3 Isolation of Salmonella spp. from suspect samples using xylose-Iysinedesoxycholate (XLD) agar and bismuth sulphite (BS) agar (24-48 h). 4 Identification of suspect colonies (24-48 h). Although this system involves the use of e:\:pensive equipment, it is much less labour-intensive than the traditional standard method and in many cases gives an indication of a positive result after incubation for 24 h, i.e. a total time of 48 h. This would give a considerable saving in the time and labour required to test samples for the presence of salmonellas. This trial was carried out to compare the effectiveness of the EasterGibson automated procedure with the British Standard method for detecting salmonellas in milk powders.

Participating Laboratories To obtain results representative of those which might be obtained in commercial practice a variety of laboratories, all with experience in food microbiology, was involved. The laboratories were also selected to allow the assessment of both the Malthus Microbial Growth Analyser (Malthus Instruments Ltd, Stoke on Trent) and the Bactometer (Bactomatic Ltd, Henley on Thames). The instruments used in the trial are shown in Table 1.

TABLE

1. The inslruments used k}' Ihe laboraton'es participating in the lrial

Laboratory

A B

Instrument Malthus 128I1 (2- and lO-ml electrode)

l\lalthus 128H (lO-ml electrode)

C

Ba<.:tomctcr M I 23

D

IVlalthus 128B (10-nll electrode)

E

Bactometcr l\1120SC

F

Malthus 128H (lO-ml electrode)

INSTRUMENTAL DETECTION

or SALIo,WNELLAS

157

Materials and Methods

Preparation of contaminated samples Skimmed milk concentrates (40°j,) w/v) prepared by dissolving skimmed milk powder in water wcre inoculated with eight different serotypes of Salmonella. Separate powders containing both Proteus mirabilis and Enterobaaer cloacae were similarly prepared and an uninoculated powder was prepared as a control. After incubating at 37°C for 6 h the concentrates were spray-dried in a Blichi 190 laboratory spray drier (Blichi Laboratory-Techniques Ltd, Flawil, Switzerland). During spray-drying the inlet temperature was 142°C and temperatures at the outlet were in the range 80-100°C. The flow rate was c. 1 1/h. After spray-drying, counts of the inoculated micro-organisms, determined using a surface plate count technique on XLD agar, were in the range 10"10 8 cful g depending on their susceptibility to drying. The stocks of spray-dried skimmed milk powder containing high concentrations of the Salmonella serotypes were then diluted in skimmed milk powder, whole milk powder or whey powder to give samples with counts low enough to be suitable for assessing dIe sensitivity of dIe detection systems. For each serotype an initial dilution was made in skimmed milk powder, whole milk powder and whey powder to give a concentration of 10" cfu/g. These were then diluted further (1: 10000) in the same types of powder to give a concentration of salmonellas of c. 1 cfu/g. In a similar two-stage process, the stock powders from the spray drier containing E. cloacae and P. mirabilis were combined and diluted with skimmed milk powder, whole milk powder and whey powder to give levels of each of dIe two micro-organisms of c. 4 cful g. The uninoculated powder from the spray drier was similarly diluted in the skimmed milk powder, whole milk powder and whey powder to be used as a negative control. The three powder types containing eaeh of me eight salmonella serotypes, together with the uninoculated control powders, were then mixed in equal quantities with the same type of powder either uninoculated or containing E. cloacae and P. mirabilis to give 'salmonella' and 'mixed flora' test samples as shown in Table 2. The powders were dispensed in 25-g amounts into sterile pots and stored at
Examination of the pre-enrichment procedure Before the main collaborative trial, one laboratory examined pre-enrichment media to enable a common medium to be used in both methods. All powders were examined with the British Standard and the Easter-Gibson preenrichment media. The ability of each of these to recover salmonellas was then examined by subsequent selective enrichment and plating according to the British Standard procedure. Triplicate samples of each were examined. A flow chart of the eA1Jerimental procedure is given in Fig. 1.

158

G. A. PRENTICE ET AI. TABLE

2. The microbiological ((mtmt of samples*" 'Mixed flora' powder

'Salmonella' powder

Salm. Salm, Sa/tn. Salm. 5alm. Salm. Salm. Salm.

agona eastboumf illfimtis kedougou kubaclw mo1ltevideo

Salm. Salm. Saltn. Sa1m. Sa/m.

Sa1m. Sa1m. Sa 1m.

~}'plzimurium

virchow

'Mixed flora' control (E. cloacae

'Salmonella' control (uninoculated)

*' Target levels

agona + E. cloacae + P. mirabilis eastboumf + E. cloacae + P. mirabilis infantis + E. cloacae + P. mirabi/is kedougou + E. cloacae + P. mirabilis kubacha + E. c!oa£ae + P. mirabilis montevideo + E. cloacae + P. mirabilis typhimurium + E. cloacae + P. mirabi/is vircltow + E. dotUae + P. mirabilis

+ P. mirabilis

only)

of contamination were Salmmulla spp. 0.5, cfu/g; EllterobaCler cloacae and Proteus

mirabilis, 2 cfulg cacho

Powder sample (50g)

/"

Add 25g* to 225ml of British Standard pre-enrichment medium. Incubate at 37°C for 16-20h

~

Add 25g* to 225ml of EasterGibson pre-enrichment medium. Incubate at 3rC for 16-20h

~

~

Add 10ml of culture to selenite cystine and Muller-Kauffmann tetrathionate broths (100m!). Incubate selenite broth at 37° for 24h and tetrathionate broth at 43° for 24h

~

Streak on bismuth sulphite agar and xylose lysine desoxycholate agar plates. Incubate at 37° for 20-24h

l

Select 5 colonies showing reactions characteristic of salmonellas and streak on MacConkey agar NO.3. Incubate at 3r for 24h

~

Inoculate Into tubes of lysine iron agar. Incubate at 37° for 24h. Record tubes giving reactions typical of salmonellas as positivet *Samples were rehydrated by the 'soak' technique, ie. they were added to the pre-enrichment broth and allowed to soak at room temperature for 1h before shaking and incubation. This allowed the micro-organisms to rehydrate slowly, thus reducing osmotic shock which may be lethal to damaged cells. tThe reaction of lysine agar slopes was felt to be adequate confirmation ofthe identity of salmonellas as all ofthe serotypes inoculated into the powders were known to give reactions typical of salmonellas in this medium. 1. EX]lcrimental procedure to compare the performance of the British Standard and EasterGibson pre-enrichment media in the British Standard detection method for salmonellas.

FIG.

INSTRUMENTAL DETECTION OF SALMONELLAS

159

Comparison of the British Standard Method with the Easter-Gibson Method For the main trial triplicate samples of each of the powders described in Table 2 were examined by each laboratory on each occasion. Each pot was allocated a random number which was to be the sole method of identifying the sample throughout the trial. Each laboratory tested 162 samples comprising triplicate samples of eight serolypes plus one control, at two background levels of contamination in three powder Iypes. The three Iypes of powder were tested on successive weeks giving a total of 54 samples per week. This was repeated on three different occasions, immediately after manufacture and after storage for three and six months at <7°e. Six laboratories took part in the trial although not all participated in every stage. Each 25-g sample of powder was sprinkled over the surface of 225 ml of Easter-Gibson pre-enrichment broth, allowed to soak for 1 h at room temperature and then incubated at 37°e for 16 - 20 h. The pre-enrichment broth was then examined for the presence of Salmonella spp. by both the Powder sample (25g)

+

Easter-Gibson pre-enrichment broth / Incubate at 37°C for 16-20h " ' " Add 10ml to 100ml selenite cystine broth and 100ml Muller Kauffman tetrathi01ate broth

Add O.25ml to 10ml Add O.05ml to 2ml selective enrichment selective enrichment broth in Malthus cell or broth in Bactometer

Incubate selenite cystine broth at 3rC for 24h and Muller Kauffmann tetrathionate broth at 43°C for 24h

I

!

W;"

I

Incubate at 37°C for 30h and monitor conductance changes

Select sample cells showing re;ponses typical of salmonellas

Streak on bismuth sulphite agar and xylose lysine desoxycholate agar. Incubate at 37°C for 24h.

+

Subculture and purify colonies giving reactions typical of salmonellas by streaking on MacConkey agar No.3. Incubate at 3rC for 24h.

+

Inoculate into lysine iron agar. Incubate at 37°C for 24h. Tubes giving reactions typical of salmonellas are reported as positive. 2. Experimental procedure to compare the performance of the Easter--Gibson conductance method with that of the British Standard detection method for salmonellas.

FIG.

160

G. A. PRENTICE ET AL.

British Standard method (Anon. 1986) and by the instrumental method using either the Malthus or Bactometer (conductance) detection system and the medium of Easter & Gibson (1985). A flow diagram of the experimental procedure is given in Fig. 2. A typical Salmonella response had the following properties: 1 A flat base-line and a slight plateau immediately before the main detection point. 2 A sharp take-off point or elbow at the point of detection. 3 A fast rate of conductance change. 4 A large total conductance change. S A flat top (akin to a stationary phase). The starting resistance in the Malthus instrument should be c. 350 Q (lO-ml electrode) or 200 Q (2-ml electrode), The rate of conductance change should be c. 100 IlS/h with an overall conductance change of at least 300 !is. In the Bactometer a typical result is obtained with base values of c, 3800 and a step size of 6 or more units. The graphs were all checked individually to avoid misinterpretation due to the adjustments carried out automatically by the instrument software when many graphs are viewed together.

Results and Discussion Because of the scarcity of milk powder naturally contaminated with salmonellas the large numbers of contaminated samples required to conduct a collaborative trial of sufficient size to give useful information had to be prepared in the laboratory. Additionally, because the resuscitation and subsequent growth of salmonellas in the experimental samples would have to represent those characteristics in commercial products, any samples prepared in the laboratory would have to contain cells which had undergone spray drying. It was also felt necessary to assess the efficiency of the detection method in powders containing other micro-organisms which might either inhibit the growth of the salmonellas or grow preferentially and swamp the detection system with false positives. For this reason powders containing both Salmonella spp. and E. cloacae and P. mirabilis were used.

Pre-enrichme7lt tn'al Both the British Standard method and the Easter- Gibson method involve a pre-enrichment stage. This stage is essential: (1) to allow the micro-organisms which have been damaged in the drying process to recover their vitality; (2) to allow small numbers of salmonellas present in the samples to multiply to a level where they can be sub-cultured before detection either by colony formation on selective agars in the British Standard method or by electrometric changes. For the results of the comparison to be valid, it was necessary to employ a

INSTRUMENTAL DETECTION OF SALMONELLAS

161

common single pre-enrichment broth. This was to eliminate the problem of variation in results due to the distribution of organisms at these very low numbers of salmonellas. Since the electrical method required the induction of certain enzymes during the pre-enrichment stage for which the Easter-Gibson pre-enrichment medium was specifically designed, it seemed desirable to use, where possible, the pre-enrichment medium of Easter and Gibson for both methods. It was necessary therefore to ensure that this medium was as effective at recovering salmonellas from milk powder samples as the British Standard medium.

Changes in pH value during pre-enrichment Brilliant green is included in the British Standard pre-enrichment medium for spray-dried milk to inhibit the growth of Gram-positive micro-organisms, especially the spore formers, many of which may be acid producers. The Easter-Gibson pre-enrichment medium however contains no such inhibitors but it does contain phosphate buffer which provides a greater buffering capacity than the British Standard medium. Since these factors all affect the development of acidity during the pre-incubation procedure and since whey powder itself is acidic, pH values were determined before and after incubation in the pre-enrichment broths. This was to give some indication of the n1ent of acid development during pre-incubation which may inhibit the recovery of salmonellas damaged by spray drying. The changes in pH during incubation in the two different media are shown in Table 3. After incubation at 37°C for 18 h, the pH values of the Easter- Gibson pre-enrichment broths were generally slightly lower than tllOse of the British Standard pre-enrichment broths for all skimmed and whole milk powders. For whey powders there was little difference in pH value between the two pre-enrichment broths.

Recovery from dij]('rent pre-enrichment broths The recovery figures for Salmonella spp. from the two pre-enrichment media is shown in Table 4. There were no significant differences in recovery from the two broths of a variety of serotypes in three different powder types. The British Standard method allowed tlle detection of 53 and 60 salmonella serotypes in 'salmonella' and 'mixed flora' powders, respectively, whereas the equivalent rates of detection for the Easter-Gibson medium were 52 and 62. Overall, 113 salmonella serotypes were detected with the British Standard medium whereas 115 were detected with the Easter-Gibson medium. This difference was not considered to be significant. The results shown in Tables 3 and 4 therefore showed that it is possible to use the Easter-Gibson pre-enrichment medium in the British Standard method without losing any efficiency in detection.

TABLE

3. Comparison of changes in pH value upon incubatio11 of British Standard and Easter- Gibson pre-enn'chmetu media Mean* plI values and range after incubation at 37"C for 16-20 h of Whole milk powder

Skimmed milk powder Pre-enrichment medium British Standard

Easter- Gibson

Whey powder

'Salmonella'

'~1ixed

flora'

'Salmonella'

'Mixed flora'

'Salmonella'

'Mixed flora'

6.76 (6.51- 7.00)

6.57 (6.17-6.94)

6.17 (5.65-6.81)

6.10 (5.57~6.69)

5.97 (5.66-6.30)

6.00 (5.62-6.94)

5.13 (4.67-6.76)

5.03 (4.93-5.11)

5.76 (4.73-6.38)

5.70 (4.63-6.31)

6.07 (5.23-6.56)

6.06 (5.68-6.40)

* l\1ean of 24 samples (triplicate samples x 8 serotypes).

163

INSTRUMENTAL DETECTION OF SALMONELLAS TABLE 4. Recovery 11" Salmonella spp. fi'om British Standard and

Easlcr- Gibson pre-etlricllll1etlt media Number of Salmonella-positive samples"' Pre-enrichment medium

'Salmonella' powder

'ivli:xed flora' powder

British Standard Easter-Gibson

53 52

60

Total

113 114

62

"' Both pre-enrichment media were sub-cultured into Mi.illerKauffmann tetrathionate and selenite-c)'stine broths followed by plating on bismuth sulphite and Aylose-Iysine-desoxycholate agars.

Comparison of the inslrumental method with the British Standard method

The results of the comparison of the British Standard method with the instrumental method for different powders examined in all participating laboratories are given in Table 5. In skimmed milk powder the British Standard method detected 219 and 388 positives in 'salmonella' and 'mixed flora' powder, respectively, whereas the results for the instrumental method were 218 and 375. Therefore, the overall number of positives detected by the British Standard method in skimmed milk powder was 607 compared to 593 by the instrumental method. For whole milk powder, the British Standard method detected 210 positives in the 'salmonella' powder and 201 in the 'mixed flora' powder. The equivalent figures for the instrumental system were 207 and 204, respectively. Overall

TABLE 5. Comparison oj the detection oisalmonellas by the British Standard method and

instrummtal method Number of samples positive for salmonellas 'Salmonella' powder

Sample type Skimmed milk powder Whole milk powder Whey powder

'Mixed flora' powder

British Standard

Instrumental

British Standard

Instrumental

219

218

388

375

210

207

201

215

213

222

204 220

164

G. A. PRENTICE ET AL.

the detection levels were 411 for the British Standard method and 411 for the instrumental method. In whey powder, the British Standard method detected 215 positive samples in the 'salmonella' powder whilst the instrumental method detected 213 positive samples. In the 'mixed flora' powder the British Standard method detected 222 positive samples whereas the instrumental method detected 220. Overall the numbers of positives detected were 433 for the instrumental method and 437 for the British Standard method. The results for the different powders show that the ability of the two methods to recover Salmonella spp. was similar irrespective of the powder type or whether E. cloacae and P. mirabilis were present. In the complete trial, 2586 samples were tested. Of these, the British Standard method detected salmonellas in 1455 samples whereas the instrumental method detected them in 1437 samples. The difference in detection rates between the two methods is therefore 18 samples in 1455 or 1.2%. This is a very good agreement for such a complcx and subjectivc tcst involving several different stages and demonstrates that, for a variety of powder types, the Easter- Gibson instrumental method is as effective at detecting salmonellas as the traditional British Standard method.

Acknowledgements This study was supported by the United Kingdom Dairy Industry Research Policy Committee and the Joint Committee of the Milk Marketing Board and the Dairy Trade Federation. Thanks are also due to the staff of the participating laboratories for their technical contribution and to Miss Marisa Marchese for her help in preparing the manuscript.

References 1986. Jlicrobiological Examination fOr Dairy Purposes, Section 3.9, Detection of Salmonella. British Standard 4285. London: British Standards Institution. EASTER, .M.e. & GIBSON, D ..M. 1985. Rapid and automated detection of Salmonella hy electrical measurements. ]oumal of Hygimc, Cambridge 94, 245 - 262. ANON.

Rapid Salmonella Detection by a Combination of Conductance and Immunological Techniques JULIE A. BIRD,h M. C. EASTER,2 S. GAYE HADFIELD,} E. MAY! AND M. F. STRINGER-f

/ School of Biological Scimces, Portsmouth Polytechnic, King llmry I Street, Portsmouth, Hampshire POl 2DY, UK; 2 Express Foods Group Ltd, 430 Victoria Road, South Ruislip, Middlesex llA4 OIlF, UK; 3 Welkome Research Laboratories, LanKley Court, Bed::enham, Kmt BR3 3BS, UK; and'; Departmmt ojiHicrobiolo!iJl, Campden Food and Drink Research Association, Chipping Campden, Gloucestershire GLSS 6LD, UK

Traditional cultural methods for detecting Salmonella in foods generally require a minimum of 3 and 7 days to produce negative and confirmed positive results, respectively (Anon. 1986). Increasing economic pressures are strengthening the desire to shorten the holding times prior to release of foods. Consequently, there is a need for methods which are less labour-intensive, cheaper, simpler and more rapid than existing cultural procedures. Two rapid alternative procedures to the traditional cultural techniques are the enzyme linked immunosorbent assays (ELISA) and the conductance methods. The ELISA methods, based on reactions between Salmonella antigens and specific antibodies, take 1- 6 h to perform (Clayden et al. 1987). However, these immunological procedures have a detection tllfeshold of 105 -106 salmonellas/ml and consequently still require pre- and selective enrichment stages prior to assay giving a minimum total test time of 2-3 days (Mattingly et al. 1985; Hartford 1987; Jay & Comar 1988). Easter & Gibson (1985) described a conductance method for the detection of Salmonella in foods using the Leifson (1936) selenite broth modified by the addition of trimethylamine oxide (TMAO) and dulcitol. Pre-enrichment stages were still required to allow for resuscitation of sub-lethally injured bacteria

* Present address: l\licrohiology Department, Unilewr Research and Engineering, Colworth Lahoratory, Colworth House, Sharnhrook, Bedfordshire lVIK44 ILQ, UK. C0[il'rir1u

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Rapid Microbiological ;\ Iethods I"r Foods, Beverages and Pharmaceuticals

0-632-02629-4

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166

J.

A. BIRD ET AI..

and for the induction of TMAO reductase enzymes. An evaluation of this method using artificially contaminated milk powders (see Prentice et al., this volume) found that the incubation period ofthe selective broth in the instrument needed to be increased from 24 to 30 h to allow for late detections. This lengthened the minimum total test time of the conductance method to 2 days. Detection of Salmonella by the conductance method is based on changes in conductance-with-time curves, which show typical Salmonella characteristics. These are produced when there are levels of 104 -10 5 salmonellas/ml EasterGibson medium. The time taken to reach this threshold is related to the number of salmonellas inoculated into the conductance medium and their subsequent growth. Immobilization of salmonellas from pre-enrichment broth cultures on to the antibody-coated solid supports used in the ELISA procedures could result in a concentrated inoculum such that higher numbers of Salmonella would be transferred to the conductance medium. This would reduce the instrumental incubation time required, Le. time taken to reach the 104 -105 cells/ml threshold of the instrument. Concentration of Salmonella could also decrease the pre-enrichment broth incubation period, resulting in a shorter total test time. The development of the ImmunoModified Electrical Technique (lMET) is described. Hadfield et al. (1987) described a rapid confirmatory test for Salmonella using a simple coloured latex agglutination technique based on serology that gives a result in 5 min. The coloured latex test was evaluated for its ability to detect Salmonella in naturally and artificially contaminated foodstuffs and environmental samples.

Materials and Methods

Media and chemicals Commercially prepared media used were: Bismuth Sulphite Agar (BSA, 007301-1, Difco); Brain- Heart Infusion Broth (BIll, CM225, Oxoid); Brilliant green agar (BGA, CM263, Oxoid); Buffered Peptone \Vater (BP\V, CMS09, Oxoid); Muller-Kauffman Tetrathionate Broth (TET, CM343, Oxoid); nutrient Agar (NA, CM3, Oxoid); Nutrient Broth (NB, CM1, Oxoid); Selenite Cystine broth (SEL, CM699 + LI21, Oxoid) and Xylose Lysine DesoAycholate Medium (XLD, CM469, Oxoid). :Hall's resuscitation agar (HRA) and minimal medium (MM) with filtersterilized glucose (0.2% w/v, BDH Ltd, Poole, Dorset) were prepared according to Hall (1979) and Davis (1980) respectively. The pre-enrichment broth used for all the methods consisted of BHI or BPW containing 0.5°10 (w/v) dulcitol (44590, Fluorochem, Glossop, Derbyshire) and 0.1 0/0 (w/v) trime-

SALMONELLA IMMUNO/CONDUCTANCE DETECTION

167

thylamine oxide (TMAO·2H zO, 92277, Fluorochem). The Easter-Gibson conductance medium was prepared according to Easter & Gibson (1985). A protein solution was prepared by diluting Bovine Serum Albumin (BSA, 44155, BOB) in Phosphate Buffered Saline (PBS, BR14a, Oxoid) to give a 1% (v/v) solution. Binding buffer solution was prepared at time of use by similarly diluting BSA in PBS to give a final concentration of 0.1 % (vIv) BSA. The wash solution consisted of PBS containing 0.05% (v/v) Tween 20 (66368, BOH). P-nitrophenylphosphate (N2765, Sigma Ltd, Poole) was diluted in I molll- 1 diethanolamine (08885, Sigma) at pH 10.0 to give a 1 mg/ml substrate solution.

Bacterial cultures Several salmonellas and closely related non-salmonellas were used in this study, many of which were isolates from naturally contaminated foods and environmental samples. Other bacteria used were obtained from the Public Health Laboratory Service, Colindale (strains labelled BR), National Collection of Type Cultures, London (strains labelled NCTC), Colworth Culture Collection, Unilever Research, Shambrook (strains labelled CCC) and Malthus Instruments Culture Collection, Crawley (strains labelled M). The cultures used were Salmonella agona (two strains including BR 1786), Salm. anatum (two strains including NCTC 5779), Salm. enteritidis (NCTC 4444), Salm. eastbourne, Salm. hadar (NCTC 9877), Salm. indiana (NCTC 11304), Salm. inflmtis, Salm. kedougou (two strains), Salm. kurbacha, Salm. montevideo, Salm. napoli (BR 3427), Salm. stanlc.y (NCTC 92), Salm typhimurium (two strains including phage type 10, CCC 1(6), Salrn. virchow, Citrobaaer freundii (three strains including M 120), Enterobacter cloacae, Escherichia coli, Hafnia alvei (MI26) and Proteus mirabilis. Stock bacteria were streaked on NA slopes and after incubation for 24 h at 3TC were stored at 4°C. Stock cultures were sub-cultured at monthly intervals until required.

Test materials Log phase cultures of Salm. agona, Sa1m. eastbourne, Salm. infimtis, Salm. kedougou, 5'alm. kurbacha, Salm. montevideo, Salm. ~yphimurium, Salm. virchow, Ent. cloacae and P. mirabilis grown in skimmed milk concentrate (40% w/v, Milk Marketing Board (MMB), Thames Oitton, Surrey) for 6 h at 37°C were indiYiduallY spraY-drinl with a laboratory spray drier (Model 190, Biichi Ltd, Flawil, Switzerland). A control powder was also prepared by spray-drying uninoculated skimmed milk concentrate. These powders were then diluted in skimmed milk powder (SMP), whey powder (WP) and whole milk powder

168

J. A. BIRD ET AL.

(WMP) to give estimated levels of Salmonella, Ent. cloacae or P. mirabilis of I, 4, 4 colony-forming units (cfu)/g powder, respectively. The uninoculated control powder was similarly diluted in SMP, WP and WMP. The milk powders containing the salmonellas and the uninoculated control powders were mixed in equal quantities with SMP, WP and WMP which were then inoculated with the Ent. cloacae and P. mirabilis Gram-negative competing microflora or left uninoculated. The ratio of competitors to salmonellas was 8:1, and the final levels of Salmonella in the powders were in the range < 3 cfu/IOO g to 1100 cfu/g powder (see Prentice et al., this volume). These samples (n = 354) were used for the evaluation of the coloured latex agglutination test only. Milk-based powder and environmental samples (n = 104) taken from a commercial spray drier were also examined by the traditional method and the coloured latex agglutination test.

Salmonella detection methods Cultural method Following overnight incubation in BPW at 37°C, pre-enrichment cultures were inoculated into SEL and TET selective enrichment broths, and incubated at 37°C and 43°C, respectively. After incubation for 24 and 48 h both selective broths were streaked on BGA, BSA and XLD selective agars and incubated at 3rC for 24 h. Presumptive colonies were confirmed by agglutination tests using polyvalent 0 and B antisera (9620- 53, 9630- 53, Difco).

Conductance method The method of Easter & Gibson (1985) was employed, using a 128B Malthus Microbial Growth Analyser (Malthus Instruments Ltd, Crawley) or 120SC Bactometer (Bactomatic Ltd, Henley on Thames). Malthus cells, each containing 10 ml of Easter-Gibson medium, were inoculated with 0.25 ml preenrichment culture. Bactometer wells containing 2.0 ml Easter-Gibson medium were inoculated with 0.05 ml pre-enrichment culture. The cells and modules were incubated at 3rC and conductance measurements recorded for 48 h. Cultures producing typical Salmonella conductance curves were streaked on XLD and incubated at 37°C for 24 h. Presumptive colonies were confirmed by agglutination tests.

Interpretation of conductance curves Conductance curves obtained from the Malthus and Bactometcr instruments were considered Salmonella positive if they showed the following characteristics:

SALMONELLA IMMUKO/CONDUCTANCE DETECTION

169

(1) a flat base-line and slight plateau immediately prior to the main detection point; (2) a sharp elbow or 'take-oir point at tJle point of detection; (3) a fast rate ofconductance change (100 ftS/h Malthus, step size ~ 6 units Bactometer); (4) a large total conductance change (> 300 f.tS Malthus) and (5) a flat top to curve (similar to stationary phase).

ImmunoModijied Electrical Technique (IME1) The Bio-Enzabead Salmonella Screen Kit (Organon Teknika UK Ltd, Cambridge) was used as a source of ferrous-coated polystyrene beads precoated with monoclonal antibodies specific to Salmonella. A lO-ml sample of a pre-enrichment broth culture incubated at 3TC was placed in a sterile capped lO-ml Malthus tube, Bio-Enzabeads were added, and tJ1e MaltJ1us tube placed horizontally in a shaking incubator (Dynatech Ltd, Billingshurst, Surrey) for 20 min at 37°C with sufficient agitation to gently move the beads around in the broth. Following incubation a magnet positioned at the base of tJ1e Malthus tube retained the beads whilst tJ1e preenrichment culture was decanted off. The beads were washed gently by dipping once in sterile BPW before 10 ml of Easter-Gibson medium and a sterile 10 ml electrode were added. The Malthus tube and contents were placed in the Malthus instrument and conductance changes were monitored for 48 h at 37°C. Broth cultures from selected tubes were streaked on XLD and incubated at 3TC for 24 h. Presumptive colonies were confirmed by agglutination tests.

Preparation offreeze-injured Salmonella cultures An overnight culture of Salmonella grown in NB at 3TC was diluted lO-fold in fresh medium and 0.5 ml transferred to a test-tube containing 9.5 ml NB. Growth at 30°C was monitored by optical density measurements. Mid-log phase cultures (0.1 ml) were inoculated into 10 ml MM with a 0.2% (v/v) glucose supplement. Injury was induced by two cycles of freezing to - 20°C for 24 h followed by overnight thawing at 5°C. Numerical differences in colony-forming units (cfu/ml) on BRA and XLD gave an estimation of the proportion of sub-letllally injured salmonellas in tJ1e culture.

Determination of minimum pre-enrichment incubation time Injured cells and overnight cultures of Salm. enteritidis, grown in NB at 3TC, were individually inoculated into flasks containing 100 ml pre-enrichment broth such that levels of 1-10 cells/ml were attained. The pre-enrichment cultures were incubated at 37°C in a water bath. Each culture was analysed at hourly intervals by plate counts on I IRA and XLD using a spiral plate maker

170

J. A. BIRD

ET AL.

(Spiral Systems Inc., USA) and plates were incubated for 48 h at 30°C and 24

h at 37°C, respectively. Aliquots (0.25 mt) of the culture were also inoculated in duplicate into Malthus tubes containing 10 ml Easter- Gibson medium and 10 ml pre-enrichment broth. The conductance method protocol was then followed for these rubes. The IMET method was carried out on the remaining portion of the pre-enrichment culture sample. These eX'j)eriments were repeated using other Salmonella serotypes and a pre-enrichment incubation time of 7 h. The other serotypes used were: Sa/m. agona (BR 1786), Sa/m. anatum (NCTC 5779), Sa/m. hadar, Sa/m. indiana, Sa/m. napoli, Sa/m. stan/e.Y and Salm. ~yphimun'um (phage type 10 CCC 166).

Effect oj competing bacteria on the detection of Salmonella conductance methods

~y

the IMET and

Overnight cultures of Sa/m. enteritidis, C. freundii and H. alvei, grown in NB at 37°C, were inoculated into 100 ml pre-enrichment broth to give ratios of Salmonella to competing bacteria ranging from 1: 100 to 1000:1. The preenrichment cultures were incubated in a water bath at 37°C for 7 h. Aliquots (0.25 ml) were used to inoculate the selective Easter- Gibson medium used in the conductance method. The IMET procedure was also performed on these pre-enrichment cultures.

Comparison of IMET, conductance and cultural methods in the deteaion of Salmonella from artificial(y and natural(y contaminated foods

A variety of foods from different sources naturally and artificially contaminated with a range of Salmonella scrotypes at differing levels were examined using the traditional cultural, conductance and IMET methods. Antibod,y immobilization on Dynal beads Eppendorf tubes (Sterilin Ltd Hourslow) were filled with protein solution, capped and incubated at 37°C in a waterbath for 3 h. The protein solution was discarded and the tubes washed ten times by filling, emptying and rinsing each tube with the wash solution. The protein-coated tubes were then airdried and stored at 4°C until required. One millilitre of Dynal beads (14001, Dynal UK Ltd, Wirral) was added to a protein-coated Eppendorf tube and washed three times in the wash solution by use of a magnetic particle concentrator (MPC, 12001, [)yoal). The Dynal beads were re-suspended in 2.5 ml of binding buffer. Aliquots (0.7 ml) of the beads in buffer solution were dispensed into five proteincoated Eppendorf tubes. Different volumes of polyclonal '0' antibody solution (9620- 53, Difco), 0, 0.05, 0.1, 0.2 and 0.5 mt were added to each tube and

S/ILMONELIA IMMUNO/CONDUCTANCE DETECTION

171

the total volume made up to 3.5 ml with binding buffer solution. Each Eppendorf tube was wrapped in tissue paper, placed on a rotating wheel and mixed end-over-end for 24 h. Following the binding period the MPC was used to retain the Dynal beads in the tubes while the binding buffer/antibody solution was discarded. One millilitre of wash solution was added to each tube and mixed as before for 45 min at 4°C. This wash stage was repeated a further three times. The antibody-coated Dynal beads were finally suspended in 0.2 ml binding buffer containing sodium azide (0.0001 % w/v) added as a preservative, and stored at 4°C until required. The coated beads were washed several times in wash solution to remove the preservative at time of use.

Determination of amount of antibody bound on Dynal beads Goat-anti-rabbit IgG antibodies conjugated with alkaline phosphatase (Sigma Ltd, Poole) were diluted 1 in 1000 in binding buffer solution. Aliquots (0.01 ml) of antibody-coated Dynal beads were dispensed into Eppendorf tubes and 0.5 ml binding buffer and 0.1 ml conjugated antibody solution were added to each tube and thoroughly mixed. After incubation for 3 h at 37°C in a water bath, the beads were washed five times in wash solution using the MPC. The p-nitrophenol phosphate substrate solution (0.8 ml) was added and the tubes incubated at 3rC for 30 min. The enzyme-substrate reaction was stopped by adding 0.2 ml of 1 mollI NaOH (Sigma) to each tube. The supernatant of each tube (0.2 ml) was transferred to a microtitre plate (Immulon 129A, Dynatech Laboratories Ltd, Billingshurst, Sussex) and the optical density (OD) measured at 405 nm, using a plate reader (Dynatech Laboratories Ltd).

EjJect of solid support size on the minimum pre-enrichment incubation time The experiments previously described to determine the minimum preenrichment incubation time were repeated using cultures of injured and uninjured Salm. enteritidis. The IMET method was modified by dividing the remaining portion of each pre-enrichment culture sample into one subsample of 5 ml and two of approximately 2 m( Two Bio-Enzabeads were added to the 5-ml sub-sample whilst 0.2 fll of antibody-coated Dynal beads were added to each of the 2-ml samples. Additionally, at the 7-h preenrichment sample time, a control of uncoated Dynal beads was incorporated.

Evaluation of the coloured latex agglutination test The method described by Hadfield et al. (1987) was followed; reagents and equipment were supplied by Wellcome Diagnostics, Department of Research and Development (Beckenham, Kent). Artificially contaminated milk powders

172

J.

A. BIRD ET AL.

and naturally contaminated milk-based powder and environmental samples were examined, using the cultural method. Selective broth cultures and presumptive colonies on BSA, BGA and XLD agars were tested for the presence of Salmonella with the coloured latex agglutination test.

Results

Determination of minimum pre-enri[hment inmbatioll time Typical Salmonella conductance curves were obtained using the IMET method for all cultures of uninjured Salm. enteritidis that had undergone pre-enrichnlent incubations of 5 h (Table 1). Frozen sub-lethally injured Sa 1m. enteritidis cells produced similar conductance curves, but the minimum pre-enrichment incubation time was extended to 6 and 7 h for the pre-enrichment broth and Easter- Gibson medium, respectively (Table 2). Cell counts of the uninjured and injured salmonellas at these times were in the range 103 - 104 cells/ml. The bacterial counts on XLD immediately after inoculation of sub-lethally injured Salm. enteritidis cells into the pre-enrichment broth were up to 3 log cycles lower than corresponding counts on HRA. The counts on the two media became similar after 4 h incubation of the pre-enrichment culture (Table 2). The seven other Salmonella serotypes tested all produced typical conductance curves after a 7-h pre-enrichment incubation and immobilization stage. The mean detection times for these serotypes ranged from 13.8 h for Salm. agona to 35.4 h for Salm. stanley (Table 3). The average detection time for the eight scrotypes was 20.6 h.

Effict of competing baaeria on the detection of Salmonella by the [MET and conduaance methods The IMET method detected Salm. enteritidis in the presence of competing bacteria in five of the six inoculation level combinations (Table 4). Presumptive Salmonella colonies were not produced on XLD selective agar for the IMET negative cultures although salmonellas were initially inoculated into this broth. Salmonella was detected in both of the duplicate tubes by the conductance method for five of the seven combinations. The remaining two samples were found to be positive in only one of the pair of Malthus tuhes. The conductance method gave detection times up to 11.4 h shorter than those of the IMET procedure (Table 4). However, the IMET conductance curves showed greater rates of change of conductance more typical of Salmonella. The total change in conductance values were similar for both methods (Fig. 1).

SALtlONELLA I\1MUNO/CONDUCTANCE DETECTION

173

1. Delee/iou ~r Salmonella ~y lite I;HET melhod ("siug pre-eIln'dl1nelll brollt (/1) and EasterGibsoll medium (B)) (lIId baclerial UlUIl/S OIl HR~ al various iumba/ioll limes of a pre-mridltllelll brolh ill/Kulaled lI'ith uuilljured Salm. enteritidis

T\IlI.E

Pre-enrichment incubation time (h)

Number of typical Salmonel/a conductance curves t

Bacterial count* (cfu/ml)

liRA 6.0 4.2 6.0 2.1 1.3 5.2 1.3 6.9 9.6 9.1

()

I 2 3 4 5 6 8 18 24

X

10 2

X

]02 2

X X

10 103

x IO~ x IO~ x lOs X 10" x 10K x 10K

A

B

0 0 0 0 0 2 2 2 2 2

0 0 0 0 0 2 2 2 2 2

du, colony-fomling units. * Mean of two results. t Two curves examined for each sample.

2. DeIeelioll of Salmonella b)' /he IMET method (using pre-e11ricltmenl bro/h (A) alld EaslerGibson medium (B)), and ba{len'al UJlwts OIl llM alld )(LD al various illcubalioll limes ofa pre-l'1lrichmeul brulh ;'lOculaled wilh /reeze-illjured Salm. enteritidis

TMILE

Pre-enrichment incubation time (h)

0 2 4 5 6 7

8

Number of typical Salmollella conductance curves t

Bacterial count* (cfu/ml) lIRA

2.1 x 10' I.Ix 10 2 2.5 x 102 6.1 X lO2 2.1 X 101 4.9 X 101 2.1 x IO~

* Mean of four results. t Four curves examined for each sample. NT, not tested; cfu, colony-forming units.

XLD

2 6.2 x L7x 3.3 X 1.1 X 1.1 X 1.5 x

W 102 102 IO J IOJ 10~

A

B

NT NT

NT NT

0 0 2 3 4

0 0 0 2 4

174

J. A. BIRD TABLE

ET AL.

3. Deteetio11 times fOr a variety of salmonel/as

~y

IMET using the

Easler-Gibs011 medium

Serotype Salm. Salm. Salm. Salm. Salm. Salm. Salm. Salm.

Range of detection times (h)

l\'1ean detection time* (h)

11.4-16.2 15.0-20.4

13.8 17.4 24.7 15.7 14.7 30.3 35.4 17.2

agona anaturn enteritidis htUlitr indiana napoli stanley typhimun'um

23.2-26.2 13.4-17.6

11.6-18.6 26.2-34.0 t

14.8-19.4

'*' Mean of four replicates. t One value only.

TABLE

4. Effie! of competing bacten'a on the detection of Sa1m. enteritidis conductance (OW) methods

Salm. ent I 1

Cit

Hal

1

I

10

1 100

100

100 10

1

10

100

10 1

1

1000 1

Number of typical Salmonella conductance cunrcst

Bacterial count'*' after 7 h at 37°e (cfu/ml)

Ratio of inoculated bacteria

0

1 0

lIRA 5.3 x lOs

4.5 X ION 5.7 X 108 8.0 x lOs 7.6 X lOs 7.2 X 108

5.6

X

108

XLD 8.0 1.2

X

8.6 1.9 8.0 4.9

X

X

103 10('

0 X X

X

~y

107 108 107 108

the IMET and

Detection times! (h)

(MET

eM

IMET

eM

2 2

2 1

12.0 6.8

5.0 4.4 15.2 2.8 2.6 2.2 2,2

0

]

NO

2 2 2 2

2

14.2

2 2 2

12.6 13.8

13.4

Salm. ent, Salm. enten'tidis; Cil) C freundii; HaI, H. alvei. ND, not detected; cfu, colony-forming units. '*' Mean of two replicates. t Two curves examined for each sample. r Mean of two replicates.

Compart'son o/IMET, conduaance and cultural methods in the detection of Salmonella from artificially and naturally contaminated fOods Salmonella was detected by both the culntral and conductance methods for 15 of the 22 contaminated food samples, representing an agreement of 68%

SALMONELLA IMMUNO/CONDUCTANCE DETECTION

175

~_-::c

(i) 600

A B

3(I)

()

C ro

tl 400 ::l

"t:)

c:

o

U

200 I

8.0

16.0

24.0

Time (h) FIG. 1. Typical conductance curves for Salmo11ella e11/e11dus in the presence of competing bacteria by IMET (A), conductance method (8) and Salmrmella only control (C).

(Table 5). The same detection results were obtained for both the cultural method and IMET procedure in 19 of the 22 samples giving an agreement of 86%. The minced beefsamples shown as Salmonella positive by the conductance method only were subsequently found to be contaminated with Citrobaaer freundii. Only five and four of the eight whey powder samples were Salmonella positive by the IMET and conductance methods, respectively. However, salmonellas were isolated and confirmed by agglutination reactions from all of the Malthus tubes containing whey powder cultures.

Determination oj the proportion oj po(yclonal 0 antibodies bound to Dynal beads The absorbance values, at 405 nm, of the double antibody ELISA performed on the antibody-coated Dynal beads shows that the lowest antibody volume (0.05 m!) added to the uncoated Dynal beads was sufficient to produce maximum binding (Table 6).

Effict of solid support size on the minimum pre-enrichment incubation time The minimum pre-enrichment incubation time for the IMET method was reduced from 5 and 6 h for uninjured and sub-lethally injured cultures of Salm. enteritidis, respectively, when using Bio-Enzabeads of 3 mm diameter) to 2 h with the use of smaller (4.5 /lm diameter) antibody-coated Dynal beads (1' abies 7 and 8). The bacterial counts of tlle uninjured and injured Salmonella preenrichment cultures corresponding to these Salmonella-positive Dynal results

J.

176 TABLE

A. BIRD ET AL.

5. Detection of Salmonella from naturally and artificially contaminated foods by the traditional cultural (TC), conductance (C#) and IMET methods

Food

Contamination

Serotype

A N N N A

Salm. typhimurium

Cottage cheese Minced beef \Vhey powder Malaysian prawns Chocolate crumb

A Cocoa powder Chocolate Desiccated coconut Linseed

A A A N

N N

* Salm. Salm. Salm. Salm. Sa/m. Sa/m. Salm,

virchow weltvereden panama enten'tidis

Total no. Level of (cells/g) samples

No. of Salmollellilpositive samples

TC

NK

2

2

2

NK <10

2

0

8

8

2 1

2 1

}

1 I 1 1 1 1

1

}

2 4 1 1 1 I 1 1 1 1 I

22

20

17

NK 46

der~y

newport infantis

2.3

1

0.4 0.9

1 1

2.3

1 1

NK NK NK

NK

Salm. ferlac

NK

Total

A, artificial }

.. 1 contammanon. , natura NK, not known.

N

*' Citrobacter freundii.

TABLE

6. Determination oIproportion ofpolyclona/ 0 antibody bound to Dyna/ beads

Antibody volume added to Dynal beads (00)

Increase in absorbance (A,tos - A 4 0S blank)

Absorbance at 405 nm (A'f{}s)

o

0.46

0.05 0.1

0.92 0.82 O.S}

0.35

0.78

0.32

0.2 0.5

Proportion of bound antibody

0.46 0.36

= A405

eM

~

A405 blank.

IM£'1'

2 0 5

2 1 1 1

1 1 }

1 I 17

S,1LlvIONELL/1 IMNIUNO/CONDUCTANCE DETECTION TABLE

7. Effect

Pre-enrichment incuhation time (h)

0/ solid support size Oil

the detectioll 0/ sub-lethal~v illjured SalOl. enteritidis kv the IMET alld conduttance (eM) methods

2 4 5 6 7

Number of typical SalmOtlella conductance curves

Bacterial count* (cfu/ml) lIRA

3.4 x 10 1 3.4 X 10 1 2.6 X 102 1.5 X 10.1 9.0 X 10.1 3.7 X 104

0

177

IMET XLD

Bio t

Dynal t

CM§

0 0 0 0 1 1

0

0 2 2 2 2 2

1.9 1.9 6.8 1.5 2.2

X X

X X X

10 1 102 10.1 10.1 10 4

I

I I I I

cfu, colony-forming units; Bio, Bio-Enzabead; Dynal, Dynal beads. * 1Vlean of two results. t One curve examined for each sample. t One curve examined for each sample. § Two curves examined filr each sample.

TABLE

R. FJfect o/solid support size on the detectioll o/uninjured SalOl. enteritidis by the IMET alld conduclilllte (0\1) methods

Bacterial count* (cfu/ml)

Pre -enrichment incuhation time (h)

0 2 4

5 6 7

Number of typical Salmonella conductance curves

lIRA

IMET XLD

Biot

Dynal t

CM§

NT

NT

NT

NT

0 0 2 2 2

2 2 2 2 2

2 2 0 2 2

NT

2.0 x 102 2.5 X 10.1 1.1 X 104 4.4 X 104 3.9 X 10'

1.3

X

2.2 9.6 1.8 1.9

X

X X X

102 10.1 10.1 104 10'

cfu, colony-fomling units; Bio, Bio-Enzabead; Dynal, Dynal heads; NT, not tested. * "'lean of two results. t One curve examined for each sample. lOne curve examined for each sample. § Two curves examined for each sample.

J. A. BIRD

178

£1' AL.

were 10 1 -10 2 cells/m!. The IMET method using the Dynal beads produced typical Salmonella conductance curves identical to those produced using BioEnzabeads, but required the shorter pre-enrichment incubation times of the conductance method.

Evaluation of the coloured latex agglutination test The coloured latex agglutination test was applied to both selective enrichment broths and to presumptive colonies from selective agars for salmonellas during (a) an evaluation of the electrical method (see Prentice et aI., this volume) using the Bactometer 120SC and (b) routine analysis of milk powders and environmental samples using the Malthus growth analyser. The results using the coloured latex agglutination test on selective enrichment broths from both the conductance and cultural methods are shown in Table 9. In 205 selective enrichment broths shown to contain salmonellas by cultural methods, 203 (99 % ) were positive by the coloured latex agglutination test. Salmonella was absent in 288 enrichment broths (as determined by cultural methods) and 278 (96.5 %) were also shown to be negative by latex agglutination. False positive reactions occurred in 10 out of 501 samples (2%), Le. agglutination was observed but salmonellas were not detected by cultural methods. This was later shown to be due to the presence of Citrobacter where a cross-reaction with the common 6, 7 antigen gives a blue agglutination with one of the polyvalent latex reagents. Most (70 % ) of the false positives were obtained with environmental samples which are likely to contain a wide variety TABLE

9. Evalualion of the coloured latex agglutination test for the detection of Salmonella ill selective enrichment broths Numbers of selective enrichment broths examined using the coloured latex agglutination test showing

Sample Skinuned milk powder Whole milk powder 'Whey powder Other dairy powders Environmental samples Total

Salmonella

Salmonella

present

absent

False negative

False positive

Autoagglutination

96

32

0

0

0

57

74

0

48 1

75

2

2

1

54

0

0

7

43

0

7

0

278

2

10

8

203

0

SALMONELLA IMMUNO/CONDUCTANCE DETECTION

179

of bacteria from the family Enterobacteriaceae, including those species closely related to Salmonella. False negative reactions occurred in 2 out of 501 samples (0.4%), i.e. no latex agglutination in the presence of salmonellas. On further investigation these samples were also found to give longer detection times with the conductance method. This suggests that the number of salmonellas present was lower than in other samples, and may have been below the sensitivity threshold of 105 -10 6 organisms/ml for the agglutination reaction. Only 8 out of 501 samples (1.6%) could not be classified using the coloured latex agglutination test due to non-specific auto-agglutination, but on further investigation none were found to contain Salmonella. Similar results to those described above were obtained when the coloured latex agglutination test was applied to the presumptive positive single colony isolates, except that no false negatives were observed. Discussion Pre-enrichment incubation allows for the repair of sub-lethal injury, and once these cells have been restored to a stable physiological condition, the preenrichment medium may serve as a source of nutrients for the proliferation of repaired cells (Andrews 1985). Numerical differences in cell counts on resuscitative and selective agars in this work indicated that the majority of the freeze-injured Salmonella cells were repaired after a 2-h pre-enrichment incubation period (i.e. counts on the two agars became similar). A preenrichment incubation time of 7 h produced Salmonella counts in the region of 10-1-108 cellslml from an initial inoculation level of 1 celli m!. Ray et al. (1972) similarly reported that freeze-injured Salm. anatum cells in minimal broth repaired within 1 to 2 h at 25°C. Good recovery of heat-injured salmonellas was also reported from milk powder pre-enrichment for 2-6 h in BPW (van Schothorst & van Leusden 1975). There is, however, some controversy over the effectiveness of shortened pre-enrichment incubation periods (D'Aoust 1981). D'Aoust & Maishment (1979) evaluated the efficacy of seven pre-enrichment culture media and showed that reduced (6 h) incubation periods were unreliable and produced unacceptably high numbers of false positive results. La Roche & Desai (1981) modified the membrane filter-disc immobilization technique (MFDI) developed by Swaminathan et al. (1978) to concentrate salmonellas from pre-enrichment or selective broth cultures, reducing the pre-enrichment incubation time to 4-6 h. The Immuno-Modified Electrical Technique (IMET) described in this chapter using Bio-Enzabeads reduced the pre-enrichment incubation time to c. 5 and 7 h for uninjured and sublethally injured S'almonella cultures, respectively.

180

J.

A. BIRD ET AI.

Scanning electron microscopy studies (unpublished data) showed that the antibody coating on the surface of the Bio-Enzabeads was not uniform, and that the immobilized salmonellas were associated only with these antibody regions. Additionally, X-ray probe analysis (unpublished data) revealed the presence of cadmium in the ferrous coat of the Bio-Enzabeads. Cadmium is toxic to bacteria, inducing injury or death by DNA single-strand breakages (Vallee & Ulmer 1972). Therefore, the Bio-Enzabeads were not only inefficient in immobilizing salmonellas but could have produced losses in viability of the captured cells. The Bio-Enzabeads are c. 3 mm in diameter and present a fairly small surface area-ta-volume ratio (SA:V) on which to immobilize the Salmonella. The Dynal beads have a much smaller diameter of 4.5 Ilm, and therefore have a higher SA:V. The use of many Dynal beads with the IMET method immobilized a greater number of salmonellas from the pre-enrichment cultures, reducing the pre-enrichment incubation times required to produce typical conductance curves to 2 h. Uncoated Dynal beads also produced typical Salmonella conductance curves. This shows that salmonellas are capable of non-specific binding to solid supports. This could possibly be overcome by coating the beads in a blocking agent such as bovine serum albumin. Commercially available polyvalent 0 or somatic antibodies were bound on to the Dynal beads. These antibodies form stronger bonds with the antigen but are less specific than those of the II or flagella group used on the Bio- Enzabeads. The H antibodies recognize a flagellin monomer which is only exposed in large quantities when the flagella are ruptured by such treatments as boiling. Naturally occurring breaks in the flagella can also present the H antigen though these occur at a fairly low frequency. It was the broken flagella ends that most probably formed the antibody-antigen link in immobilization of cells on to Bio-Enzabeads using the IMET procedure. The flagella antigen-antibody bond, and attachment of the flagella to the cell may be easily broken by physical actions such as vigorous washing. One solution to this problem could be the use of a mixture of 0 and 1-1 antibodies. The immobilization of salmonellas from a pre-enrichment broth culture on to a solid support results in the transfer of a concentrated inoculum into the selective Easter- Gibson broth. It was expected that this would result in a shorter time to the detection point (i.e. time to reach the 104 -105 cells/ml threshold of the instrument). The detection times for the conductance method and IMET using Dynal beads were similar. However, the IMET procedure using Bio-Enzabeads gave detection times up to 11.4 h longer than those of the conductance method. This could be attributable to the inefficient immobilization of salmonellas from the pre-enrichment broth cultures using the BioEnzabeads, and inhibition by cadmium as previously discussed.

SAL/vIOVELLA IMMUNO/CONDUCTANCE DETECTION

181

The detection times of eight Salmonella serotypes ranged from 13.8 h to 35.4 h; the mean detection time was 20.6 h. The immobilization of the Salmonella cells could be serotype-variable since each strain is characterized by a different set of antigens. Although an improved algorithm and updated computer software have been produced, conductance curves on the 128H model still require visual assessment and the detection times tend to be unreliable. One of the main problems in the detection of Salmouella is that many other closely related members of the Enterobacteriaceae, e.g. C. freundii and If. alvei produce false-positive results. The curves produced with the conductance method quite often show only some of the characteristics typical of salmonellas. Immobilization of salmonellas by the antibodies in the IMET procedure introduces an additional selectivity stage into the method. The conductance curve characteristics produced by Salm. enteritidis in the presence of competing bacteria were more defined with the IMET procedure. The incidence of atypical or false positive curves was also reduced by the incorporation of the antibody immobilization stage. False-positive Salmonella results were observed for the conductance method during the IMET evaluation using minced beef thought to be naturally contaminated with Salmonella. This isolate was later identified as C. freundii. Eighty-six per cent of the artificially and naturally contaminated foods showed the same Salmonella detection results for both the cultural method and IMET technique using Bio-Enzabeads. However, Salmonella was only detected in four and five of the eight whey powder samples naturally contaminated with low levels of stressed Salm. virchoTV by the IMET and conductance methods, respectively. This is most probably attributable to the formulation of the Easter-Gibson medium which fails to detect dulcitol-negative salmonellas. It is believed that substitution of antibody-coated Dynal beads for BioEnzabeads in the [MET procedure would improve the agreement in the detection rates between the cultural and IMET methods. The coloured latex agglutination test can be used as a rapid test to demonstrate the presence of salmonellas in selective enrichment broths and from isolated colonies within 10 min. The agglutination test shows significant promise as it is simple to usc and can be applied directly to cultural methods. The latex agglutination test can be used in conjunction with the IMET method to give a more rapid detection system for Salmonella. Such a combination offers advantages of speed, ease of examination, automation and improved selectivity. It has been shown that a more rapid and selective procedure for the detection of salmonellas in food can be achieved by immobilizing the bacteria on antibody-coated supports and subsequently detecting them by conductance methods. A further decrease in the overall detection time can be obtained by

182

J.

the use of shortened periods coloured latex agglutination analytical techniques could particularly those with long high degree of specificity.

of pre-enrichment and rapid confirmation by the technique. The concept of combining different be applied for the detection of other bacteria, protracted procedures (e.g. Listeria) requiring a

A. BIRD ET AL.

Acknowledgement The authors are grateful to the Joint Committee of the Milk Marketing Boards of England and Wales and the Dairy Trade Federation for the preparation and supply of spray-dried milk powders containing Salmonella, and to other colleagues for samples of naturally contaminated foods. Dynal UK Ltd are thanked for the supply of Dynal beads. We arc also indebted to our other colleagues for technical assistance, especially Mrs Carol Wallace (Express Foods Group Ltd) and Dr Naresh Patel (CFDRA). Part of this work was conducted under an extra-mural contract from the Ministry of Agriculture, Fisheries and Food Awarded to Portsmouth Polytechnic.

References \V.H. 1985. A review of culture methods and their relation to rapid methods for the detection of Salmonella in foods. Food Tedmolog}l, March, 77 -1 08. ANON. 1986. Microbiological examination for dairy purposes. Section 3.9. Detection of Salmol1ella. British Standard 4285. London: British Standards Institute. CLAYDEN, lA., ALCOCK, S.]. & STRINGER, l\1.F. 1987. Enzyme Linked Immunosorbent Assays for the Detection of Salmonella in Foods. In Immunological Techniques in Microbiology ed. Grange, ).1\1., Fox, A. & Morgan, N.L. pp. 217-229. Society for Applied Bacteriology Technical Series 24. Oxford: Blackwell Scientific Publications. D' AOUST,j.V. 1981. Update on pre-enrichment and selective enrichment conditions for detection of Salmonella in foods. Joumal of Food Protection 44, 369-374. 0' AOUST, J.V. & .MAISHMENT, C. 1979. Pre-enrichment conditions for effective recovery of Salmonella in foods and feed ingredients. Joumal ofPood Protection 42, 153-157. D;WIS, B.D. 1980. Bacterial nutrition and growth. In Microbiology 3rd edn, cd. Da..is, B.D., Dulbecco, R., Eisen, II.N. & Ginsberg, B.S. p. 63. New York: Harper and Row. EASTER, M.e. & GIBSON, D.M. 1985. Rapid and automated detection of Salmonella by electrical measurements. Joumal of Hygiene. Cambridge 94, 245 - 262. HADFIELD, S.G" JOUY, N.r. & .Mch.LMURRAY, M.B. 1987. The application of a novel coloured latex test to the detection of Salmonella. In bmnu71ologiml Tedmiques ill A1icrobiolob.'Y, ed. Grange, ).M" Fox, A. & lVlorgan, N.L. pp. 145 -151. Society for Applied Bacteriology Technical Series 24. Oxford: Blackwell Scientific Publications. IIALL, L.P. 1979. The isolation of Esc/un'chia coli from frozen food. A new direct plating method. Campden Food Preseroali011 Rest'arch Association Technical iWemoralldum No. 220. HARTFORD, J.P, 1987. An evaluation of a commercially available enzyme immunoassay test for the rapid detection of salmonellae in food and environmental samples. Epidemiology and ANDREWS,

JAY,

IllftttiOt199, 127-136. L.S. & COMAR, D. 1988. Comparative study of TECRA Salmonella visual immunoassay and

S/ILMONELLA IMMUNO/CONDUCTANCE DETECTION

183

Australian standard cultural methods for analysis of salmonellae in foods. Food Technology in

Austratia 40, 186 ~ 191. L:\ ROCHE, R.N. & DESAI, V. 1981. Field evaluation of the membrane filter-disc inununoimmobilization technique in the detection of salmonellae in egg products. Poultry Science 60,

2265-2269. LElFSON, E. 1936. New selenite enrichment media for the isolation of typhoid and paratyphoid (Salmonella) bacilli. American Joumal o/Hygiene 24,423-443. l'vLUTINGLY,].A., ROBISON, BJ., BOEHM, A. & GEHLE, W.O. 1985. Use of monoclonal antibodies for the detection of Salmol/ella in foods. Food Technology 39, 90--94. R, Y, 8., JANSSEN, OW. & BUSTA, 1".1". 1972. Characterisation of the repair of injury induced by freezing Salmollella anatum. Apptied Microbiology 23,803-809. SllAvllNATIlAN, B., DENNER, ].M. & AYRES,].C. 1978. Rapid detection of salmonellae in foods by membrane filter-disc immunoimmobilization technique. Joumal 0/ Food Scimce 43,

1444-1447. V"LLLE, 8.L. & UL:lILR, D.o. 1972. Biochemical effects of mercury, cadmium and lead. Annual

Review

0/ Bioclll7nistry 41, 91- 128.

VAN SCHOTHORST, !'vi. & VAN LWSDEN, F.M. 1975. Further studies on the isolation of injured salmonellae from foods. Zelltmlblau fur Bakteriologie, Mikrobiologie und Hygit1/e, 1. AbteilUllg

OngillaleA 230,1041-1045.

Automated Conductimetric Detection of Salmonellas in Confectionery Products S. J. PUGH! ]\ND M. L. ARNOTT 2 I Cadbury Ltd, Technical Laboratories, Boumville, Birmingham, B30 2LU, UK; and 2 Cadbury Schweppes PLC, Group Research, The Lord Zuckerman Research Centre, The Universil;}1 oj Reading, Reading RG6 2LA, UK

The confectionery industry world-wide has a good record for consumer safety, but over the last two decades a small but significant number of outbreaks of salmonellosis has resulted from the consumption of contaminated chocolate, cocoa and other dry foods (Oden-Johanson 1972; Craven et al. 1975; Gill et al. 1983; Rowe et al. 1987). These have dramatically emphasized the importance of salmonellas to the industry. It is now widely recognized that end-product testing for salmonellas is statistically of very limited use and that the way forward lies with good manufacturing practice, including the application of the concept of Hazard Analysis and Critical Control Point (lIACCP; Simonsen et al. 1987). Testing food samples for contamination by salmonellas will always be necessary, however. For several reasons these tests are normally the most complex and lengthy routine examinations. It has been shown that the infectious dose of salmonellas in chocolate products is very low indeed (D'Aoust & Pivnick 1976; Greenwood & Hooper 1983) and furthermore, the manufacturing processes usually stress the bacteria through desiccation and heat. It is vital, therefore, that the samples are preenriched in a non-selective broth. Selective enrichment is then done in two broths followed by plating on at least two differential media (D'Aoust 1977); eA'Perience in our laboratories has shown that during initial isolations, cultures may be positive in just one brod1 or agar, even though subsequent pure culture gives positives in all of them. This may be due to initial physiological condition or to competitive inhibition from other organisms also present in the food sample. As a result, the minimum test time is normally 3 days but this is often extended by confirmatory tests necessary to eliminate false positives. Copyri}!;h/ Rapid

~licrobiological

Methods for Foods,

Beverages and Pharmaceuticals

185

©

1989 by the Solidi' jilr Applied Bac/eriIlIIlKl' All righls of rcpmdMtitm ilt all)' jimll n·.,,·rod 0-632-0262')-4

186

S.

J. PUGH AND M. L. ARNOTT

This has led to great interest in the development of rapid methods to replace these conventional techniques. A variety of such tests has been reported (Ibrahim & Fleet 1985; Pugh 1987) based on immunology, serology and genetics, etc. but none offers the right combination of low cost, simplicity and high sensitivity required for routine use in our quality assurance laboratories. Enzyme-linked immunosorbent assays (ELISA), for instance, are available at £5-6 per test and require at least 10s-106 cells/ml for a positive result. The measurement of impedance, conductance and capacitance changes due to microbial gro\\th (Firstenberg-Eden & Eden 1984) is becoming widely accepted in Europe as a rapid method. The Bactometer Microbial Monitoring System (Vitek Ltd, lfenlcy-on-Thames), which can monitor all three types of change, is one of two commercially available instruments which work on this basis. The other is the Malthus Microbiological Growth Analyser (Malthus Instruments Ltd, Crawley) which can only measure conductance. In these electrical techniques, specially developed media are required for detection of salmonellas because conventional broths, such as tetrathionate, selenite-cystine and those of Rappaport, do not give satisfactory signals. Some suitable media have been developed by Firstenberg-Eden & Eden (1984) and Easter & Gibson (1985) hut most have significant shortcomings. For example, the selenite-cystine-trimethylamine oxide-dulcitol medium (SC/T ID) of Easter & Gibson (1985) performs well but does not detect strains unable to ferment dulcitol, and so cannot represent, on its own, a complete test. Most salmonellas are able to decarbo~1'late lysine (Farmer et al. 1985) and a selective broth based on this characteristic has been developed as reported here. A test which has a completely different basis was adapted for use in the Bactometer by Firstenberg-Eden & Eden (1984) and Stannard (1984). This compares the response produced by a sample inoculum with and without Salmonella-specific bacteriophages. The lysis ofany salmonellas present causes a delay in the instrument detection. The combination of SC/T /D, modified lysine decarboxylase broth (MLD) and phage tests gives a complete protocol for the rapid detection ofsalmonellas in food samples.

Description and Principles AU experiments were done on a Bactometer 1\1123 with incubation of modules at 35°C for 24 h. One mt of each medium was used for each test and the inoculum was 0.1 ml of diluted culture or pre-enrichment broth. All cultures were from the Cadbury Ltd collection and were maintained on nutrient agar slopes at ambient temperature. They were from a variety of sources, mainly

CONDCCTIMETRIC DETECTION OF SAU\10NELLAS

187

food, and include salmonellosis outbreak strains, such as Salm. napoli. When tested in pure cululre, strains were grown overnight in Brain Heart Infusion Brotll (BIll, Oxoid, diluted 10- 4 in Maximal Recovery Diluent (MRD, Lab M, Bury) and 0.1 ml inoculated directly into wells or into brilliant green-milk broth (BGMB, containing ultra-heat treated liquid skimmed milk, and brilliant green at 0.002%). To simulate our typical routine situation salmonella-free cocoa powder was included to 10% (w/v). Many samples of chocolate, ingredients and dried products were artificially inoculated with salmonellas and tested after drying and storage. Naturally-contaminated samples were included where possible.

Sclenite-cystine-trimethylamine oxide-dulcitol medium This medium (SC/T10) contained (gil): bacteriological peptone (Oxoid L37), 5.0; Na zHP0 4 ·2H zO (BDH 10393 4G), 10.0; dulcitol (Sigma 0 0256), 5.0; trimetl1ylamine oxide hydrochloride (BOH 30517), 5.6; sodium biselenite (Oxoid LI21), 4.0. Adjustment of pH to 7.2 ± 0.1 was done after prefiltration (Whatman No.6) but before membrane filtration (Minisart 0.2 !lm, Sartorius, Belmont). Steaming for 10 min is an alternative sterilization metl1od. The medium is stable for at least 7 days at 4- rc. Before use add L-cystine stock solution at the rate of 0.1 ml per 10 rol. This is made up by dissolving 0.1 g L-cystine (Sigma C 8755) in 15 ml of 1 molll NaOH solution, filter-sterilizing and adding 100 ml of sterile distilled water. Generally tl1is medium performs well in our laboratories although biphasic conductance curves are sometimes produced which are difficult to interpret. It requires tl1e ability to ferment dulcitol and to reduce trimethylamine oxide in the presence of 0.4% sodium bisclenite. Most salmonellas give good results but we have found some strains, such as Salm. typhimurium ATCC 14028, which are dulcitol-negative and so are missed. This characteristic occurs in up to 5 Oft) of clinical strains (Farmer et al. 1985) and those isolated from food are likely to be similar. "\Then non-salmonellas do grow tl1ey produce generally weak responses and so curve criteria indicating fast growth (rate of change) and e:\1ensive growth (magnitude, or total change) can indicate salmonellas (Fig. 1). Many trials in our laboratories have shown that tl1e latter nearly always produce curves exceeding 25 unitslh rate and 250 units total change. Easter & Gibson (1985) recommended supplementing buffered peptone water (the pre-enrichment broth) with dulcitol and trimethylamine oxide hydrochloride (TMAO.HCl) when using SC/T10. Table 1 shows some of our data which suggest that tl1is is nol necessary when BGMB is used as the pre-enrichment broth.

188

S.]. PUGH AND M. L. ARNOTT

170

~

schwarzengrund

150 130 D u 110 c co u 90 :J Q)

...

c

\)

C

0

u

70 50

---

A

30

Escherichia coli

10 0

2

4

6

8

10

12

14

16

18

20

22

24

26

28

Time (h)

FIG. I. Conductance curves from Salmonella schwarzengrund and Esdzen'cllia coli in sdenitecystine-trimethylamine oxide-dulcitol medium (SC/T/D). Total change is from DT, A, to peak B, calculated as value on conductance scale at B minus value at A, multiplied by the step. Rate of change is slope for 1 h calculated as conductance value at D minus value at C, multiplied by the step. Positive curves exceed 250 units and 25 units/h, respectively. Base = 3668, step = 3.90.

TABLE 1. Efficts of incorporating dulcitol and trimethylamine oxide hydrochloride in pre-mrichment broth on subsequent Salmonella detection itl selenite-rystine-trimethylamine oxide-dulcitol broth

Detection rime (h) for

Strain

Salmonella sp. (SIS) (S33) enteritidis (SI9) guinea (5L46) houtm typlzimurium (517) typhimurium (5L34)

BG~lB

BGi\1B/D

BG~IB/D/T

13.5 6.8

11.6 6.2

13.9 5.8

7.6 NDT 7.8

6.9 NDT

6.6 NDT

6.2 NDT

6.4 NDT

NDT

Pre-enrichment media: BGMB, brilliant green - milk broth; BG1\ilB/D, BGMB plus 0.5% dulcitol; BGMB/D/T, BGMB/D plus 0.5% trimethylamine oxide. NDT, no detection in 24 h.

CONDUCTL\lETRIC DETECTION OF SALMONELLAS

189

Modified I)lsille daarbo;r)lIase broth A range of basal media containing a single carbohydrate (sucrose, rhamnose, :-.ylose, mannitol or dulcitol) or one amino acid (ornilhine or lysine) were screened for performance complementary to SC/TID. Any showing promise were furlher tested after inclusion of selenite, tetralhionate or malachite green. In each case, the effects of oxygen tension and pH on the impedance, conductance and capacitance signals were also studied. A lysine decarbo:-'ylase broth gave excellent results and as about 96(7'0 of salmonellas arc able to decarbm,ylate lysine, this medium was developed further. The tinal version (MLD) was prepared as follows. Make up the following aqueous wlv stock solutions: sodium biselenite, 8%; ferrous ammonium sulphate (Sigma F 3754), 0.75'10, sodium thiosulphate (Sigma S 8503), 0.75'10; bromcresol purplc (Sigma B 4263), 0.16'10, pH 7.0. (If the ferrous ammonium sulphate solution precipitates it should be discarded and fresh made up after adjusting lhe water pI I to 5.0.) Add 1 ml of each stock solution to 80-90 ml de-ionized water in a 100-ml volumetric flask, mixing after each addition, and then add furlher water to 100 ml. Mix well and use this solution to dissolve yeast extract (Oxoid/Oifco), 0.3 g; dextrose (Oxoidl Oifco), 0.1 g and L-lysine monohydrochloride (Sigma L 5626), 0.5 g. Filter as SC/T10 but adjust pH to 6.1 ± 0.05. Most interfering organisms (mainly Esdlerichia coli and Klebsiella spp.) are suppressed in MLO without adversely affecting salmonella curves. With the same curve characteristics as for SC/T /0 but values of 10 unitslh rate and 100 units total change, salmonellas are clearly detected (Fig. 2).

Phage test Cherry et al. (1954) reported the use of the 0-1 phage of Felix & Callow (1943) for the identification of salmonellas. The phage was applied to a culture of the suspect strain on the surface of an agar plate. Visible plaques were formed within 6-18 h if the test was positive. Fey et al. (1978) quantified the specificity of a modified 0-1 test and found that 96.1 ok, of 22800 Salmonella strains (representing 304 different species) were lysed. FirstenbergEden & Eden (1984) modified this test for use in the Bactometer and shared their knowledge with us, which is the basis of the method we use. A second phage, G~7' has now been obtained, propagated and included, as recommended by Glidel & Fey (1981) because the 0-1 tends not to lyse salmonellas in o groups E I to E~. In the Bactometer test, duplicate wells of a slightly selective dulcitol medium, DULE, (containing (gil): Bacto-Tryptone (Difco), 34.0; BactoSoytone (Difco), 6.0; dulcitol (Signla D 0256), 5.0; K2HPO~ (Sigma P 5504),

190

S.

J.

PUGH AND M. L. ARNOTT

170

B

150

Salmonella sch warzengrund

130 ~

c

110

0

ro

+J

u J

90

"0

C

c 0

u

70

50

~----

Escherichia coli

30 10

o

2

4

6

8

10

12

14

16

18

20

22

24

26

28

Time (hl

2. Conductance curves from Salmonella Sdlll'arzl'11grund and EStherichia coli in modified lysine decarboxylase medium (MLD). Curve criteria defined as \\ith selenite-cystine-trimethylamine oxide-dulcitol medium, but using positive threshold values of 100 units total change and 10 units/h rate of change. Base = 1210, step = 2.70. FIG.

2.5; MgCl z (Sigma M 0250), 0.4; phenol red (BDlI 20091 3V), 0.5; sodium desoxycholate (Oxoid), 0.1; bile salts No. 3 (Oxoid), 1.0) are prepared and 0.1 ml of phage suspension is added only to the second well. Both wells then receive identical sample inocula and any salmonellas present, if sensitive, are lysed by the phage. This usually causes a delay in detection time (DT) of at least 1 h (Fig. 3). In practice, we have seen phage-induced conductance curve effects supplementary to DT delay with some strains or mixed cultures, as have previous workers (Stannard 1984, 1985). These are a premature reduction of rate of change (or plateau) and a delay in the onset of gas production (Fig. 4). The lattcr effcct is seen most dearly if, with the aid of a Pasteur pipette or wire loop, a small drop of molten paraffin wax is placed on the well bottom bctween the electrodes (Fig. 5). This is done in all wells for the phage test before adding the media. The gas bubbles produced by dulcitol fermentation stick to the wax until they become too large and suddenly float up to the surface. This fluctuation in the conductance between the electrodes causes visual disruption of the signal. Two factors are important in the phage test if consistent results arc to be obtained. Firstly, to obtain lysis-from-without the multiplicity of infection needs to exceed 100: 1 (phages:bacteria) and so the phage stock must contain

191

CONDUCTIMETRIC DETECTION OF SALMONELLAS

170

......-._ _~ No phage __ No phage . With phage

150 130 Ql

u

110

,)

i

c:

i i i

....'"U 90 ::J

"tl

c: 0

u

j

70

i

i

i.

/1 _ _I

50 30

I

I..

I.

.....s-~'=-':::;::!..j.... -I

o

2

Salmonella typhimurium __ - - - - - - - - - - - - - -

-------- ~------

6

4

8

10

12

14

16 18

20

22

W'Ith phage

24

26

28

Time (h)

3. Conductance curves from Salmonella typhimurium and Escherichia eoli in the phage test. The salmonella shows a typical positive reaction of dclay in detection time (DT), here at its most extremc with complete elimination of growth response in the well with phage. Base = 2540, step = 7.13.

FIG.

170 150

Ql

110

/-A--'- ----

u

c:

~

<.>

c:

0

/

/

/

/

_/

90

,

::J

"tl

,

B •

130

70

/

u

r/ I

50

I I

f

30

I

I f

I

10 0

--=-==---.,. ./ 2

4

y

I

6

8

10

12

14

16

18

20 22

24

26

28

Time (hl

FIG. 4. Visual-positive conductance curves from the phage test. Premature plateau, i\, and delay in onset of gas production, B to C, shown in positive curves with DTs differing by less than 1 h. Base = 2687, step = 7.13.

192

S.

J. PUGH AND

M. L. ARNOTT

FIG. S. Bactomerer module showing spots of paraffin wax (one arrowed) between the electrodes in wells 3 r 4, 7, 8, 11, 12, 15 and 16 rcady for phage tests.

at least 109 pfu/ml lllinimum. Secondly~ the metabolites produced by the host bacteria must be renloved to prevent non-specific inhibition. Phage stocks produced as below Ineel these criteria.

Phage preparation and pu nji'attion Grow at 35°C an 18-h culture of the host sahnonella (any strain that !,rives good plaques on plates will do) in 10 ml of basal dulcitol tnedium (BDJ\'I, containing (gIl): Bacto-Tryptone (I)ifco), 34.0; Bacto-Soytone (Difco), 6.0;

dulcitol (Siglna f) 0256), 5.0; KlJ-IPO.. (Sigma P 5504), 2.5 and i\!lgC1 2 (Sigma ~1 0250), 0.4). Inoculate 0.1 ml of this culture per 100 ml of fresh BD~1 and incubate at 35°C on a shaker or in a shaking water bath at about 100 rev/min until a light swirling turbidity is observed. This equates to about lOi cfu/nl1 and should takl~ 2-3 h. \vben the culture reaches this point add 0.2 nil of existing phage preparation (containing at least 109 pfu/mI) per 100 rnl. Continue shaking at 35°C lor 6-18 h. Centrifuge the lysate twice for 1 h at 3000 g to relllove much of the cell debris and viable cells. Decant and save supernatant fluid. Add ] Ill] of chlorofonn per 100 ml of lysate and incubate at 35°C for 1 h, then decant the lysate, taking care to exclude all the chloroform, and filter-sterilize. This is most easily done using a series of

193

CONDUCTIMETRIC DETECTION OF SALMONELLAS

filters with decreasing pore size, e.g. 0.8 ~lm, 0.45 /lm and then 0.2 ~tm. A titre may be done at this point to check that the phage concentration is at least 109 pfu/ml (Fih'S 6 and 7). Metabolite filtration and phage concentration is performed with an ultrafiltration cell (Model 8200, Amicon, Stonehouse) and PM30 membrane with a molecular weight cut-off of 30000 Daltons (Fig. 8). The lysate is thereby reduced to about 20'Yo of its original volume. It is possible at this stage to dilute the concentrated lysate 1 in 10 with sterile BDM, as long as the final titre still exceeds 109 pfu/ml. It is then filter-sterilized once \more and refrigerated. If Ule 0-1 and G+7 phages are both to be used, they are

9 ml MRD diluents

LysatH

10

1

10"

10 3

10 4

10 5

10 6

10'

10 8

"'"'I "' m'l "m'j "m'l "' m'j '"" " ' ' ' ' ' ' " ,>"C ",,,,

o 1 ml host salmonella

!d IItJ lj I

II

"T~

l 8 lj r

~

!!

1r

1r

j j

0 0 () 0 0

BHIA plates Ibase layer) 10·

5

10

6

10 7

10 8

10 9

Control

Incubate at 37°C from 6 h to overnight

FIG. 6. Determination of the titre of phage lysate. MRD, Maximal Recovery Diluent; BI 11i\, brain - heart infusion agar; O.7'j(, BI IIi\, BI ILl,. containing 7 gil agar ('sloppy' agar). .'\fter incubation, plaques on suitablt· plates are counted and multiplied hy the dilution factor to give the titre in pfu/ml.

194

S.

J.

PUGH AND M. L. ARNOTT

FIG. 7. ~Sloppy' BHIA plate of host Salmonella with added diluted phage, as prepared during the dctemlination of lysate titre. Lysis of the bacterial cells in a plaque is seen as a dear zone 1- 2 ml11 in diameter.

produced separately and combined 9 final filtcr-steriliz3 tion.

pa11s

0-1 to 1 part G·47 just before the

Inoculum dilution During protocol development we stopped using lhe 1:40 inoculum-ta-medium

ratio specified for SC/T ID by Easter & Gibson (1985). vVith a 50-~tl inoculum this had ~quated to a 2-ml volume of medium in the wells and this frequently caused leakage during handling. The largest inoculum possible has to be used to ensure detection even when pre-enrichment leads to abnormally low cell concentrations but interference from pre-enrichment broths must be avoided. A sinlple protocol which did this and also coped wit.h the normal high cell concentration was desired. \Ve found that optimal results are obtained by diluting the BCilVIB culture 1: lOin MRI) for the SC/T/D and fvlLD wells, and 1:1000 for the phage test.

CONDUCTlMETRIC DETECTION OF SALMONELLAS

FIG.

195

8. Ultrafiltration cell ready lor use with magnetic stirrer and nitrogen cylinder.

Method Evaluation

Pure wIll/res A total of 81 salmonella strains and 39 non-salmonellas were tested in the protocol, as shown in Tables 2 and 3. In general, the negative reactions were evenly scattered amongst the salmonella cultures, but Sainz. guinea was an exception. All five strains tested were phage-negative and this adversely distorts the results. Although one sub-genus 4 su'ain (Sainz. houten) was detected only in one of the three tests (MLD) the protocol as a whole did not miss any salmonellas. Blackburn & Ellis (1973) reported that 15.6% of 552 salmonellas isolated from dried milk products and milk-drying plants were able to ferment lactose. These organisms are easily missed by conventional methods, particularly if they are unable to produce hydrogen sulphide. The irrelevance of these two

196

s. J.

PUGH

2. Reactions of salmonellas

TABLE

Organism

Salmonella sp. alachua anatum anatum arizonae braenderup campinense choleraesuis choleraesuis eastbourne enteritidis enteritidis ftrlac gallinarum guinea hatfield houten infantis italy johannesbu rg kentucky leeuwarden litchfield litchfield litchfield livingstone london montevideo montevideo napoli newport panama paratyphi para~yphi

saintpaul schwarzl71grund senflenberg senfienberg tennessee letmessee thompson ~yphimurium

(yphimurium

AND M. L. ARNOTT

No. of strains*

SC/T/D

MLD

Phage test

13 I 1 1

+ + + +

+ +

+

2 1 1 1 I 1 3

1 1 1

5 1 1 3

I 1 1

I I 1 1 1 1

4 1

1 1 3 I

1 2

+ + + + + + + + + +

+ + + + +

+ +

+

+

+

+

+ + +

+ +

+ +

+ +

+ +

+

+ + + +

+ + + + +

+ + + + + + + + + + +

+ +

+

+

+

+

+ + + + + +

+ + +

1 2

+

7 2

+

3 1

+

+ + + + + + + + + + + +

+ + + + + +

1 2

+ + +

+ + +

+

+ + + + + + +

"* ,",bich gave the same pattern of results. SC/T/D, selenite-cystine-trimethylamine oxide-dulcitol medium. MLD, modified lysine decarboxylase broth. All cultures: Cadbury Ltd Culture Collection.

CONDUCTIMETRIC DETECTION Of SALMONELLAS T.'IIlI.E

Organism

Badlllls eerells Cilmba{/er allla/mlii/ims diverslls ji'CIl11dii jYeliudii E/llt'robacler c!oawt saka;:;akii Fsclierieliia wli

afrugiuosa

Serratia

3. Rm{/io/ls o( /lO/l-sa/ullmd/as !\o. of strains*

SCiT/D

martc.,\(.:fllS

Shif!,d/a jlexneri SlapIiY/lJam'llS allrells epidennidis

[,,[l.D

Phage test

I I I

5 5 2

+

I ')

3

w!i K/cbsid/a p/lell/lll!lliat Pmltlls sr. vil/}!.aris Pselldm/lonas aerll}!.iulJsa

197

2 I I I I 2 I

+ +

+

I I

* Which gaye the same pattern of results. Abbreyiations: sec f()()tnote to Table 2. All cultures: Cadbury [.td Culture Collection.

characteristics in the Bactometcr proIocol means that such organisms are detected without problem. A lactose-positive IlzS-negative strain of Saltlt. 1011dol1 was included in the pure culture screen and was positive in all three tests. Citrobacter freundii gives false-positive reactions in many conventional salmonella tests and indeed five strains did so in SC/T10, but a further five did not. The false-positive reactions in MLD were given by E. coli (three strains), Proteus vulgaris (I) and Pseudomonas aeruginosa (1).

I/loculated samples After pre-enrichment in BGMB, the inoculated samples were tested by conventional and Bactometer methods. The former involved selective enrichment in tetrathionate and mannitol-selenite broths followed by streaking-out on XLO and Hektoen Enteric agars (all Oxoid). The results are given in Table 4.

Confirmatory procedures Presumptive positive reactions were confirmed by plating the contents of wells on XLO and IIektoen Enteric agars. This can be done before the 24-h test is

198

s. J.

PUGH AND M.L.

TABLE

ARNOTT

4. Results from inoculated samples* Bactometer tests

Sample and inoculum Skimmed milk powder Salmonella eastbourne illfimtis euteritidis napoli ~yphimurium

Conventional test

+ +

SC/T/D

MLD

Phage test

+

+ + + + +

+ + + +

+

+ + + +

+

+ +

+

+

+

Uninoculated control Egg powder Salmonella cas/bourne in/antis enteritidis napoli typhimurium Uninoculated control Milk chocolate erumb Salmonella eastbourne il1fimtis enteritidis napoli ~yphimurium

+

+ + + +

+ + + + +

+ + + +

+ +

+

+

+

+

+

+ +

+

+

+

+ + +

+ +

+

+ + +

+

+

Uninoculated control Milk chocolate Salmonella easlbourne itlfimtis enteritidis napoli ~yphimun'um

+ + + +

+

+

+

+

+

+

+

+

+

+

+

+

t-

+

Uninoculated control Dark chocolate Salmonella eastboume ill/antis enteritidis napoli typhimurium Uninoculated control Fondant-filled chocolate egg Sal11l01u:llil eas/boume ;njil11tis ttlterilidis

199

CONDUCTIMETRIC DETECTION OF SALMONELLAS

Ilapoli (yphimurium Uninoculated control Cocoa powder Salm(mella illfanlis e1l1erilidis llapoli lyphimurium Uninoculated control

+

+

+

+ + +

* Bactometer tests which gave positive curves but which did not contain recoverable salmonellas are not included. Abbreviations: see footnote to Table 2.

over if the module is briefly removed from the Bactometer processing unit. If this is done within a few hours of the test starting then the plates are available for examination at the same time as the finished Bactometer curves. Otherwise an extra day is added to the test duration. Confirmation of suspicious colonies was by serology and the use of biochemical test strips (API-20E, API BioMcrieux, Basingstoke or Micro-ID, Organon Technica, Cambridge. On some occasions, positive MLD curves were produced but no colonies appeared on the confirmatory plates - the salmonellas appeared to die out relatively quickly. To avoid this it was suggested that all three tests (excluding the phage-containing well) should be plated if any were positive. However, as usually only one well is presumptive positive, it would now appear sensible to do the following: Presumptive positive SC/TID Presumptive positive MLD Presumptive positive phage test

plate out SC/TID and phage-free DULE well plate out MLD and phage-free DULE well plate out SC/TID and phage-free DULE well

Should more than one test be presumptive positive then all three should be confirmed. We are keen to reduce the time taken by confirmation; novel rapid methods, such as ELISA and modified antisera, are currently under evaluation. These would reduce the test time to 2 days. Conclusion We have found that the Bactometer protocol is a reliable, effective replacement fl1r conventional methods of detecting salmonellas. It significantly reduces the test time and so allows earlier release of raw materials and finished goods.

200

S.

J.

PUGH AND M. L. ARNOTT

The method has a higher sensitivity but a lower cost (disposables amounting to about £1 per test) than the ELISA and DNA - DNA hybridization techniques. Laboratories that test a large number of samples can on this basis justifY the high capital cost of the Bactomcter for salmonella testing alone, but the instrument is becoming increasingly popular because it can be used for many other purposes as well.

Acknowledgement Mr C. Davda and Mrs Y. Watts arc gratefully acknowledged for their technical assistance; thanks are also extended to Dr G. Meier, University of Bern (G.t? phage and host), to Bactomatic Inc. (0 - 1 phage, original phage methodology and some strains) and to the many people who have donated strains to our culture collection.

References RD. & ELLIS, £.M. 1973. Lactose-fermenting Salmonella from dried milk and milk-drying plants. Applied /l1icrob;olob.'Y 26, 672-674. CHERRY, W.o., DAVIS, R.B., ED\VARDS, P.R. & HOGAN, R.B. 1954. A simple procedure for the identification of the genus Salmonella by means of a specific bacteriophage. ]ounlal vf Lahortltory and Clinical Medicine 44, 51- 55. BLACKBURN,

CRAVEN,

P.C,

MACKEL,

D.C.,

BAINE, W.B., BARKER,

\V.H.,

GANGi\ROSA,

Ej.,

GOlDFIELD,

N1.,

11., ALT'\1AN, R., LACHAPELU:, G., DAVIES, ].W. & SWANSON, R.C. 1975. International outbreak of Salmot/ella castboume infection traced to contaminated chocolate. Lancet I, 788-793. D'AOUST, j.Y. 1977. Salmonella and the chocolate industry. A review. ]oumal of Food ProJection ROSENFIELD,

40, 718-727. j.Y. & PIVNICK, H. 1976. Small infectious doses of Salmouella. Lancet 1, 866. EASTER M.C. & GIBSON, D.M. 1985. Rapid and automated detection of Salmonella by electrical measurements. Joumal of Hygiene, Cambn'dge 94, 245-262. FAR.i\1ER, J.J., DA\'IS, B.R., HICKMAN-BRENNER, F.W., MCWHORTER, A., }-IUNTLEY-CARTER, G.P., ASBURY, .M.A., RIDDLE, c., WATHEN-GRADY, ILG., ELIAS, c., F.t\NNING, G.R., STEIGERWALT, A.G., O'HARA, C.M., MORRIS, G.K., S.\1ITH, P.B. & BRENNER, Dj. 1985.

D'AOUST,

Biochemical identification of new species and biogroups of Enterobacteriaceae isolated from clinical specimens. Journal of Clil11l:aJ A1icrobiology 21, 46-76. FELIX, A. & C,\LLO\V, B.R. 1943. Typing of paratyphoid B bacilli by means of Vi bacteriophage. Bn'tish Medical }ouma[ 2, 127-130. FEY, I-1., BURGI, E., MARGADANT, A. & BOLLER, E. 1978. An economic and rapid diagnostic procedure for the detection of Salmonella and ,Shigella using the polyvalent salmonella phage

0-1. Zmlrallblilll ./iir Bakler7oJogie, A1ikrobioJogie ulld Hygiene, J Ableilrmg Or;ginale A 240, 7-15. F1RSTENBERG-Em':N, R. & EDEN, G. 1984. Impedalla: }1'1iaubio!v!fY. Letchworth: Research Studies Press. BARTIJ'TT, C.L.R., VAILE, M.S.B., ROWE, B., GILBERT, R.j., fl.C. & SAl.lv1ASO, S. 1983. Outbreak of Salmonella napoli infection caused by contaminated chocolate bars. Lancel 1, 574-577.

GILL,

a.N.,

DULAKE,

SOCKETT,

c.,

P.N.,

MURRELL,

CONDUCTIMETRIC DETECTION OF SALMONELLAS

201

GREENWOOD, M.H. & HOOPER, W.L. 1983. Chocolate bars contaminated with Salmouella uapoli: an infectivity study. Brilish Medical Joomal 286, 1394. GOIlEl., K. & FEY, I I. 1981. Improvement of the polyvalent salmonella phage's 0- I diagnostic value hy addition of a phage specific for the 0 groups E 1 - E 4 • Zmtrallblall fur Bakleriologie,

Mikrobiologie and Hygime, I Ableilong Origillale A 249,220-224. G.F. & FI.EET, G.H. 1985. Detection of salmonellae using accelerated methods. IlIlemalional Joomal ofFood /'vlicrobiolo/,,'Y 2, 259-272. ODEN-JOHANSON, B. 1972. An epidemic of Salmonella dorham caused hy contaminated cocoa. Liikartidingm 69, 5335 - 5338. PUGH, S.]. 1987. Rapid Microbiological Ivlethods. In Food Tedl1lolo/,,'Y IUlematiOllal Europe, cd. Turner, A. pp. 259-261. London: Sterling Publications Ltd. ROWE, R, BEGG, NT., HUTCHINSON, D.N., DAWKINS, I I.e., GILBERT, R.]., JACOB, M., lIAI.ES, B.H. RAE, F.A. & JEPSON, M. 1987. Salmouella ealiug infections associated with consumption of infant dried milk. Lancel 2, 900-903. . SI\10NSEN, B., BRYAN, F.L., CHRISTIA"J, ].H.N., ROBERTS, T.A., TOMPKIN, R.B. & SIl.LIKER, J .II. 1987. Prevention and control of food-borne salmonellosis through application of Hazard Analysis Critical Control Point (IIACCP). IlIlematiolla! Joomal of Food MiL'robioiogy 4, 227-247. STANNARD, c:.]. 1984. Development and usc of rapid microhiological methods in food quality assessment. PhD lhesis, University of Surrey. STANNi\RD, e.]. 1985. Development of a Rapid EICl1rical Detectioll Melhod for ~aI11lollella. Research Report 511. Leatherhead: British Food Manufacturing Industries Research IIJRAHI~I,

Association.

A Medium for Detection of Lancefield Group D Cocci in Skimmed Milk Powder by Electrometric Methods P. NEAVES, M. J. WADDELL" AND G. A. PRENTICE Techniral Division, Milk Marketing Board, Thames Ditton, Surrey KT70EL, UK

In the food industry, there is a movement away from traditional microbiological techniques towards instrumental methods for the detection of micro-organisms. This change has occurred largely because developments in the application of computers have made easier the automation of microbiological investigations. During the last decade, electrometric instruments have become relatively sophisticated and increasingly abundant in factory laboratories. The Bactometer and Malthus instruments, which are the most common, have both found applications for monitoring production processes and for testing finished products. Their use is restricted, however, because of a lack of suitable selective media. Apart from coliform media, there are few commercially-available media for indicator organisms and pathogens. The commercial production of skimmed milk powder requires strict microbiological control. After pasteurization, milk is concentrated by evaporation and spray- or roller-dried. Under poor production conditions, Lancefield Group D cocci may grow during the manufacture of dried milk powders. A number of major food companies include tests for them in their specification for skimmed milk powders. This work was therefore undertaken to develop a medium for the detection of Lancefield Group D cocci in skimmed milk powder with elcctrometric instruments. Preparation of Skimmed Milk Powder Contaminated with Lancefield Group D Cocci Commercially-produced skimmed milk powder was re-constituted with sterile distilled water to give 30% (w/v) total solids. Actively-growing cultures in Nutrient Broth (Oxoid), of Enterococcus Jaecalis (NCIB 775), Ent. Jaecium " Present address: Dale Farm Foods, Progress Drive, Flash Lane, Bramley, Rotherham, S. Yorkshire 566 OTU, UK. Copyright Rapid l'vlicrobiological Methods for Foods, Beverages and Pharmaceuticals

© 1989 by the Society fiJr Applied BtUurioloJ(Y All rights '1 reproduction in a".y jimn resaved 0-632 -02629-4

203

204

p . NEAVES E1' AL.

(NCIB 662), Streptococcus bovis (NCDO 597) and Strep. equinus (NCDO 1037) were separately inoculated into 1-1 batches of the reconstituted milk. After inoculation, the numbers of micro-organisms were c. 103 cfulml. After incubation at 37°C for 7-10 h, the numbers had increased to c. 108 cfu/ml. The cultures were then spray-dried using a laboratory-scale spray-drier (Biichi Ltd, Flawil, Switzerland). The inlet temperature was 182 - 185°C and the flow ratc was c. 1 I/h. Colony counts of Lancefield Group D cocci from the concentrates after incubation and from the powders were made using KF Streptococcus Agar (Difco) according to the British Standard method (Anon. 1984). From these, the death rate during spray-drying was determined. Survival of Lancefield Group D cocci during spray-drying

The survival of Lancefield GTOUp D cocci during spray-drying is shown in Table 1. The expected levels of Lanccfield Group 0 cocci in powder were derived by correcting the observed colony counts in skimmed milk concentrate for the reduction in volume due to evaporation of water. The differences between actual and expected levels in powder thus represent the death during spray-drying. Since all of the species were fairly resistant to spray-drying, powders containing high levels of contamination were prepared. These results suggest that spray-drying has little effect on Lancefield Group 0 cocci. This may be attributable to the short residence time or to the product temperature which remains relatively low due to cooling by evaporation of water. TABLE

1. Suroival of Lancejidd Group D cocci in pmvder spray-dried in the labora/ot)' Colony count during processing (loglO cfu/g or ml)* Ent. ftetalis

Skimmed milk concentrate (30% w/v total solids) Expected level in powder Level detennined in powder Net loss during spray-drying (log (.'}"des)

£11/.

faeciurn

S/rep. b(fVis

Strep. equinus

8.11

8.04

8.11

8.00

8.63

8.56

8.63

8.52

5.45

738

7.23

6.97

3.19

1.18

1.40

1.55

'*' Colony counts of Lancefield Group D coed for artificially inoculated powders.

INSTRUMENTAL DETECTION

or

ENTEROCOCCI

205

Use of Conventional Selective Media in Electrometric Instruments A variety of media formulations, containing various selective agents, has been proposed for detecting Lancefield Group 0 cocci by enrichment or colony count techniques. Broth versions of four of these media were chosen as a basis for an electrometric medium. The initial assessment and development were undertaken using the Bactomeler instrument (model MI23). The media employed were: KF Streptococcus Broth (Difco); Kanamycin AesculinAzide Broth (Oxoid); Slanetz & Bartley Medium (Oxoid); and thallous acetate broth (Barnes 1956; modified). The thallous acetate broth was modified by omitting the tetrazolium salt and agar. It contained (gil) : Proteose peptone (Difco), 10; Lab-Lemco (Oxoid), 10; glucose, 10; thallous acetate, 1.0; pH 6.0. The sterilized media were dispensed aseptically into Bactometer modules (2 ml/well) and separate wells were inoculated with actively-growing pure cultures of the Lanceficld Group D cocci. Conductance and capacitance signals were monitored during incubation at 37°C.

Quali~y

of response curves with conventional media

Since all lour species grew on KF streptococcus agar, all would be detected if present in a sample. Their responses in the electrometric medium must therefore be similar. The detection times for the same inoculum level and the magnitude and rate of conductance change should be similar for all species, otherwise only that organism which produces the strongest or fastest response may be detected. This may occur even when it is not the predominant species. In each of the four media, capacitance responses for the different species varied considerably; Ent. jaecalis produced strong capacitance curves in all formulations with 'Step' values (which indicate the scale of the .)I-axis) in the range 9-18 whilst the remaining three species produced much poorer curves. Thus, these would probably not be detected if Enl. faecalis were present in a sample. The conductance responses were generally similar lor all four species although 'Step' values were lower and in the range 3 -4. Streptococcus equinus failed to produce a conductance response in either Sianetz & Bartley medium or kanamycin-aesculin-azide broth. Thallous acetate broth showed the shortest detection times, strongest accelerations and least variations between the four species. These curves are shown in Fig. 1. It was decided that the conductance signal in thallous acetate broth was the most appropriate for further development.

206

P. NEAVES ET AL. -170 (J)

... ..... -..

Base = 1992 Step:::: 3.27

+'"

'§ 150

.....

>-

~ 130 :0

.e 110

, , .,-' t

I

90

,• ,,,

co

"5

70

,,

Q,)

g co

50

I

+oJ

U

.g c o

~

,

Q,)

g>

...

30 ~ ~........._...,--

U

2

4

6

8 10 12 14 16 18 20 22 Incubation time (h I

FIG. 1. Conductance responses for broth cultures of Streptococcus bovis (--), Stnptococcus equinus ( ), Enterucoccus fteaum (- - - -) and Enterococcus jaecalis ( ) in thallous acetate broth.

Medium Development for Electrometric Detection The effect of medium composition on conductance curve profiles was investigated for all species. Initial experiments employed double-strength media inoculated (50:50) with pure cultures in reconstituted skimmed milk. Subsequent investigations used powders containing Lancefield Group D cocci and spray-dried in the laboratory. A 10·,·,1 dilution of each powder was prepared directly in the conductance medium by adding 1 g of powder to 9 ml of broth, incubating at 45°C for 15 min and shaking vigorously to dissolve the sample. Bactometer modules were then inoculated with 2 mllwelL. Three aspects of the medium composition were investigated. Because the major conductance responses may be the result of the metabolism of nitrogen compounds, the peptone and yeast extract content of the medium was modified to promote conductance curves showing a rapid change with a large amplitude. The effect of pH on detection time was investigated because this affected the rates of detection for the different species. The concentration and types of selective agent were adjusted to maintain selectivity without destroying the curve quality. Details of the experimental procedures are shown in Table 2. Colony counts of Lancefield Group D cocci were also made on the same sample dilutions, to enable detection time and inoculum level to be compared. From the results of this series of experiments, a medium was derived which produced conductance curves of a high quality.

INSTRUMENTAL DETECTION OF ENTEROCOCCI TABLE

207

2. Concentrations 0/ companmts employed in modifying tlte conductana: medium

Component Nitrogen source Proteose peptone (Oxoid) Special peptone (Oxoid) Trypticase peptone (BBL) Skimmed milk powder Urea Yeast extract Selective agent Sodium azide ThaUous acetate pH value

Concentration range (0;;, w/v)

1-7 1-3 5

10 1

1-10 0.02-0.04 0.1-0.4 6.0-9.0

Effect of Medium Composition on Curve Quality In pure culture studies the final concentration of milk solids was 5% (w/v), the selective agent was thallous acetate (0.1 % w/v) and the pH before adding the sample was 6.0. Increasing the concentration of proteose peptone failed to affect either the gradient or the magnitude change. The addition of yeast extract did not increase the height of the curve. Substituting proteose peptone for trypticase gave poorer curves, showing increased base-line drift and a lower gradient, whilst substituting with special peptone failed to change the curve profile. In subsequent experiments, where Lancefield Group 0 cocci spray-dried in the laboratory were used, the final concentration of milk solids in the medium was 10% (w/v). This resulted in improved conductance curves for Eut. jaecium, Strep. bovis and Strep. equiuus which showed higher overall conductance changes and steeper gradients (Fig. 2), 'Step' values for these species increasing to 6. This suggested that a component of milk might be responsible for the curve quality. The lab-Iemco and glucose components of the medium were eliminated without affecting the conductance curves. The effect of pH on detection times for all species is shown in Fig. 3. All times increased with decreasing pH over the range pH 6.6-7.0. At pH 7.07.8 detection times generally remained constant except for that of Strep. bovis which showed a reduction with increasing pH value. The colony counts for powders containing Eut. jaecium, Strep. bovis and Strep. equiuus were similar whilst that of Ellt. jaecalis was lower. The colony counts correlated most

208

P. NEAVES ET AL. (i)

Base = 2537 Step = 6.16

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Base == 2570 Step = 6.55

~

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Fl(j. 2. Conductance responses for laboratory-prepared skinuned milk powder contammg Streptomccus bflVis ( a __, ), ,Strt:ptococcus equinus ( a ~ ~ - -, ), Enterococcus Jaecium ( b __, ) and Enterococcus jaecalis ( b - - -, ). The medium contained (% w/v): skimmed milk powder, 10; proteose peptone, 1; thallous acetate, 0.2; pH 7.0.

closely with detection times at pI-I 6.9. It was found that if the initial pH of the medium was 7.0, the final pII after addition of the sample was c. 6.9. To maintain selectivity of the medium without destroying the high quality of conductance curves it was necessary to employ two selective agents. The modified medium was prepared with additions of sodium azide and thallous

INSTRUMENTAL DETECTION OF ENTEROCOCCI

209

20 18

16

" 14

.§

§ 12

B

~ 10 u

o"

8 6 4'--......._ 6.4 6.6

....._ .....- ' _......_ .....-'_..l 6.8 7.0 7.2 7.4 7.6 7.8 8.0

pH value after addition of sample

Ftc;. 3. Effect of pI I value on detection times for lahoratory-prepared skimmed milk powder containing Ellleroam:m ji/Cl,dis ( ., 5.45 10glO du/g), Ellleyomaus jiJeliulIl (., 7.38 loglO cfu/g), .)'lnplllmCClis bmJis (", 7.23 loglil cfll/g) and SlreplII(lIcWS l'ifllilllls ( 0, 6.97 loglil cfu/g): the medium composition was as descrihed in Fig. 2.

acetate at a range of concentrations. The effect of sodium azide on conductance profiles was found to be critical; minor increases in concentration delayed detection time, reduced the overall conductance change and produced poor acceleration. At the concentration found in KF streptococcus broth (0.04% w/v), the profile of conductance curves was very poor. Thallous acetate had a less detrimental effect on curve profile. At the concentration fOl!nd in Barnes medium (0.1 % w/v), detection times were delayed but the curve profiles were unaffected. Detection times were derived with various mixtures of sodium azide and thallous acetate and compared with colony counts for naturally contaminated powders containing high and low levels of Lancefield Group 0 cocci. The best correlation was obtained with a conductance medium containing 0.02% (w/v) sodium azide and 0.1 % (w/v) thallous acetate. Calibration of Malthus and Bactometer Instruments The initial development of the medium was undertaken with the Bactometer instrument. It was intended, however, to develop a medium which would give comparable results with either the Bactometer or the Malthus instruments. The Malthus 128B instrument was operated at 37°C with 2-ml electrodes; the cells were inoculated with 5 ml of the primary dilution of the sample in selective media. Calibration curves of detection times against colony counts were constructed for both instruments with the artificially-inoculated, laboratory

210

p . NEAVES ET AL. TABLE

3. Comparison of calibration curoes obtained using the Bac/omeler or Mallhus inslrument

Linear regression analysis

Regression line parameters

Slope Intercept Correlation coefficient

Bactometer

Malthus

-0.34 7.82

-0.34 8.25

-0.89

-0.89

10 -0.371 +8.90 + 1.48 corr. coeff. "" +0.927

slope

intercept std. error

9

:§ 8 ~

~

7

0 ~

6

OJ

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5

0 u

~ 0

4

0

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\ 2

6

10 14 Detection time (h)

18

22

26

FIG. 4. Calibration curve for Lancefield Group D cocci in the Malthus instrument. The medium contained (% w/v): skimmed milk powder, 10; proteose peptone, 1; urea, 1; thaUous acetate, 0.1; sodium azide, 0.02; pI I 7.0.

spray-dried powders and naturally-contaminated powders. The results were subjected to linear regression analysis (Table 3). For both sets of results the colony counts ranged from 1.3 x 102 to 3.2 X 107 cfu/g. The regression analyses showed no marked differences between the calibration curves for the two instruments. Although the correlation coefficients of -0.89 were satisfactory, it was felt that these could be improved. With the Malthus instrument, an improvement in correlation coefficient was obtained when 1% (w Iv) urea was added to the medium. This did not affect curve profiles but modified the detection times for some powders containing low levels of Lancefield Group D cocci. These results (Fig. 4) gave a linear correlation coefficient of -0.93. The medium was not examined with the Bactometer.

INSTRUMENTAL DETECTION OF ENTEROCOCCI

211

This medium has the following formulation (gil): Proteose Ppptone (Difco), 10; urea, 10; sodium azide, 0.2; pH 7.0. The medium is prepared and autoclavcd at 121°C for 15 min. Immediately before use, an aqueous solution (10% w/v) of thallous acetate is added to give a final concentration of 0.1 % (w Iv). The final medium is warmed to 45°C and a 10- 1 suspension of the skimmed milk powder sample is then prepared in the medium. This suspension is tempered at 45°C for 15 min to assist the sample to dissolve. After further mixing, this dilution is dispensed into a Bactometer module (2 mllwell) or Malthus cell (5 mllcell). The instrument is operated at 37°C for 24 h and the conductance signal is recorded. Acknowledgements The authors are indebted to Bactomatic Ltd for the loan of the Bactometer instrument and to Miss Marisa Marchese for her invaluable assistance in preparing the manuscript. References AI\ON. 1984. Microbiological Examination jor Dairy Purposes. Part 3.11. Detet'ii011 a1ld E1Iumerati01l of F'aeml Streptococci (E1Iterocoai). British Standard No. 4285. London: British Standards Institution. BARNES, E.M. 1956. Methods for isolation of faecal Streptococci Lancefield Group D from bacon factories. ]oumal IIfApplied Bacteriology 19, 193-203.

BIOCHECK - a Mediated Amperometric Microbial Activity Monitor A. SWAIl'\,

M.

ALLEN, B. H. SCHNEIDER, AND A. P. F. TURNER

F.

TAYLOR

Bioelectronics Division, Biotechn%gy Centre, Cranfield II/stitute of Techn%gy, Cranfield, Bedfordshire MK43 OAL, UK

There is a substantial market requirement for a simple, inell.'pensive and rapid on-site method of monitoring microbial activity. Such a method would serve to complement the alternative laboratory-based approaches which tend to be more elll'ensive, non-portable and require trained personnel for their operation. Harris & Kell (1985) have produced a comprehensive review of methods used to estimate microbial biomass, categorizing them according to their principle of detection, i.e. physical, biochemical, biological or mathematical (computational). Such laboratory-based techniques using physical detection methods include for example the electrometric instruments marketed by Bactomatic Ltd (I Icnley-on-Thames, Oxon), and Malthus Instruments (Crawley, Sussex). The Bactometer measures impedance, conductance or capacitance changes in the sample, whilst the Malthus instrument measures conductance changes only, caused by the presence of actively metabolizing bacteria. An example of a biochemical detection method is the Lumac microbial testing technique (Lumac BV, 6370 AC Schaesberg, The Netherlands), which relics on the detection of adenosine triphosphate (ATP) by bioluminescence. This method is based on the assumption that living cells of a given type contain a reasonably consistent amount of ATP which is lost rapidly upon cell death. The direct epifluorescence filter technique (DEFT) was developed at the National Institute for Research in Dairying (NIRD) and is an example of a direct biological approach. Here cells captured on a polycarbonate filter are stained with acridine orange and then examined with an epifluorescence microscope. All these cited above serve as examples of laboratory-based rapid methods

Copyrq,hl Rapid .~.ticrobiological :\'1cthods for Foods, Beverages and Pharmaceuticals

© 1989 b)' Ihe SOdd)' IilY Applied Boaaiolog)' A /I rixhts (!l rcprodlltl;ml i" all)l./iJnn n.'sl'n1nl O~632··02629-4

213

214

A. SWAIN ET AL.

for estimating microbial biomass and are described 1n more detail elsewhere in this volume. In contrast the Biocheck is being developed primarily as a portable, singletest instrument, which can be used by the layman. Such a coarse screening method would be useful in guiding treatments, e.g. whether or not to add a farmer's milk to a tanker load, whether or not to dose a cooling water system with biocide or to detect unacceptably contaminated raw foodstuffs. Such a method could lead to savings in both time and money. With this aim in mind the following design specifications were outlined: 1 Rapidity of operation - around 10 Inin to obtain a result. 2 Portable, hand-held and battery operated. 3 Inexpensive - basic hardware, using disposable materials for each test. 4 Accurate to within an order of magnitude of bacterial contamination. S Robust and easy to use by unskilled personnel. 6 Minimum of sample handling - a pre-concentration step only being necessary for lower bacterial concentrations. The basis of the bioelectrochemical method for the assessment of microbial activity involves the use of low-molecular-weight redox compounds (mediators) to trap electrons produced from catabolic reactions and shuttle them to an electrode, thereby diverting· them from the natural electron acceptor. The electrochemical cell employed is operated in an amperometric (potentiostatic) mode, and the current measured at the working electrode is directly proportional to the concentration of elcctroactive material present in the stirred solution. In the presence of bacteria, reduction of the mediator molecules gives rise to an increase in current which can be related to the number of bacterial cells present in the sample and to their metabolic activity (Turner et al. 1983; Ramsay et al. 1987).

Requirements for Amperometric Biomass Sensing Three-elatrode, constant-potential system

In a three-electrode configuration the potential of the working electrode is maintained at a preset voltage with respect to a reference electrode which does not participate in redox reactions. A counter (or auxiliary) electrode is used to complete the electrochemical cell. Figure 1 shows a scheme for a three-electrode system suitable for biomass detection. The bioelectrochemical cell (BEe) itself can be a pyrex beaker combined with a lid designed to hold the electrodes in place. In this type of BEC, thorough mixing of the solution is necessary in order to ensure the efficient mass transport of those mediator molecules reduced by the bacteria in the bulk solution to the electrode surface. This is done by a standard magnetic stirrer coupled to a stirring bar inside the BEe.

BIOCHECK -

A MICROBIAL ACTIVITY MONITOR

I

Chart recorder

I

~ ---- Load

I

Potentiostat

215

resis tance

I

Nitrogen

I I I

1

Glucose

I I

Working electrode

\

J

Mediator\ (~tion)

E

'" 'cm

Ol

e-

0

o 0

0 00 0 0 00

t; ~

Oxidation products

Counter electrode

e-

~

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f--=---.

-+ Mediator (Oxidation)

Luggin capillary

V ~~----

(

>----- ---

Electrolyte

Magnetic flea

FIG. 1. Schematic diagram of a three-electrode system for amperometric determination of microorganisms. In this example the potential is controlled by a potentiostat and the current change with time measured on a chart recorder.

The working electrode may be constructed from either a suitable metal such as gold or platinum or a carbon material with low resistivity. From a research point of view the metal electrodes offer the advantage of relative chemical inertness. Thus a polishing with alumina slurry is generally sufficient to restore the electrode surface to a clean condition. Carbon materials, on the other hand, are affected by their use as an electrode. This is due to both chemical absorption of material and redox activity of surface groups on the carbon. Highly ordered carbon materials such as pyrolytic graphite and glassy carbon may be returned to pristine condition with either electrochemical treatments or by polishing with alumina slurry or fine emery paper. More novel electrode materials such as graphite felts and cloths, graphite foil and carbon fibres are not easily re-used but are more applicable to a commercial device incorporating a disposable cell. A number of different carbon materials suitable for use as electrodes in the BEC may be obtained from Le Carbone (UK) Ltd (Portslade, Sussex) or Union Carbide (Sheffield, UK) amongst others. The counter or auxiliary electrode is usually made of platinum wire or

216

A. SWAIN ET AL.

gauze, although other electrode materials could be employed. The reference electrodes commonly used are the saturated calomel (SCE) or silver/silver chloride (Ag/AgCI) electrodes. The potential between the working and counter electrodes is then fixed using a suitable potentiostat (e.g. from H.B. Thompson & Associates, Newcastle, UK) which holds the potential of the working electrode constant compared to the reference whilst current is flowing. The reference electrode is usually placed in a l..uggin capillary filled with saturated KCl. This allows the SCE to monitor the solution adjacent to the working electrode whilst being physically distant. The potential employed in the BEe depends on the redox potential of the mediator used. In order to ensure that the electrochemical reaction does not become rate-limiting, it is usual to work at a voltage about 100 mV more positive than the mediator's redox potential (Eo values at same plI). A standard arrangement of the three electrodes in the BEC is shown in Fig. 1. Care must be taken to avoid the working and counter electrodes coming into physical contact. If the cell volume used is small the counter electrode may be placed in a separate compartment which is in ionic contact with the working solution (e.g. via a Luggin capillary or borosilicate plug). A record of the changes in current occurring during the eX'Periment may be obtained by connecting the potentiostat to a chart recorder. In this case it is necessary to connect a load resistance in parallel (using a variable resistance box) with the chart recorder. The test procedure

With the BEe organized as shown in Fig. 1 the changes in current due to bacteria may be measured. The BEe is filled with an electrolyte containing buffer, an exogenous substrate (glucose) and the mediator. If an auto-oxidizable mediator (the reduced form being rapidly oxidized by molecular oxygen) such as thionine or N,N,N'N' -tctramethyl phenylenediamine (TMPD) is used then the solution needs to be thoroughly sparged with nitrogen or argon both before and during the experiment (in the latter case above the surface of the liquid). This precaution is unnecessary, however, if a mediator such as potassium ferricyanide or potassium ferricyanide in combination with benzoquinone (Turner et al. 1987) is used. After applying the desired voltage to the system the current is allowed to decrease to a stable level at which point an addition of bacterial sample is made. In this type of experiment 0.5 ml of bacterial sample is added to a working volume in the BEC of 9.5 ml, giving a 20-fold dilution of the sample. The current-time profile thus obtained can be related to the number of bacteria in the sample by measurement of either the initial rate (!lA/min) or the final maximum current (peak height).

BIOCIIECK -

1\ ""'lICROBIAL ACTIVITY MONITOR

217

Two-electrode configuration When highly accurate control of the potential applied to the working electrode is unnecessary the functions of the reference and counter electrodes may be accomplished by a single electrode. The Ag/AgCl electrode does this well provided that the current density generated at the electrode does not become too large. Chloridization of a piece of silver foil may be accomplished electrochemically. The silver is employed as the working electrode with a platinum gauze counter and SCE reference electrode in a stirred vessel containing 0.1 molll KCI. Typically a voltage of +400 mV vs. SCE and a resistance load of 1- 5 kQ are employed, under which conditions an even coating of silver chloride (dark brown) is formed after several minutes. A two-electrode system with a working electrode and Ag/AgCl reference electrode can also be operated via a potentiostat as described above for the three-electrode system. One requirement of the two-electrode arrangement is for the inclusion of KCI in the electrolyte. This prevents too severe a loss of silver chloride when large currents (mA/cm:!) are generated. A final concentration in the BEC of 0.1 molll KCI is acceptable. It should be noted that the potential of the Ag/AgCl electrode changes with the chloride ion concentration, although only in the order of millivolts per 100 molll increase.

Computer-controlled multichannel

~ystem

Although a potentiostat allows a fine control of the applied potential with a wide range of currents being generated at the working electrode, (from nanoamps to milliamps) it has the severe limitation of only being able to operate one BEC at a time. It was evident at an early stage in the work at Cranfield that computer control of an electrochemical cell would lend itself admirably to the study of amperometric biomass detection, simplifying the evaluation of mediators, electrodes and cell designs with the facility for multichannel operation. Interfaces suitable for running either 4 or 16 BECs with a BBC microcomputer (Acorn Computers Ltd, Cambridge) have been developed (Artek, Lavendon, Buckinghamshire). This system requires a twoelectrode BEC (Fig. 2). A software package developed at Cranfield allows the variation of current with time to be followed on a VDU and a hard copy obtained on a compatible dot-matrix printer. Measurement of the bacterial responses obtained with this system were calculated as initial rates within the program. Evaluation of Suitable Mediators In considering the basic requirements of a suitable mediator lor amperometric biomass sensing, it is useful to list the desirable characteristics for mediators

218

A. SWAIN ET AL.

Printer

I Computer and VDU

Disc drive

I

I Computer interface Nitrogen in

~ r--

Silver/silver chloride r eference electrode 00

0~08°

PI atinum working el ectrode

'--

~-

I I !

J

t---~-

Bioelectrochemical cell

I--

Water bath Magnetic stirrer

FIG. 2. A two-electrode system for the amperometric detennination of microhial biomass. A BBC microcomputer was employed to maintain a potential across the working and Ag/AgCl reference electrodes \'ia an interface and, at the same time, to monitor the current from a number of bioelectrochemical cells.

in general (Cardosi & Turner 1987). A good mediator should: 1 Readily participate in redox reactions with both the biological component and the electrode, effecting rapid electron transfer with both. 2 Be stable under the required assay conditions. 3 Not participate in side reactions during the transfer of electrons, e.g. the reduction of dioxygen. 4 Have an appropriate redox potential away from those of other electrochemically active species that may be present in samples. 5 Be unaffected by a wide range of pH values. 6 Preferably be non-toxic, especially for in vivo applications and for monitoring food processes on-line; 7 Be amenable to immobilization. Some of the above criteria are not requirements for a biomass sensor based on the methodology outlined in this contribution. For example, the

I3IOCIIECK -

A ;\1!CROBIAL ACTIVITY MONITOR

219

TABI .I: 1. List oj'lhe best medialors jiJUnd for some oj'the

micro-organisms tested in lite bioelalrodlem1cal tell Organism

Mediator

Pseudomonas aeruginosa Al(l/ligenes ji/emlis Nocardia (NClB 8863) EnterobaCler aerogenes Adlromobacler album Cladosporium resinae StreptococflIs lattis Lacwbadllus bulgaritus Lactobacillus planlarum Saccharomyces cen"Visiae

PES Ferricyanide Ferricyanide Ferricyanidc DCPIP Fe rricyanidc Fcrricyanide Ferricyanide

PES Fcrricyanide

toxicity question is less critical because the present technology requires the removal of a sub-sample for testing rather than on-line monitoring. Rather than being able to immobilize the mediator, this particular amperometric technique relies on the mediator being sufficiently water-soluble to shuttle electrons from the bacteria to the electrode. A number of commonly used mediators such as phenazine ethosulphate (PES), dichlorophenol indophenol (DCPIP) and potassium ferricyanide were tested with a range of micro-organisms tllat are often present as contaminants in real samples. Table 1 lists each micro-organism tested together with the mediator that gave the best BEC response with it. In a majority of cases potassium ferricyanide was found to give the highest response. This, coupled with the fact that it is not auto-oxidizable, led to its use in a number of eXlJeriments on a range of bacteria. One major drawback of ferricyanide, however, was the poor response obtained with pseudomonads which are found in a wide range of industrial and food samples. Further optimization of the mediator employed in the BEC therefore appeared necessary if the amperometric approach to biomass sensing was to become a commercial proposition. A number of different mediators were considered, including a range of ferrocene derivatives, thionine and various quinones, and a breakthrough was obtained when p-benzoquinone was mixed with ferricyanide into a mediator 'cocktail' (Turner et al. 1986, 1987). This mixture, for example, increased the response with Pseudomonas aeruginosa by over 100-fold compared to ferricyanide alone. Assessment of Electrodes With the discovery of a mediator (cocktail) that gave relatively even responses from a range of bacteria it was important to assess the sensitivity of the

220

A. SWAIN ET AI..

technique. This was primarily concerned with the evaluation of different working electrode materials (an Ag/Agel electrode was used throughout as the reference in a two-electrode system). In ·particular, high-surface-area carbon and graphite materials were looked at as these appeared to be a better option for a prototype instrument based on a disposable cell than a noble metal working electrode. To begin with, these materials were examined by the electrochemical technique of cyclic voltammetry in the presence of potassium ferricyanide to obtain an indication of their properties as an electrode. Figure 3 shows the types of voltammograms obtained. Solid graphite materials such as a spectrographic graphite and a highly pure compressed graphite (Fig. 3A) gave voltammograms with sharply defined peaks for ferricyanide electrochemistry and typical peak potential separation (for oxidation and reduction). With a number of cloth-like materials supplied by Le Carbone, such as thc graphite felt (RVG 1000) and cloth (TGM) less well-defined electrochemical activity was observed (Fig. 3B). Carbon-fibre electrodes (Fig. 3C) gave similar cyclic voltammograms to the TGM cloth. \Vith a number of activated charcoal cloths, however, no ferricyanide electrochemical activity was observed

(Fig. 3D)..

All these electrode tnaterials (except the charcoal cloths) were successfully used as working electrodes in the BEC. It was clear that the larger the surface area of the electrode dIe larger the BEC response, although the nature of the electrode material also had an effect. Using the RVG 1000 as the working electrode enabled the detection of about 5 x 105 Escherichia coli/ml (Fig. 4). It should be noted that the concentration of the E. coli sample actually added to the BEC was 20 times higher. Prototype Development The development of a prototype instrument to test the amperometric biomass sensor with a view to designing a commercial product has progressed through two stages. The first Biocheck model, which was described by Turner et al. (1987), used a disposable BEe based on a glass vial incorporating freezedried reagents (phosphate buffer, KCI and glucose) with a moulded plastic holder retaining a graphite working electrode and an Ag/AgCI reference electrode. This instrument is shown in Fig. 5 and the BEe itself shown in diagrammatic form in Fig. 6. Examination of the Biocheck revealed a problem which had not been evident with the computer-based system, namely the polarization characteristics of the working electrode. It was necessary for the working electrode to display the following properties: 1 Rapid decay of the charging current upon application of the voltage. 2 A stable background current in the absence of bacteria which does not drift over the time required to make a measurenlcnt.

BIOCIIECK -

A MICROBIAL ACTIVITY MONITOR

221

IlmA

d £'IG. 3. Cyclic voltammograms of four different carbon materials in a buffered solution of potassium ferricyanide at a scan rate of 100 III V/ s. A, highly pure compressed graphite; B, TGM (carbon) cloth; C, carbon-fibre bundle; D, charcoal cloth.

3

Low levels of background current (electrochemical noise). To achieve these objectives a compromise on the absolute sensitivity of the instrument was necessary within the constraints laid down by the BEC design. With this prototype instrument the lower limit of sensitivity corresponded to c. I X 107 E. coli/m!. This sensitivity was not improved when samples such as industrial cooling water, raw or pasteurized milk were spiked with the same concentration of bacteria as shown in Table 2. Certain limitations in the software and hardware of the Biocheck were found when it was used with samples allowing a high level of background current, or at very

high cell concentrations; this

also meant that evaluation of

222

A. SWAIN ET AI-.

Time (min) FIG. 4. Computer print-out of current against time for dIe detection of low levels of bacteria using a high-surface-.uea brraprute fdt (RVG 1000) electrode. The following additions were made to the bioelectrochernk.ll {'ell: A, addition of 0.9% $aline; B & C, addition of a suspension of Escheridlia (oN to give a final concemrarion of 5 x 105 organisms/tnl.

FlU.

5. The Bjochcck prototype instrument Jc\'eJoped for the amperometric detection of microbial

activity.

BIOCHECK -

A 'V11CROBIAI. ACTIVITY MONITOR

223

-I---Moulded plastic holder for electrodes Glass vial Silver/silver chloride reference electrode JIIt;-""-"i+--- Carbon

working electrode

.-,---,H---Electrolyte/sample

------ -Magnetic stirring bar

FIG. 6. Diagram of the disposable bioelectrochcmical cell employed with Biocheck prototype instrument.

TABLE 2. Results obtained with the Biocheck protozype instruml'11t jOr samples of raw milk dvsed with bacteria. Background readings measured with the uncontamllzated ran' milk lVere subtracted to give the mrrected values listed bdow

Micro-organism

Viable count (cfu/ml)

Escherichia mli

X

108 107

1488 162 30

X

lOR

69

8

117

X X

Pseudomonas aeruginosa Lactobacillus plantarum

4

104

Biocheck reading

X

10

larger electrodes (to improve the sensitivity) was not possible. The updated version of the instrument, Biocheck II, incorporates a number ofmodifications to both the software and the hardware which have improved the sensitivity to below 5 x 106 E. coli/ml at room temperature. Additionally, expansion of the current range that the microprocessor can detect, without loss of resolution, has enabled the prototype to be tested with a number of sample types and working electrodes. A good correlation between the viable counts and the readings of the instrument has been obtained with E. coli at room temperature using a disposable BEe based on elements manufactured on a large scale (electrodes,

224

A. SWAIN ET AL.

holders and vials of freeze-dried reagents). Figure 7 shows a calibration curve obtained with this instrument on shake-flask-grown E. coli at room temperature. An excellent relationship is obtained between the viable counts and the logarithm of the Biocheck reading.

Biocheck calibration

7

9

10

7. A calibration curve obtained with the Biocheck prototype instrument using Esdzerichia coli samples. The results were obtained over a number of days using overnight cultures resuspended in 0.9% saline. Each point on the graph represents an average of three replicates. This log-log plot of Biocheck reading against viable counts gave a correlation coefficient of 0.993. FIG.

Considerations for the Examination of Real Samples During the course of the present study, a wide range of real samples have been examined by this mediated amperOlnetric approach. Various food samples such as meat washings, vegetable washings, milk and beer have been studied as well as samples of industrial origin such as cutting tluids, cooling and process waters. A single clinical sample type, urine, has also been examined. For the amperometric test method the sample must be introduced in a liquid foml. Solids such as meat, therefore, were prepared as they would be for a routine microbiological plate count - homogenized in a stomacher. Samples such as milk, beer and urine were introduced directly into the BEC. Not unexpectedly, differing degrees of background interference due to the reduction of the mediator by chemical constituents of the sample have been observed. The magnitude of the interference problem is variable, for example no interference problems have been experienced with any of the

BIOCHECK -

A MICROBIAL ACTIVITY MONITOR

225

cooling water, river water or swimming pool water samples examined to date. Some background interference is present in milk but it can be tolerated as it has tended to be reproducible. The backgrounds observed in urine samples, however, were extremely variable. This variation is perhaps not surprising, as the chemical composition of anyone sample will change with time throughout the day, with diet and with the state of health of the individual in question. At present, means of overcoming the backgTound interference in sewage taken from a treatment plant are being investigated. An obvious approach is to filter the sample, thereby entrapping the bacteria and eliminating soluble interference-generating substances in the filtrate. Such a pre-filtration may also provide a simple and rapid means of pre-concentration of more dilute samples. An alternative test approach may be to introduce the idea of a reference blank, where the bioelectrochemical response has been eliminated by treatment of the sample with a biocide, for example. It is envisaged that the Biocheck prototype will form the basis of a range of instruments targetted to meet specific market requirements. In all the models produced however, the design specifications listed earlier for the production of a rapid, simple, on-site microbial activity monitor will remain. Acknowledgements The authors gratefully acknowledge the generous sponsorship of de la Pena Biotechnology Ltd (Racecourse Road, Pershore, Worcestershire WRIO 200) throughout the course of this developmental project. AS and BS are Teaching Company Associates, sponsored by de la Pena in conjunction with the SERC and the DTI. References CARIlOSI, ;v1.F. & TURNER, A.P.F. 1987 The realisation of electron transfer from biological molecules to electrodes. In Hiosensoys: FUlidammlals (md Applicaliolls, cd. Turner, A.P.F., Karube, I. & Wilson, G.S. Pl'. 257~275. Oxfl)l"(l: Oxtonl Uni\'ersity Press. II.'\RRIS, c.l\l. & KEI.L, D.B. 1985 The estimation of microbial biomass. Biosensors I, 17~84. RA\lS.'\Y, G., TURNER, A.P.F., FR>\NKLIN, A. & I liGGINS, I.J. 1987 RapiJ methods for the dett'etion of li\ing microorganisms. Proceedings oflhe lsi IFAC Con[erClltt: Oil Ihe Modellillg alld COlltrol ofBiotedmo!ogica! Processes, Nordwikjerhollt, December 1985, ed. Johnson, A. Oxford: Pergamon Press. TURNIOI{, A.P.F., C'\RDOSI, M.F., FR·\"KI.lN, A. & R·\\IS,W, G. 1986 ,IVlediators filr bioelectrochemical cells. Brilish Palmi Applimlioll 8606831. TURNIR, A.P.F., CARDOSI, M.F., R.\\ISilY, G., SUINE1i>ER, l.UI. & S'L\IN, A. 1987 Biosensors Illr use in the fllod industry: a new rapid bioactidty monitor. In Bioledmo!(jgy ill Ihe Food I/lduslry. Pinner: Online Publications. TURNER, A.P.F., R.\\IS.'\Y, G. & IlK,GINS, I.]. 1983 Applications of electron transfer between biological systems and electrodes. Biodll'l/liw! SoddY "I'm/1sMlioIlS 11, 445~448.

Detection of Electron Transfer for the Assessment of Bacterial Contamination R. G. KROLL,l R. A. PATCHETT, I STEPHANIE E. LEAROY02 AND C. F. THURS'f01\'2 Department ofMicrobiology, AFRC Institute of Food Research, Reading Laboratory, Shinfield, Reading RG2 9AT, UK; and 2 Department ofMicrobiology, King's College, Universi~y ofLondon, Campden Hill Road, Londml W87AH, UK J

The microbial contamination of perishable items needs to be frequently and reliably assessed. Ideally, the methods used should be cheap, simple to perform and interpret, and provide the necessary information within defined limits of precision and repeatability. More important, such methods need to be rapid to allow the efficient management of food supplies. To achieve this, new methods must target some unique structural, biochemical or functional properties of micro-organisms that distinguish them from their surroundings. One possible approach is to e),:ploit the electron-carrying ability of cellular redox components. Indeed, the dye reduction tests, used for decades for assessing the hygienic quality of raw and pasteurized milk, fit this general description but they are now of little value for a variety of reasons (Luck 1982). Throughout this century, and especially Witll the oil crisis, alternative methods of producing electricity have been sought. Of particular interest has been the development of microbial fuel cells to convert organic wastes directly into electricity (Benetto 1987). Such cells were found to be efficient and capable of producing sustained currents but only if soluble mediator compounds were included (Delaney et al. 1984; Roller et al. 1984; Benetto et al. 1986). The principles of operation of microbial fuel cells is that chemoheterotrophic organisms derive their energy (and carbon source) to drive biosynthetic processes by oxidizing reduced carbon substrates. Energy is conserved by controlling the flow of electrons from these substrates to an oxidant, such as ox')'gen, and coupling the energy released to the synthesis of ATP. In microbial

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228

R. G. KROLL ET AL.

fuel cells the flow of electrons to the natural oxidant does not occur but is diverted by the mediator compound which is reduced directly by the bacteria and subsequently oxidized by transferring the electrons to an electrode. The electrons pass through an external circuit where their rate of flow, Le. current, may be measured. The electrons flow to the other half of the fuel cell where a suitable oxidant, e.g. ferricyanide, is the terminal electron acceptor. Electroneutrality is maintained by an ion-exchange membrane which separates the anode and cathode compartments and allows ions to move to balance the charge. The current generated is directly related to the rate of mediator reduction and hence the rate of metabolism. It should also be dependent on the biomass present and this has led several workers to investigate the use of fuel cells as a means of rapidly estimating microbial nunlbers. The exploitation of the reducing power of micro-organisms for their detection and/or quantitation is attractive in principle because it is quantitatively amongst the largest chemical activities of heterotrophic organisms. It can also be directly transduced into an electrical signal, for which a vast array of modem measuring and signal-processing apparatus is available. These theoretical advantages will only be realized if redox-mediator!electrode combinations are devised or identified with the appropriate properties of sensitivity and specificity. Nishikawa et al. (1982) investigated a variety of redox dyes and found dichlorophenol indophenol performed the best in conjunction with their electrode. A linear relationship between current output and plate count above 10-l organisms!ml was observed but this induded a large pre-concentration of organisms from water samples. Turner et at. (1983) used phenazine ethosulphate as a mediator and found a good relationship with cell numbers above 4 X 106 / ml. Of the many mediators tested, thionine was shown to be reduced by a wide range of bacteria (Roller et al. 1984) and with high efficiency in fuel cells (Delaney et at. 1984; Benetto et al. 1986). We have been investigating a modification of this design of thionine-mediated fuel cell for rapidly assessing microbial numbers (Patchett et al. 1988) and we report here some of the characteristics of this system for enumerating pure cultures of bacteria and bacteria in foods. An alternative electron transfer reaction is that carried out by the cytochrome c - cytochrome c oxidase terminal portion of the electron transport chain which is the site of reduction of molecular oxygen to water 0 ones 1980). This activity can be simply and cheaply assayed by the oxidation of the redox dye N, N, N', Nt -tetramcthyl-p-phenylene-diamine (TMPD). This is of interest because the Gram-negative psychrotrophic rods that are primarily responsible for the spoilage of pasteurized Iuilk and cream (Phillips et af. 1981; Schroeder et al. 1982) possess this terminal oxidase while in general the other common, milk-associated, non-psychrotrophic bacteria lack or have

DETECTION OF ELECTRON TRANSFER

229

weak or undetectable TMPD oxidase activity (Kroll 1985). The possible applications of this method will be discussed. Materials and Methods Thionine, TMPD, platinum wire and cation exchange membranes were purchased from BDH (Poole, Dorset), and benzalkonium chloride from Sigma (Poole, Dorset). Reticulated vitreous carbon (RVC) was supplied by]. Tipple, Imperial College, London. All other chemicals were of analytical grade.

Growth of cell suspensions Pure cultures of a variety of Gram-positive and Gram-negative food-associated bacteria were grown in 100-ml portions of Nutrient Broth (Oxoid No.2) supplemented with 0.2% (w/v) glucose or, for Lactomccus lactis, M17 broth (Difco) supplemented with 1.0% (w/v) glucose, in 250-ml sterile conical flasks. After overnight shaking at 30°C, cell suspensions were harvested by centrifugation (5000 g for 10 min), washed twice and resuspended in 100 mmolll Tris-HCI, pH 7.5 and stored on ice. In some experiments, cell suspensions were used immediately from the flask culture or diluted in buffer to give the required cell density.

Fuel cell design The microbial fuel cells were based on the design described by Roller et al. (1984) and Delaney et al. (1984). The design was modified by using a Rank ox}'gen electrode (Fig. 1) as the fuel cell body (Rank Bros., Cambridge, England). The bottom electrode compartment was filled with 100 mmolll potassium ferricyanide in 100 mmolll Tris-HCI, pH 7.5. This cathode compartment was isolated from the water-jacketed (30°C) anode compartment by a piece of cation exchange membrane sealed by a rubber 'O'-ring. The liquid in the anode compartment was stirred by a magnetic flea and isolated from the atmosphere by the lid of the oxygen electrode. The reaction mixture was rendered anoxic by bubbling with ox}'gen-free nitrogen through a small needle inserted through the lid. The anode consisted of a small (12 x 5 mm) cylinder of RVC connected to a platinum wire with conducting cement (Union Carbide, Chicago). The anode was boiled in distilled water prior to immersion to remove trapped air bubbles and eliminate vegetative bacteria. The platinum wire protruded through the electrode lid and an electrical circuit was made by connecting the anode wire through a 560 Q resistor to the cathode which was the platinum electrode of the 'oxygen electrode' base. The annular silver electrode of the o"}'gen electrode base was not used.

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1. Rank oxygen electrode adapted for use as a microbial fuel cell.

nut

Cathode compartment containing ferncyanlde

~

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DETECTION Of ELECTRON TRANSfER

231

The current generated by the fuel cell was measured as the voltage drop across the resistor using a digital voltmeter (Model 19SA, Keithley Instruments, Reading). Four fuel cells were monitored simultaneously with a multiplexing scanner (Model 705, Keithley Instruments) which connected the digital voltmeter to each fuel cell at 0.35-s intervals. The digitized data were collected using a BBC model B computer linked to the IEEE 488 interface of the scanner and voltmeter via an Aries B488 interface (Aries Computer, Cambridge, England). In some eXlJeriments voltages from two fuel cells were measured, using two separate digital voltmeters (Model 177, Keithley Instruments) with the analogue outputs going to a two-ehannel'yltime chart recorder (Model CR600, J.J. Instruments, Southampton). In initial eXlJeriments the anode compartment contained 1.0% (w Iv) glucose, 20 ~lmol/l thionine and 100 mmol/l Tris-HCl, pH 7.5 to 4 ml. The anode compartment was made anoxic by bubbling with oxygen-free nitrogen and the voltage readings were allowed to stabilize. Before bacteria were added to different final cell densities with a glass microsyringe the needle supplying the nitrogen was withdrawn above the level of the buffer to minimize the noise due to bubbling and to continue the exclusion of oxygen from the headspace. In later experiments, different densities of cell suspensions were incubated anaerobically with glucose and thionine in the anode compartment. In these experiments, however, the electrical circuit was not completed and after different lengths of incubation time the circuit was closed and the current produced recorded for a few minutes. Samples of raw and pasteurized milk and braising steak were obtained locally and examined either immediately on receipt or after incubation at 5°C for several days. Low-count raw milk «100 bacteria/ml) was obtained by hygienic hand milking of mastitis-free cows. Laboratory-pasteurized milk was prepared by heating low-count raw milk at 63°C for 30 min. Meat samples (10 g) were homogenized in a stomacher in 90 ml of 10 mmol/l Tris-HCI (pH 7.5) for 2 min. Suspensions from meat samples or milk samples were incubated with glucose and thionine in the open-circuit fuel cell for various lengths of time before the circuit was closed and the current generated recorded. In some experiments food samples were spiked with pure cultures.

Assa'y of C)'tochrome c oxidase activizy in milk and cream This has been described (Kroll 1985; Kroll & Rodrigues 1986a,b) and will be only summarized here. One ml of 1% TMPD was added to 4 ml of a sample of milk or cream in a clean sterile test-tube and mixed gently by tilting the tube a few times. With cream samples 1 ml of 0.1 % (w/v) Tween-80 in 0.1 mmol/l citric acid/NaOH buffer (pH 6.6) was added before the addition of 'I'MI'D to assist mixing. The tubes were incubated at 25°C for 5 min in a

232

R. G. KROLL ET AL.

water bath. Reference scores of zero were obtained by adding 1 ml of distilled water without TMPD to a sample. The degree of blue coloration was assessed by eye with reference to a set of lOPB Munsell Colour Standards on a scale of 1(9/1), 2(8/2), 3(8/4), 4(7/6), 5(7/8), 6(6/10) and 7(5/10). Half scores could easily be made. Solutions of TMPD must always be freshly prepared before each assay as TMPD auto-oxidizes slowly.

Pre-incubation of pasteurized milk and cream To inhibit the growth of Gram-positive organisms a filter-sterilized solution of benzalkonium chloride (0.05% w/v, final concentration) was added to 20-rot volumes of milk or cream. Samples were pre-incubated for 18 h, usually at 20o e, before assaying for oxidase activity.

Spectrophotometn'c assay of TMPD oxidation rates

~y

pure cultures

The method adopted was basically that described by Jurtshuk & Liu (1983). Pure cultures of a variety of milk-associated bacteria (Table 1) were grown as lawns on the surface of Yeastrel Milk Agar (Oxoid) for 24 h at 30°C. The lawns were scraped into 20 mmol/l potassium phosphate, pI-I 7.0, with a sterile glass hockey stick. Cell suspensions were washed once by centrifugation (5000 g for 10 min) and finally re-suspended in the phosphate buffer and stored on ice until used for analysis. Cell suspensions were added to 2 ml of fresh buffer at room temperature in a plastic cuvette to give an 00650 of about 0.2. The reaction was initiated by the addition of 0.5 ml of 10 mg/ml of TMPD. After rapid mixing by inversion of the cuvette the initial rate of formation of Wiirsters blue was measured by the increase in absorbance at 610 nm. This was measured using a Cecil Model CE272 spectrophotometer linked to a Model CR600 chart recorder (J.J. Instruments, Southampton) with a full-scale deflection of 0.5 A units and chart speed of 1 or 2 em/min. The specific rate of TMPD oxidation was calculated using a molar extinction coefficient of 12000 (Jurtshuk & Liu 1983). Auto-oxidation of TMPD in phosphate buffer is significantly higher than in distilled water (due to pH effects and traces of catalytic amounts of metal ions) and this background rate of oxidation was measured and subtracted from the values presented.

Plate counts All plate counts were performed by serially diluting cell suspensions in 1/4strength Ringer solution and plating in Yeastrel Milk Agar (Oxoid). Colonies were counted after incubation at 30°C for 2- 3 days.

DETECTIOI'\ OF ELECTRON TRANSFER

233

Results and Discussion The dye reduction methods used tor much of this century by the dairy industry tor grading the quality of raw and pasteurized milk were ideal as they were cheap and simple to perform and interpret. They relied mainly upon the direct reduction of soluble dyes by the bacteria, although a decrease in the redox potential of the sample itself was a contributory factor (Wilson 1935; Luck 1982). Because of the improved hygienic quality of raw milk and the introduction of refrigeration of milk supplies, however, these tests are now of little value as they are insensitive and refrigeration has changed the milk flora. This now makes the psychrotrophs the dominant flora and these are often found to be poor at dye reduction (Luck 1982). Dye reduction tests rely on a gross colour change and theretlJre a large shift in the relative concentrations of the oxidized to the reduced form of the dye. As the rate of dye reduction can be used to produce a direct electrical output (Nishikawa et al. 1982; Turner et al. 1983; Delaney et al. 1984) this otTered the promise of a more sensitive method of measuring the rates of dye reduction, most particularly in opaque samples like foods. The direct electrical output also offered the opportunity for simple automation if this was desired. Wnen a bacterial suspension was added to the anode compartment of a fuel cell an immediatc and rapid increase in current output was seen (Fig. 2). The initial rate of current increase and the final steady-state current generated were proportional to the number of bacteria added. The relationship with cell numbers was good for several ditTerent organisms and the minimum number of bacteria that could be detected was 10 51ml (Patchett et al. 1988). Obviously this sensitivity is encouraging but it is not good enough for many applications although results are obtained very quickly, i.e. in a few minutes. This mcthod of adding a small quantity of a sample to the fuel cell has limitations, however, as it results in a dilution of the sample and reduces overall sensitivity. An alternative method which would not necessitate the dilution of a sample was therefore needed. One method we have investigated has been placing the sample directly into the fuel cell, making the compartment anaerobic and then adding the thionine. This method did work in principle but when the thionine was added a large initial spike current was ohserved, even at low cell numbers, which then tailed off to give a steady-state current which was cell number dependent, but only at high cell densities (> 107 /ml). In ordcr to increase the sensitivity we reasoned that if the bacteria were incubated with the dye, without the dye being re-oxidized by the electrode, a bacteria-dependent pool of reduced dye should be built up which could tllen be measured. Pure cultures of bacterial cell suspensions were incubated in the anode compartment anaerobically with thionine but with the external circuit open. After various lengths of incubation,

234

R. G. KROLL £1' AL.

8 1.65 x 106 /ml

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FIG.

on completing the circuit, a rapid peak of current was produced which then settled to a steady value. With a constant cell density, with various incubation times, the peak current generated was found to increase with length of opencircuit incubation, but the final steady-state current generated was constant. With different cell densities incubated open circuit for a constant length of time (e.g. 8 min), the peak current upon dosing the circuit was dependent on cell concentration. This system proved to be relatively insensitive, however, and the lowest cell densities that could reliably be detected were 106 / ml (Fig. 3). The method does suggest that by lengthening the open circuit incubation period to a few hours sensitivity could be substantially increased. It should also be noted, however, that we have as yet been unable to make operation of this design of fuel cell totally reliable. On several occasions the responses expected on past experience were not obtained. In some instances this could be attributed to either improper sealing by the gaskets which separate the anode and cathode compartments (Fig. 1) or poor electrical

DETECTION OF ELECTRON TRANSFER

235

25

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contact between the RVe anode and the platinum wire: on other occasions no obvious cause for poor response was discernible. Despite these problems we investigated this system for measuring bacteria in samples of food. This was severely hampered by the large bacteriaindependent signals produced by both raw and pasteurized milk (Fig. 4) and meat (not shown). Even in very low count milks, large currents were generated

236

R. G. KROLL ET AL.

(Fig. 4) against which any bacteria-dependent signal was not discernible (not shown). This signal was probably caused by the presence of endogenous chemical reductants, e.g. ascorbic acid or the somatic cells or mitochondria in samples of raw milk and meat. There are several possible approaches to solving these problems which we are currently investigating. Efficient means of separating the bacteria from the food would be an obvious step. Othcr electrochcmical devices \-vith different electrode configurations and using altcrnative mediators have been developed (Turner et al. 1986) but these methods are all currendy limited by not being sensitive enough for many applications and liable to interference by the large non-microbial sources of reducing power present in many foods and other types of sample. Furthermore, the rate of redox mediator reduction will not simply be related to bacterial numbers; rates of metabolic activity will be a substantially contributory factor. Information on bacterial activity rather than number should be of more value in many situations and there is clearly much room for improvement in this technology.

Cytochrome c oxidase test jOr predicting the keeping and cream

quali~y

ofpasteurized milk

The statutory tcst for predicting the keeping quality of pasteurized milk in England and Wales is currently the methylene blue test (Anon. 1983) after overnight storage of milk samples. Like the resazurin test for raw milk, however, the usefulness of this method has been questioned (Luck 1982). This is mainly due to its insensitivity and poor reduction of the dye by the psychrotrophic Gram-negative rods which are primarily responsible for spoilage of the products. Furthermore, these organisms often fonn only a fraction of the initial population and several alternative methods have been proposed which require pre-incubation of the samples with inhibitors of Gram-positive organisms followed by rapid assay of microbial ATP or the DEFT (Griffiths et al. 1984; Rodrigues & Pettipher 1984). We were interested in finding a simple test for this purpose. The cytochrome c oxidase test was found to be generally selective for the Gram-negative psychrotrophs but only in samples containing over 104 organisms/ml (Kroll 1985). However, after pre-incubation for 18 h at 20 with benzalkonium chloride (0.05% w/v) the test can reasonably assess this post-pasteurization contamination; a reasonable relationship with plate count can be seen (Fig. 5) and the method therefore has potential for predicting the keeping quality of pasteurized milk and cream (Kroll & Rodrigues 1986a,b). The method is not without its problems: psychrotrophic coliforms are all oxidase negative, for instance, and this could be problematic in some particular dairies. Some other Gram-negative psychrotrophs are all oxidase negative, D

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DETECT[O~

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e.g. Acinetobacter. Furthermore, it was not known whether the oxidase activity of different oxidase-positive psychrotroph strains are of the same order so we have measured the specific rates of TMPD oxidation in a variety of milkassociated bacteria (Table 1). This activity falls into three fairly distinct categories. The high oxidase activity organisms are all Gram-negative psychrotrophs and their activities are very similar - between 1 and 2 f.!mol/mg/min. There are some notable exceptions of important psychrotrophs that have low oxidase activity, however, e.g. Pseudomonas putida, Ps. stutzeri and Flavobacterium odoratum (variations in Pseudomonas spp. are well known (Jones 1980)). Of the few Bacillus species we have tested, these can be intermediate (l50-200 nmol/mg/min) or low «70 nmol/mg/min, confirming the variations shown by Jurtshuk & Liu (1983). The majority of genera with low TMPD oxidation rates were not Gramnegative psychrotrophs. This test is simple and cheap to perform and may well be of use but confirmation from use in dairies with 'real' samples is needed. The method may also have application to other products where spoilage by Gram-negative psychrotrophs predominates. The method is not directly applicable to nonvacuum-packed meats, however, because of the large rates of non-bacterialassociated TMPD oxidation, presumably by the meat mitochondria (R.G. Kroll, unpublished results). This does not appear to be a problem in milks (Kroll 1985) but could interfere with the test in high somatic cell count raw milks. We are currently investigating some other simple tests relying on different principles as well as refinement of the methods described here.

238

R. G. KROLL £1' AI. TABLE

1. Specific rates of TMPD oxidation by milk-assiciated bacteria

Organism

High rates of TMPD oxidation Pseudomonas fragi NCDO 2755 GI (b) G3 (b) G5 (b) P3 (b) (Pseudomonas sp.) P4 (b) PS (h) Intemlediate rates of TMPD oxidation C3 (c) Bacillus cereus 818 (d) GP5 (e) Low rates of TMPD oxidation Escherichia coli NCDO 744 Esdzeridlia coli NCDO 1989 Proteus vuIgan's NCTC ] 0020

Al (0 A3 (f) Escherichia coli K12 Flavobaflerium odt)ralum HRI G 12/6 (g) PseutWmonas putida lIRI 893 (g) Pseudomonas stut.::::eri HRI B414 (g) Chromobacterium sp. HRI 12 (g) Acinetobacter sp. IJRI 155 (g) Enterobacter agglomerans lIRI 1274 (g) Alcaligem:s faecaIis HRI/G27 (g) Bacillus pUlnulis KRl\1 029 (g) Micrococcus Juteus NCOO 947 A1icrococcus roseus NCDO 767 111icro(occuS sp. M3 (h) Staphylococ£us len/us (i) Staphylococcus aureus NeOO 1499 Streptococcus sp. S1 (h) Streptococcus sp. 53 (h) Pediococcus pmtosaceus NCDO 122] LeUcol1ostoc mesC1lteroides NCDO 548 subsp. mesmleroides Microbacterium lactieuln NCDO 1103 Camobacten'um piscicola lVIT35 (j)

Specific rate of Tl\IPD oxidation (a)

2055 1033 1420 1268 1679 1130

± 817 (4)

± 401 ± 35 ± 442 ± 134 ± 210 1514 ± 85

(4) (2) (2)

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o (1)

5.0 ± 2.8 (2) 52.9 ± 1.9 (2) 14.8 ± 6.2 (2) 14.1 ± 0.6 (2) 48.9 ± 2.2 (2) 65.1 ± 0.9 (2) 0.7 ± 0.7 (2) 69.7 ± 3.0 (2) 3.7 ± 3.7 (2) 0.7 ± 0.7 (2) 12.9 ± 0.4 (2) 2.7 ± 2.7 (2) 2.1 ± 2.1 (2) o (2) 14.7 ± 8.4 (3) 39.6 ± 4.4 (2) (2) 2.5 ± 2 o (2)

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DETECTION OF ELECTRON TRANSFER

239

(a) Given as nmol/ng/*min. The second figure is the standard deviation and the number in parentheses the number of individual separate experiments. (b) Unidentified Gram-negative psychrotroph isolated by U.1\1. Rodrigues, IFR, Reading Laboratory. (c) Unidentified Gram-negative psychrotroph isolated by R.G. Kroll, IFR, Reading Laboratory (possibly Acirletobacter). (d) Isolated by J.G. Franklin, NIRD. (e) Unidentified Gram-positive psychrotrophic spore formerly isolated by II. Underwood, IFR, Norwich Laboratory. (f) Unidentified coliforms isolated by U.M. Rodrigues, IFR, Reading Laboratory. (g) Kindly supplied by M.RW. Griffiths, Hannah Research Institute, Ayt. (h) Isolated by U.M. Rodrigues, IFR, Reading Laboratory. (i) Isolated by CH. IVIcKinnon, NIRD. (j) Supplied by RA. Phillips, IFR, Reading Laboratory. " Assuming I mg dry weight

= 0.0'650

2.0.

References ANON. 1983. Paslean~'il1g Plant Mallaal, 3rd edn. Huntingdon: Society of Dairy Technology. BENETTO, 11.1'. 1987. Microbes come to power. New Scimtisl No. 1556, PI'. 36-39. BENETTO, 11.1'., DILANE\', G.M., MASON, J.R., ROLLER, S.D., STIRUN(i, J.L. & THURSTON, Cl". 1986. The sucrose fuel cell: efficient biomass conversion using a microbial catalyst. Biotcehllolo!,:)' Letters 7, 699-704. DELANEY, G.M., BENETTO, 11.1'., MASON, J.R., ROLl.ER, S.D., STIRLING, J.L. & THURSTON, CF. 1984. Electron transfer coupling in microbial fuel cells. 2. Performance of fuel cells containing selected microorganisms-mediator-substrate combinations. Joarnal of Chemical Tcehnolob,), f5 Biotechnology 34B, 13-27. GRIFFITHS, J\1.W., PHILIPS, J.D. & MUIR, D.o. 1984. Methods for rapid detection of postpasteurisation in cream. Joamal of/he Society o/Dairy TecJl1lology 37,22-26. JONES, CW. 1980. Cytochrome patterns in classification and identification including their relevance to the oxidase test. In Microbial Classificatioll al1d ltkntification, ed. Goodfellow, M. & Board, R.G., Society for Applied Bacteriology Symposium No.8, PI'. 127-138. London: Academic Press. jURTSHUK, p. & Lru, J.K. 1983. Cytochrome oxidase analyses of Bacillas strains: existence of oxidase-positive species. lrttemational Joamal of Systematic Bacten'ology 33, 887-891. KROLL, R.G. 1985. The cytochrome c oxidase test for the rapid detection of psychrotrophic bacteria in milk. Joamal ofApplied Bacteriology 59, 137-141. KROLL, R.G. & RODRI(iUES, UM. 1986a. Prediction of the keeping quality of pasteurised milk by the detection of cytochrome c oxidase. Joamal ofApplied Bacteriology 60, 21-27. KROLL, R.G. & RODRIGUES, U.M. 1986b. The direct epifluorescent filter technique, cytochrome c oxidase test and plate count method for predicting the keeping quality of pasteurised cream. Food Microbiolob')' 3, 185-194. LOo.:, H. 1982. Reduction tests for determination of the bacterial quality of raw milk. Kieler ,WilchwinscJlajliche Fomhal1gsberichte 34, 104-116. NISHIKAWA, S., SAKAI, S., KARUBE, I., MATSUf'AGA, T. & SUZUKI, S. 1982. Dye-coupled electrode systems for the rapid deternunation of cell populations in polluted water. Applied and Environmental Microbiology 43, 814-818.

240

R. G. KROLL ET AL.

PATCHETT~ RA., KELLY~ A.F.

& KROLL, RG. 1988. Cse of a microbial fuel cell for the rapid enumeration of bacteria. Applied Microbiology and Biotechnology 28, 26- 31. PHILLlPS,J.D.~ GRIFFrrHS~ M.\V. & fvluIR, D.O. 1981. Factors affecting the shelf-life of pasteurised double cream. Journal of the Society of Dairy Technology 34, 109-112. RODRIGUES, U.M. & PETTIPHER, G.L. 1984. Use of the Direct Epifluorescent Filter Technique for predicting the keeping quality of pasteurised milk within 24 hours. Journal ofApplied Baiten'ology 57, 125-130. ROLLER, S.D., BENETTO~ H.P., DELANEY, G.M., MASON~ J.R. STIRLING, J.L & THLJRSTON~ C.P. 1984. Electron-transfer coupling in microbial fuel cells. 1. Comparison of redoxmediator reduction rates and respiratory rates in bacteria. Journal of Chemical TedmoloJ...'}' and

Biotedmology 34B, 3-12. MJ.A., COUSINS, eM. & McKINNON, C.II. 1982. Effects of psychrotrophic postpasteurisation contamination on the keeping quality at II ° and SoC of HTST -pasteurised milk in the UK. Journal of Dairy Research 49, 619-630. 'I'URNER, A.P.F., CARDOSI~ M.F.~ RAMSAY, G., SCHNEIDER~ RH. & SWAIN, A. 1986. Biosensors for use in the food industry: a new rapid bioactivity monitor. In Biotechnology in lhe Food Industry, pp. 97-115. London, New York: On Line Publications. TURNER, A.P.F., RAMSAY, G. & HIGGINS, I.J. 1983. Applications of electron transfer between biological systems and electrodes. Biochemical Socie~y Transactions 11, 445-448. WILSON~ G.S. 1935. The Bacteriological Grading of Milk. Medical Research Council. London: HMSO. SCHROEDER,

Computer-Assisted Identification of Moulds A.

P. WILLIAMS AND ANIA BIALKOWSKA

Leatherhead Food RA, Rantlalls Road, Leatherhead, Surrey KT22 7RY, UK

In an age of rapidly advancing scientific research, food mycology remains almost totally neglected by microbiologists. Many reasons can be found to explain this (Pitt & Hocking 1985; Jarvis & Williams 1987; Williams 1989) and, indeed, it is pertinent to ask whether the neglect in fact reflects the true sib'l1ificance of the subject. This introduction aims to show that changing circumstances in food manufacture and distribution are leading to an increase in mould spoilage. Historically, human food could be divided into two rather distinct categories: 1 Foods made shelf-stable by drying or by use of three major natural preservatives: sugar, salt and vinegar. 2 Perishable foods. The former were not usually subject to bacterial spoilage and, if mould spoilage occurred, it was considered to be a function of age and the food was discarded. The latter foods were usually subject to rapid bacterial spoilage and the slower-growing moulds rarely caused a problem, except in fruit and vegetables. In the last few years, the distinction between these categories has virtually ceased to exist. Sugar and salt, now in medical disfavour, are used in lower concentrations, and the public has been convinced that it prefers mild pickles. The public also wishes to shop less frequently and perishable foods are required to last longer. The manufacturers' and retailers' answer has been to establish a chill chain, which, when operated efficiently, controls the growth of most food-poisoning bacteria and retards the growth of spoilage bacteria. In the process the conditions are set for mould growth to become a major source of primary spoilage, because moulds grow over such a wide range of nutritional and environmental conditions. Moreover, such mould growth is now accepted as having the potential to form toxic metabolites. In order to control mould growth in food it is essential to limit the level of

Cr,pyright Rapid Microbiological Methods for Foods, Beverages and Pharmaceuticals

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1989 by the Society for Applied Bacteriotol{)' All rights of reproduction in any fOrm reserved

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242

A. P. WILLIAMS AND A. BIALKOWSKA

contamination as far as possible and to und(~rstand the conditions that govern rate of growth. In each case the fundamental need is to identify the species present (Pitt & Hocking 1985; Williams 1989). With accurate identification it is possible to recognize and control sources of contamination, as well as to predict spoilage rates in different classes of food, and under different storage conditions.

The Evolution of I\!lould Identification Mycology originated as a branch of botany and mould identification followed the same precepts, using morphological criteria to define species and dichotomous keys to distinguish between them. This approach served the science well, although over the years, as more and more species were described, the language used became so complex as to be incomprehensible to all but the specialist taxonomist. Bacteria, on the other hand, have little morphological variation, and identification procedures have been based on physiological, biochemical or immunological differences. This has given bacteriologists morc information about individual organisms, and dichotomous keys have, therefore, been almost totally abandoned in favour of identification profiles, as used for example by Cowan (1974). Several mycologists have recognized the need for improved identification procedures and have introduced standard plating regimes and physiological measurements (Pitt 1973, 1979; Frisvad 1981; Pitt & Hocking 1985). Much of this work has centred on the genus Penicillium and especially on the threestage branched species, because they are so common as spoilage agents of agricultural materials and of foods, especially refrigerated foods. Pitt & Hocking (1985) retained the use of dichotomous keys but greatly simplified their language in order to make them comprehensible to microbiologists without specialist experience. Pitt (1979) used both dichotomous and synoptic keys in different sections of his monograph on Penicillium. The disadvantage of both synoptic keys and more particularly of dichotomous keys is that results may be obtained from a variable number of answers, depending on the location of the organism in the key. In both cases, the misreading of a result will often lead to a failed identification. There is an obvious need for schemes that obtain identification by using a standard range of tests to compare a strain with a group of related species. A microcomputer is of great value in making these comparisons. The scheme described below was devised for use by microbiologists who do not have a specialist knowledge of mycology. The moulds described arc the three-stage branched penicillia, which are among the most difficult common moulds to identify by conventional methods, because they are very closely

COMPUTER-ASSISTED MOULD IDENTIFICATION

243

related to each other (Williams et al. 1985). Other groups of moulds could equally well be identified by substituting an appropriate database in the computer program and adjusting the identification criteria as necessary. Fourteen easilyread physiological and morphological criteria are compared with a standard database and all are given equal weight. This means that a single failed test or misreading will rarely give a failed identification. Because this is an identification scheme, and only 14 criteria are used, it must be emphasized that taxonomic completeness is not intended. Rather, the aim is to encourage food microbiologists to identifY some of the more common moulds for themselves and, by doing so, to recognize the most likely sources of contamination. This will help us to improve and monitor the quality of our food.

Methods

Isolation and storage of cultures of sub-genus Penicillium (species with three-stage branched heads) Methods for isolation of moulds are described in detail by Pitt & Hocking (1985). Sources include agar plates that have been used for colony counting or food samples with visible mould. Moulds are examined under the microscope and species of Penicillium with three stages of branching in mature heads arc selected for identification. Mould growth is streaked on a plate of a suitable antibacterial growth medium such as Rose Bengal Chloramphenicol Agar (Oxoid CM549) and incubated in air at 25°C for 5 - 7 days to check for purity. If storage is necessary, a slope of malt extract agar (Pitt & Hocking 1985) may be inoculated with a loopful of spores and incubated as above before storage in refrigeration.

Method jor obtaining data A spore suspension is prepared from an actively growing 5 - 7-days culture, by the method of Pitt & Hocking (1985). The following media are inoculated with triple points (three equidistant spots) of the spore suspension: Czapek yeast agar (CYA) Malt Extract agar (MEA) Yeast extract-sucrose agar (YES) Creatine-sucrose agar (CREA) Nitrite-sucrose agar (NSA)

(Pitt & Hocking 1985) (Pitt & Hocking 1985) (Frisvad 1981) (Frisvad 1981) (Frisvad 1981)

All plates are incubated in air at 25°C for 7 days.

244

A. P. WILLIAMS AND A. BIALKOWSKA

Recording of results After plates have been incubated, results arc recorded for each of the 14 following criteria. The range of colours, in particular, has been simplified to the fewest possible categories. 1 Mean colony diameter (mm) on eYA. 2 Mean colony diameter (mm) on MEA. 3 Mean colony diameter (mm) on YES. 4 Growth on CREA (-, -VV, +, ++)*. 5 Acid production on CREA (-, W, +, + +)*. 6 Growth on NSA (-, W, +)*. 7 Colour of spores on CY A: white; pale to mid-grey; olive brown; blue-green or blue-grey; grey-green or dull-green; yellow-green or bright green; dark green or dark blue-green. 8 Reverse colour on CYA: pale or dull; orange, brown or red; bright yellow or yellow-green; dark green or blackish green. 9 Reverse colour on YES: pale or dull; brownish orange or brown; orange-yellow or orange; bright yellow; blackish green with some orange. 10 Texture of stipe surface: smooth; finely roughened; rough. 11 Details of spore: round or oval, smo~th; round or oval, finely roughened; round or oval, rough or spiny; cylindrical, smooth or rough. 12 Shape of head: *-, negative; w, weak; +, moderate; ++, strong.

COMPUTER-ASSISTED MOULD IDENTIFICATION

245

fan-shaped; two or more lower branches; squat; 1- 2 lower branches; complex; very short phialides; normal. 13 Detachable crusts of spores on MEA (YIN). 14 Texture of colony surface on CYA: 'drumsticks' more than 2 mm high; feathery yellow tufts (especially on MEA); very tufted; deep and fluffY; gramy; velvety.

Computer and database This work was done on an Apple lie Microcomputer (64 k) and the program was written in Apple Basic. It will also soon be translated into IBM Basic.

The structure of the computer

k~y

Data are requested by menu and entered from the keyboard. From the input data a string containing 14 sub-units is constructed. The full string comprises 93 characters, each of which is a class of value 'yes' (Y), 'no' (N), or 'no data available' (Z). The input string is sequentially compared with 23 unique strings, prepared from our own database and from published data (Pitt 1979; Frisvad 1981, 1985a,b), representing each major species in sub-genus Penicillium. For each sub-unit of the string, agreement between the unknown isolate and all of the named species is scored as follows: Agreement Disagreement A slightly atypical result

1.0 0.0 0.5

Agreement with each of the 23 species is expressed as a percentage. The results, as percentages, are then sorted in descending numerical order and displayed on the screen. Finally, a main branching menu offers the following options: Re-scan the results List any atypical results Check input data Print a results sheet IdentifY another isolate End

246

A. P. WILLIAMS AND A. BIALKOWSKA

A feature of this method of scoring the results is the ability to take into account results that are atypical. When penicillia are isolated from natural environments individual strains are occasionally found that differ from published accounts in one or two characters only. Examples from our own collection include Penicillium roquefOrtii with very small colonies or totally smooth stipes, P. chrysogenum with dark green spores and no yellow pigments, and P. ita/icum that forms detachable crusts of spores on MEA. In the first two cases the identification would fail if conventional keys were used and in the last there would be confusion with P. crustosum, the only species in the sub-genus that regularly produces spore-crusts. Furthermore, even with simplified schemes, some characters, such as colony texture, are occasionally difficult to interpret. Again, it is possible to make allowance in the database for results that are open to misinterpretation. By taking such discrepancies into account, the success of identification is greatly enhanced: this is the objective, but it has to be at the expense of taxonomic accuracy. The ability of the key to take into account atypical results is illustrated below. Table 1 is a printed results sheet for a typical strain of P. roquefortii and TABLE

Isolate Date

I. Identification of a typical isolate of Penicillium roquefortii

Specimen 23/Aug/87

Identification (using 14 answers): 1 Similarity to Penicillium roquefOrtii (species 19) is 100%

Characteristics of the strain: 1 Colony diameter on CYA (nun) 2

3 4

5 6 7 8

9 10

II 12

13 14

Colony diameter on MEA (nun) Colony diameter on YES (nun) Strength of growth on creatine . . . . . . . . . . . . . . . . . . . . Acid production on creatine Growth on nitrite. . . . . . . . . . . . . . . . . . . . . . Colour of spores on CYA Reverse colour on eYA Reverse colour on YES Texture of stipe surface Detail of spore Detailofhead Crusts of spores on MEA Colony texture . . . . . . . . . . . . . . . . .

The next nearest three species are: 2 Penicillium ita/hum (species] 7) 3 Penicillium commune (species 6) 4 Penicillium verrucosum (species 21)

58 62 68

++ -

+ Grey-green or dull green Dark or blackish-green Blackish green + orange Rough Smooth Normal

No Vc1vety

(62%) (58%) (54%)

COMPUTER-ASSISTED MOULD IDENTIFICATION TABLE 2. Idmtifiwtiol1 of 01/ atypical isolate Isolate Date. .

247

of Penicillium roquefortii

Specimen 23/Aug/87

Identification (using 14 answers): I Similarity to Pmidllium roquifortii (species 19) is 8 I %

Charal'tenstics of the spedes: I Colony diameter on CYA (mm) . 2 Colony diameter on MEA (mm) . 3 Colony diameter on YES (mm) 4 Strength of growth on creatine .. 5 Acid production on creatine 6 Growth on nitrite. . 7 Colour of spores on CYA 8 Reverse colour on CYA 9 Reverse colour on YES 10 Texture of stipe surface 11 Detail of spore .. 12 Detail of head . 13 Crusts of spores on 1\1EA 14 Colony texture .. 771e followil1g Near miss: Near miss: Near miss:

23 24

. .

............ 27

.

++

.

.

. .

.

+ Grey-green or dull green · .Dark or blackish-green · .Blackish green + orange · . Rough · .Smooth . .... Normal .No . .... Grainy

dwracteristic(;) did 110t agree: 1 Colony diameter on CYA 2 Colony diameter on MEA 3 Colony diameter on YES 14 CYA colonies should not have been grainy

The l1ext Ilearest three isolates 2 Pmidllium verruwsum 3 Pmidlliam solitum 4 Penicillium atrammtosum

are: (species 21) (species 20) (species I)

.

(69%) (69%) (69%)

shows the sort of result that is normally obtained. Table 2 is the result for an atypical strain of P. roquejOrtii with a very slow growth rate. The position is complicated by a misreading of the colony teA1ure on CY A as 'grainy'. If a person unfamiliar with P. roquefOrtii were to attempt to identifY this strain by using the scheme of Raper & Thorn (1949), a successful identification would be impossible because of the misreading of colony texture. If he were using that of Pitt (1979) the same position would apply because of the atypically slow growth rate. The computer key, however, selected P. roquefOrtii as the most likely species, although only with an 81 % agreement. The printed results sheet shows which criteria did not fit those of a typical P. roquejortii. Colony diameters were too small, although 'near miss' indicates that this does

248

A. P. WILLIAMS AND A. BIALKOWSKA

sometimes happen. It is unlikely, however, that the colony texture of this mould would be interpreted as other than velvety. This character is, therefore, recorded as a disagreement, alerting the user to the need to check the result. The overall identification, however, remains as P. roquejOrtii.

Conclusion The methods and results illustrated above demonstrate that the use ofcomputerscan greatly assist the development of simplified identification methods. It is planned that this scheme will shortly be used in field trials and, if these are successful, be made more widely available. Such identification schemes, usable by microbiologists without mycological eXl'crience, should help to resolve some of the mould-spoilage problems that result from changing practices in the manufacture and distribution of our food.

References CmrAN, S.T. 1974. Cowan and Steers /Wanual for the ldetltification of/Hedical Btllteria. Cambridge: University Press. FRISVAD, j.c. 1981. Physiological criteria and mycotoxin production as aids in classification of common asymmetric penicillia. Applied and Environmental A1icrobiology 41, 568-579. FRISVAD, J.c. 1985a. Classification of asymmetric penicillia using expressions of differentiation. In Advtltlces in Penicillium tltld Aspergillus Systematics, ed. Samson, R.A. & Pin, J.1., pp. 327-335. New York & London: Plenum Press. FRISVAD, Jc. 1985b. Profiles of primary and secondary metabolites of value in classification of Pmicillium viridicatum and related species. In Advances in Penicillium andAspergillus Systematil's, cd. Samson, R.A. & Pitt, ).I., pp. 311-325. New York & London: Plenum Press. JARVIS, B. & \VILLIA.\1S, A.P. 1987. Methods for detecting fungi in foods and beverages. In Food and Beverages iMycology, ed. Beuchat, L.R. pp. 599-636. New York: AVI, Van Nostrand Reinhold Company Inc. PITT, J.I. 1973. An appraisal of identification methods for Penicillium species: novel taxonomic criteria based on temperature and water relations. iHy£ologia 65, 1135-1157.

PITT,].1. 1979. The Genus Pnlicillium and its Teleomorphic Statts Eupmicillium and Talaromyces. London: Academic Press. J.I. & HOCKING, A.D. 1985. Fungi and Food S'poilage. Australia: Academic Press. RAPER, K.B. & THOM, C. 1949. A iVIallual of tlu Penicillia. Baltimore: \ViJliams and Wilkins. \VIl.LJAMS, A.P. 1989. Fungi in foods - rapid detection methods. In Progress in Industrial Microbiology, Vol. 26, Rapid Alethods in Food Microbiology, ed. Adams, l\tR. & Hope, C.F.A. Amsterdam: Elsevier Science Publishers. [0 press. Wn.L11\MS, A.P., PITT, ].1. & HOCKING, A.D. 1985. The closely related species of subgenus Penicillium - a phylogenetic exploration. In Advances in Penicillium and Aspergillus Systematics, ed. Samson, R.A. & Pitt,].I. pp. 121-128. New York and London: Plenum Press. PITT,

Immunological Detection Methods for Salmonellas in Foods C. DE W. BLACKBURN AND CATHERINE]. STANNARD'"

Applied Microbiology Section, Leatherhead Food RA., RanJalls Road, Leatherhead, Surrey KT22 7RY, UK

Numbers of outbreaks of Salmonella food poisoning are increasing. It is, therefore, important for manufacturers to detect and control contaminated foodstuffs. Conventional cultural methods are labour-intensive and expensive, but their major problem to the food industry is that it may take five days or more to determine whether a sample is positive for Salmonella. The expense, inconvenience and loss of potential shelf-life of products while being tested have prompted the development of more rapid methods (Wood & Gibbs 1982). Immunological methods (Ibrahim & Fleet 1985) offer several advantages over other techniques, including the inherent specificity of the antigen/antibody reaction and the low capital expenditure necessary. Four kits based on the immunological detection of Salmonella are now available and are outlined and briefly evaluated in this paper; these evaluations were not concurrent. Bio-Enzabead Screen Kit

Principle The Bio-Enzabead Screen Kit (Organon Teknika Ltd, Cambridge) is a sandwich enzyme-linked immunosorbent assay (ELISA, see Fig. 1). It utilizes two monoclonal antibodies, which are specific to Salmonella flagellar antigens (structural flagellin, rather than the specific H antigens) and which show no cross-reactivity with other enteric species. The Salmonella antigens, if * Present address: Applied Research Department, Pedigree Petfoods, Melton Mowbray, Leicestcrshire LE 13 I BB, UK. COf!yri"hl Rapid Microbiological Methods for Foods, Beverages and Pharmaceuticals

© 1989 b)' the Socie/I' fOr Applied Baaeriol01O' All nghts of "production in any jimn reserved

0-632-02629-4

249

250

C. DE W. BLACKBURN Ar\D C.

~

J.

STANNARD

Antibodv adsorbed to solid phase

}<~o

Add sample containing antigen and incubate

}<<> }<<>;

Wash to remove unbound antigen

E

E

yE

Add enzyme-labelled antibody and incubate

}-<<>>-E

Wash to remove unbound enzymelabelled antibody

~O>--!-E ~

Add enzyme substrate (0 l and incubate. Measure coloured enzyme product (e)

O. •

0

Amount of antigen ex [enzyme product] FIG.

1. Principles of a sandwich enzyme-linked inununosorbent assay.

IMMUNOLOGICAL DETECTION OF SALMONELLAS

251

present, bind to the antibodies, which are coupled to a plastic-coated ferrous bead. An antibody-conjugated enzyme (horseradish peroxidase) then binds to the bound antigen. A colourless substrate, which is then added, is cleaved by the bound enzyme. The quantity of coloured product can be read spectrophotometrically and compared with negative controls and a substrate blank. Washing procedures are aided by use of a magnet to transfer the antibodycoated ferrous beads from one microtitre plate to another.

Procedure The procedure is outlined in Fig. 2. The manufacturer recommends the preenrichment procedures outlined in The Food and Drug Administration's Bacteriological Ana(ytical Manual (BAM), or the pre-enrichment procedure normally performed in the user's laboratory. However, the manufacturers specify that raw and highly contaminated foods should be pre-enriched prior to selective enrichment. Portions of the selective enrichment broths and unheated M-broths should be stored at 4°C for confirmation of ELISA-positive results by plating on selective agar media.

Brief evaluation Washed cells of Salm. ~yphimun'um (Leatherhead Food R.A. culture, resistant to nalidixic acid) were subjected to acid stress (37°C for 60 min in 0.3 molll sodium acetate buffer, pH 4.2), two heat stresses (54°C for 40 min in dried skimmed milk reconstituted in fresh distilled water, 40% solids (w/v), or 51°C for 40 min in 0.1 moll I potassium phosphate buffer, pH 6.0), and freeze-drying stress (in dried skimmed milk or cocoa powder reconstituted in fresh distilled water, 40% solids (w/v); using the Speedivac centrifugal freeze dryer, Edwards Hi Vacuum Ltd). Cells were added to samples of minced beef, cocoa powder and dried skimmed milk in Buffered Peptone Water (BPW, Oxoid; samples diluted 1:10 (w/v» to a level of c. 2/g of food material. The same pre-enrichment broths were used tor both the ELISA (Fig. 2) and conventional cultural procedures. The same selective enrichment broths, namely Selenite-Cystine (SC; Difco) Broth and Rappaport- Vassiliadis (RV; Difco) Broth, were also used for bOtll methods but the incubation time for the conventional cultural procedure was 18-24 h regardless of the food type. Selective enrichment broths were streaked on to Bismuth Sulphite Agar (BSA; Oxoid) and Xylose Lysine Desoxycholate Agar (XLD; Oxoid) plates, and incubated at 3rC for 24-48 h. Suspect colonies were presumptively identified as Salmonella spp. using the ApiZ rapid screening kit (API Systems, France).

252

C. DE W. BLACKBURN AND C. J. STANNARD

Low-moisture materials

High-moisture materials

Pre-enrichment

Pre -enrichment

1

1

24 ± 2 h

18 h

Selective enrichment

Selective enrichment

1 M-brr~~ ,:Il

118 - 24 h

6-8 h

M-broth (10 mil

1

h

I

20 min

'"

Remove 0.5 ml and heat in boiling water bath

Resuspend in phosphate-buffered saline (1 mil and heat in boiling water bath

~

20 min

6 h

Centrifuge (1500 g, swinging bucket)

~

/ 0 min

Cool to 25° ~ 37°C. To wells of a 96-well microtitre plate, add 200 ~I heat-treated samples and control antigens Add antibody-coated beads 20 min, 37 DC, agitation

1

(10-100 rev/min)

Wash beads and add antibody - enzyme conjugate solution (200 /il) .20 min, 37 DC. agitation (10 -- 100 rev/min)

1

Wash beads and add enzyme substrate solution (200

1,0

/il)

min, room temperature

Add stop solution (25 1J1) and swirl plate to disperse coloured product. Remove bead and read absorbance at 405 nm or 690 nm Absorbance values > O. 20 constitute a positive result FIG. 2. Bio-Enzabead Screen

Kit procedure.

The Bio-Enzabead Screen Kit was easy to use, although fairly labourintensive. The centrifugation, re-suspension, heat treatment and assay of small numbers of M-broth cultures were easily completed within 2 h. To handle larger nunlbers without the use of a large-capacity centrifuge took considerably longer. At the time of the evaluation the centrifugation step was recommended for all foods. Since then, the ELISA protocol has been altered such that dry foods do not require the centrifugation step, thus saving time. Results were obtained late on the third day, although it was often more convenient to refrigerate the heat-treated M-broths from high-moisture foods, and assay on the fourth day.

lM:\1UNOLOGlCAL DETECTION OF SALMONELLAS T.~lJLL

I. Summary ol results usillg the Bio-EIlZilbetui Screm Kit alld the cOIlvmtiollal cultural method

Food type

Salmollella stress

Dried skimmed milk

None None None Ileat stress - milk Ileat stress - milk Freeze-drying stress

Cocoa powder

None None None Heat stress - milk Freeze-drying stress

?lileat

253

None None None I Ieat stress - buffer Heat stress - huffer Ileat stress - huffer Acid stress Acid stress Acid stress

ELISA result

Conventional cultural method result

-/-/-/-/-/-/-

-/-/-/-/-/-/-

-/-

-/-/-/-/-

-/-/-

t -/-

-/-/-/-/-/-

-/-/-/-

- (0.17)*

* Negative result was of a visually higher optical density than the negative controls (i.e. 0.0. <: 0.20). t Samples transferred directly into selective enrichment broths (all positive by ELISA also).

Using the ELISA method, 19 of the 20 samples were pOSItIve for Salmonella, the other being positive by eye but negative spectrophotometrically. Twelve of the 20 samples were positive by the conventional cultural method, seven of the eight negatives being meat samples (Table 1). This failure of the conventional cultural method to detect Salmonella could be due to the presence of large numbers of mesophilic micro-organisms (c. I x 10 7 cfu/g) that may have overgrown the salmonellas. The fact that the three meat samples that underwent direct selective enrichment (i.e. no pre-enrichment, not shown in Fig. 2) were positive by both ELISA and conventional cultural methods would appear to be in agreement with this explanation.

TECRA Salmonella Visual Immunoassay Principle The TECRA Salmonella Visual Immunoassay (BioEnterprises Pty Ltd, Australia), like the Bio-Enzabead Screen Kit, is a sandwich ELISA (Fig. 1). It

254

C. DE W. BLACKBURN AND C.

J.

STANNARD

differs, however, in that it utilizes a polyclonal antibody and instead of ferrous beads, the well surfaces of a microtitre plate are used as the solid phase.

Procedure The procedure is outlined in Fig. 3. The manufacturer recommends that food samples should be prepared according to the user's normal procedure and that the standard pre-enrichment and selective enrichment media described in the Food and Drug Administration's BAM should be used. ELISA positives are confirmed in the same way as with the Bio-Enzahcad Screen Kit.

Brief evaluation Cells of each of the following Salmonella serotypes were subjected to heat stress (51°C for 40 min in Maximum Recovery Diluent; Oxoid) and freezeProcessed foods

Raw foods

Pre-enrichment

Pre-enrichment

1 1

~

16- 20 h

Selective enrichment

16- 20 h

Selective enrichment

~ 16 - 20 h M-broth

6 h M-broth

16~

~~

Heat in a boiling water bath

115

min

Add 200 fJl heated M-broths and control antigens to antibody-coated wells of microtitre plate

1 1

30 min, 37°C

Wash wells and add antibody-enzyme conjugate solution (200 /AI)

30 min, 37°C

Wash wells and add enzyme substrate solution (200 /AI)

110- 15

min, room temperature

Add stop solution /20 ~l) and gently mix well contents. Read absorbance visually at 414 nm. Absorbance values> 0.30 lor its visual equivalent) constitute a positive result

FIG. 3. TEeRA Salmonella Visual Immunoassay procedure.

I'\1MUNOLOGICAL DETECTION OF SALMONELLAS

255

drying stress (in skimmed milk powder or cocoa powder reconstituted in fresh distilled water, 40% solids (w/v); using the Speedivac centrifugal freeze dryer, Edwards Hi Vacuum Ltd): Salm. ~yphimurium NCTC 74, Salm. stanley NCTC 92, Salm. virchOll} NCTC 5742, Salm. lle1Vport NCTC 129 and Salm. enteritidis var. danysz NCTC 4444. Cells were added to samples of chicken, prawns and chilli-eon-came (diluted 1 in 10 (w/v) in buffered peptone water) cocoa powder (diluted I in 10 (w/v) in UHT milk + 0.002'Yo (w/v) brilliant green) and skimmed milk powder (diluted 1 in 10 (w/v) in distilled water + 0.002% (w/v) brilliant green) to give two inoculum levels (namely 0.04-0.2 and 0.4- 2 cells/g of food material). Uninoculated food homogenates were also prepared. The same pre-enrichment broths were used for both the ELISA (Fig. 3) and conventional cultural procedures. In addition to SIC and RV selective enrichment broths, as used in the conventional cultural procedure, Muller- KauHinann Tetrathionate (TET; Oxoid) broth was also used in the ELISA procedure. The selective enrichment incubation time was 18- 24 h regardless of the food type. The selective plating media used were BSA and XLD agar; colonies after 24-48 h were presumptively identified with the API 20E kit (API Systems) and confirmed to species level with 0 and H antisera (Wellcome). The TECRA Salmonella Visual Immunoassay was easy to use and not particularly labour-intensive. The heat treatment and assay of large numbers of samples could be completed within 2 1/2 h. Using the ELISA method, presumptive results from processed foods were obtained on the third day compared with 4- 5 days required when using the conventional cultural method. With raw foods the 6-8 h M-broth incubation meant that ELISA results could be obtained late on the third day. In practice, however, it was more convenient to refrigerate the boiled samples overnight and assay them on the fourth day. All the artificially contaminated samples of processed foods (Table 2) were found positive for Salmonella at both inoculum levels by both the ELISA and the conventional cultural methods. Only one ELISA-positive sample was not confirmed by selective plating from either the selective enrichment broths (at the time of sub-culture to M-broths) or the M-broths. This sample, however, was positive by the conventional method, which specifies a longer period of selective enrichment than the ELISA procedure. There was also 100% agreement between the ELISA and conventional methods for the detection of Salmonella from prawns. Using the conventional method, six of the eleven chicken samples were positive while the ELISA gave nine out of eleven Salmonella-positive results. In two samples, including the uninoculated control, the ELISA-positive samples were not confirmed by selective plating and the corresponding conventional cultural method results were also negative. The absorbance readings of these false-positive results were only marginally higher (0.31-0.39) than the requirements for a positive sample (> 0.30). These

256 TABLE

C. DE W. BLACKBURN AND C.]. STANNARD 2. Summary of results from the TECRA Salmonella Visual Immunoassay Kit and the conventional cultural method No. of positive results

Food type

Salmonella Slress

No. of samples

ELISA

Conventional cuhural method

Chilli-con-carne

Heal Slress

11

10 (9)*

10

Skimmed milk powder

Freeze-drying stress

11

10 (10)

10

Cocoa powder

Freeze-drying stress

11

10 (10)

10

Prawns

None

11

9 (9)

9

Chicken

None

11

9 (7)

6

* Number of samples confirmed as Salmonella positive by selective plating of stored seleclive broths and/or ~l-broths shown in parentheses.

false-positive results may have been caused by an enhanced competing flora due to the extra period of non-selective enrichment, because they did not occur with uninoculated chicken placed directly into selective enrichment broth (results not shown). In such cases an alteration in the absorbance value required to indicate an ELISA -positive result may be appropriate. Alternatively, direct selective enrichment could be recommended. It is possible, however, that the false-positives were in fact Salmonella-positives that the conventional cultural procedure failed to detect.

Kirkegaard and Perry Salmonella ELISA

Principle Like the TECRA kit, the Kirkcgaard and Perry Salmonella ELISA (Dynatech Laboratories, Billingshurst) is a sandwich ELISA (Fig. 1) utilizing a polyclonal antibody bound to the well surface of a microtitre plate. This kit differs from the other two in that the adsorption of the capture antibody to the solid phase is done by the user. Also, the recommended assay uses time-lapse sampling, in which colour difference is measured behveen a sample taken at the time of medium inoculation and a sample taken after culture growth. 'I"he choice of medium and enrichment conditions are largely left to the user.

Procedure The procedure is outlined in Fig. 4. The manufacturer states that the use of time-lapse sampling allows users to modify their Salmonella detection pro-

IM:'V1lJNOLOGICAL DETECTION OF SALMONELLAS

257

Enrichment (unspecified)

!

Add enrichment broth f 100 1'1) to 10 ml nutrient broth or selective media and mix ____ ~

~ overnight

Immediately remove 2 ml I'IN' sample} and heat in boiling water 15 . min

1

Store refngerated

Remove 2 ml rOUT' sample) and check pH is 5 - 7. Adjust with sodium hydroxide or acetic acid if necessary. Heat in boiling water 15 min

1

~

Add capture-antibody solution (1001'1) to wells of a 96-well microtitre plate

Bring to room temperature

1

60 min, room temperature

Empty wells and add bacterial diluent/ blocking solution (300 Jil)

jl0

mia. worn 'em"",","

Empty wells and add 1001'1 'IN' and 'OUT' heat-treated samples and control antigens

1 1

60 min, room temperature

Wash wells and add antibody-enzyme conjugate solution (1001'1) 60 min, room temperature

Wash wells and add enzyme substrate solution (100 1'1) AHer sufficient colour development

1

Read absorbance visually or at 405 nm or 410 nm. Cultures where the absorbance of the 'OUT' sample is greater than the 'IN' sample are considered positive

FIG. 4. Kirkegaard and Perry ELISA procedure.

cedures to achieve the most rapid results. The procedure, therefore, does not specify pre-enrichment or selective enrichment conditions. Portions of appropriate enrichment media should be stored at 4°C for confirmation of ELISApositive results by plating on selective agar media.

258

C. DE W. BLACKBURN AND C.

J.

STANNARD

Bn'if evaluation Pre-enrichment suspensions of foods, inoculated with stressed and uninjured salmonellas, were prepared as for the TECRA Kit evaluation. The same prcenrichment broths were used for both the ELISA and conventional cultural procedures and incubated for 18-20 h. For the ELISA procedure I-ml volumes ofeach pre-enrichment broth were used to inoculate 10-ml quantities of SC, RV and TET broths. Separate selective enrichment broths were used for the conventional cultural procedure (see TEeRA Kit evaluation). The Kirkegaard and Perry ELISA was easy to use, although labourintensive, requiring numerous manipulations. The assay required at least 4h and even longer if the binding of the capture antibody and the pII checking and heating of the broths were not performed simultaneously. It was found most convenient to use pH paper to check the broths prior to heat treatment but only very occasionally did they require alteration with acid or alkali. Because the manufacturers claim that absorbance readings between duplicates can commonly vary by 100/0, a difference of more than about 20% between 'IN' and 'OUT' sample readings was taken to constitute a Salmonella-positive result. All the artificially contaminated saolples of chilli-con-canle, skimmed milk powder, cocoa powder and prawns were found positive for Salmonella by both the ELISA and conventional methods (Table 3). Only two ELISA-positive samples were not confirmed by selective plating from the selective enrichment broths. These samples, however, were positive by the conventional cultural

TABLE

3. Summary of results usitlg the Kirkegaard and Perry ELISA Kit aud the conventional cultural method No, of positive results

Food type

Salmonella stress

No. of samples

ELISA

Conventional culrural method

Chilli -coo-came

Heat stress

11

10 (9)*

10

Skimmed milk powder

Freeze -drying stress

11

10 (10)

10

Cocoa powder

Freeze- drying stress

II

10 (10)

10

Pravvos

None

11

10 (9)

10

Chicken

None

11

8 (6)

6

* Number of samples confirmed as Salmonella-positive by selective plating of stored selective broths and/or M-broths shown in parentheses.

IMMUNOLOGICAL DETECTION OF SALMONELLAS

259

method. The ELISA method detected two more positive chicken samples than did the conventional cultural method, although neither of these was confirmed by selective plating. Whether growth of salmonellas (as seen by an absorbance increase between 'IN' and 'OUT' samples) occurred in the selective enrichment broths appeared to be dependent on the Salmonella serotype and the food sample. In many cases Salm. zyphimurium and Salm. newport ELISA-positives were from SIC broth only. Most of the chicken samples positive for salmonellas were from RV broth. It may, therefore, be preferable to take 'IN' and 'OUT' samples from a non-selective broth, e.g. M-broth. The manufacturer's instructions emphasize the development of the enrichment protocol by the user and the results of this brief evaluation show the importance of the choice of broth in detecting the growth of certain Salmonella serotypes in different foods.

Salmonella 1-2 Test Principle The Salmonella 1- 2 Test (BioControl Systems, Inc., USA) is based on enrichment serology. The test is conducted in a disposable plastic device with two chambers: the inoculation chamber containing selective enrichment broth (tetrathionate-brilliant green-serine broth) and the motility chamber containing a peptone-based, non-selective motility medium to which is added polyclonal Salmonella flagella antiserum. The pre-enriched food sample is added to the inoculation chamber and, after about 4 h, motile Salmonella, if present, move into the non-selective motility medium where they encounter the diffused flagellar antibodies. They become immobilized to form a characteristic U-shaped band (Fig. 5).

Procedure The procedure is outlined in Fig. 6. Positive 1- 2 Test results should be confirmed by plating from the inoculation chamber on to selective agar media. Longer incubation than the recommended 8-14 h does not interfere with interpretation of the test result.

Brief evaluation Skimmed milk powder was diluted 1: 10 in buffered peptone water (180 ml) containing 0.001 'Yo (w/v) brilliant green and incubated at 37°C for 24 h. The concentration of Enterobacteriaceae organisms was determined by plating on Violet Red Bile Glucose Agar plates (Oxoid). Appropriate dilutions

260

FIG.

C. DE W. BLACKBURN AND C.]. STANNARD

5. Positive (left) and negative (right) Salmfmella 1-2 Tests.

Processed foods

Non-processed foods

Pre-enrichment

Selective enrichment in tetrathionate both + 0.001 % m/V brilliant green

~

1~~ ~minimum

Remove inoculation chamber screw cap of 1 - 2 Test. Add iodine·iodide solution (1 drop). Replace cap and gently shake.

~

Remove motility chamber screw cap. Cut off gel-former plug from cap. Add antibody preparation 11 drop) to gel void. Replace cap.

1

Remove inoculation chamber screw cap. Remove chamber plug. Add enrichment culture (0.1 miL Replace cap.

i 1

8 h, 35°C

Examine for positive results (U-shaped band in motility chamber). Reincubate negative 1 - 2 Tests 6 h (minimum). 35°C

Re-examine for positive results

FIG. 6. Salmonella 1 ~ 2 Test procedure.

261

IMMUNOLOGICAL DETECTION OF SALMONELLAS TABLE

4. Results oI Salmonella 1-2 Test method Cell concentration in pre-enrichment broth

1-2 Test ,'ialmoneUa serotype

S. Iyphimurium

S. t'nteritidis

S. stanley

Salmonellas*

Enterobacteriaceae

result

+ ct +c

1.5 X lOx 1.5 X lO6 1.5 X 104 1.5 X lO2

+c +c +c

1.2 1.2 1.2 1.2

X

108

X

106

X

104

+

X

10 2

+

+c +c

1.3

X

IOH

1.3

X

lO6 104 102

1,3

X

1.3

X

+c c c

+c

+c

None * Salmonellas added to skimmed milk powder pre-enrichment after 24 h incubation at 37°C. t Confirmed by selective plating from inoculation chamber.

of overnight cultures of Salm. typhimurium NCTC 74, Salm. stanley NCTC 92 and Salm. Ctlteritidis var. danysz NCTC 4444 were added to portions of the skimmed milk powder pre-enrichment broth after incubation to provide cell concentrations of each serotype of c. lOR, 106 , 104 and 102 cfu/mt. The preenrichment broths were then used to inoculate 1- 2 Test units according to the method previously described (Fig. 5). The Salmonella 1-2 Test was quick and easy to usc, each test taking only a matter of minutes to set up, with a minimum of manipulations. The 1-2 Tests detected all three Salmonella serotypes at all inoculum levels (Table 4). The test could detect a concentration of salmonellas as low as 102 cfu/ml in the presence of c. 107 competitors. Whether this level of cells could be detected from a non-processed food likely to contain a larger number of competitors after pre-enrichment is questionable. The 1- 2 Tests were examined after 8 h of incubation but only those inoculated from pre-enrichment broths containing c. 108 salmonellas/ml were positive. The other tests required further incubation, in this case overnight, before a positive result was seen. It is, therefore, more likely that a Salmonella test result from naturally contaminated foods would be obtained on the third day.

262

C. DE W. BLACKBURN AND C.

J. STANNARD

Discussion The aim of this work was to outline and to evaluate briefly four commercially available, immunologically-based kits for the detection of salmonellas in foods. All four methods provide a presumptive Salmonella result more rapidly than the conventional cultural nlethod, although they all require some form of sample enrichment. The Bio-Enzabead and TECRA ELISA kits generally provide presumptive Salmonella results late on the third day. Both ELISAs take a similar length of time to perform, although the centrifugation step for high-moisture foods may significantly increase the assay time for the Bio-Enzabead kit. The Kirkegaard and Perry ELISA differs from the Bio-Enzabead and TEeRA kits in a number of ways. This ELISA utilizes time-lapse sampling and allows the user the flexibility to modifY the enrichment procedure with dIe potential of obtaining a more rapid Salmonella detection. Time-lapse sampling, however, results in twice as many broths to heat in comparison wim the BioEnzabead and TEeRA kits. In addition, although no centrifugation step is required, me pH of the broths must be checked and altered if necessary. This, together with the fact that the user is required to bind the capture antibody to the solid phase, means that perfonning the ELISA takes longer and requires more manipulations than do the Bio-Enzabead and TECRA kits. The Salmonella 1-2 Test is based on enrichment serology and consequently samples require only a period of pre-enrichment prior to inoculation as the selective enrichment stage is incorporated in the test. Once inoculated, the test requires a minimum of 8 h of incubation, making presumptive results feasible on the second day but more likely on the third day. None of the ELISA kits failed to detect salmonellas in food samples when the corresponding conventional cultural method gave positive results. All three ELISA kits did, however, detcct salmonellas in samples that were negative by the conventional cultural method. In many cases these presumptive-positives were confirmed by selective plating from the selective broths and/or Nt-broths. Although ELISA-positive samples that were not confirmed would be considered to be false positive, this situation could result from the failure of the conventional cultural procedure to detect the organism. Most false-positive results occurred with chicken and minced beef samples. Better agreement between ELISA and the conventional cultural method results were obtained when these samples underwent direct selective enrichment, probably due to the suppression of the competing flora. The Salmonella 1- 2 Test could detect low levels of salmonellas in the presence of competitors from skimmed milk powder. It was found in evaluating the Kirkegaard and Perry ELISA kit that Salm. typhimurium NCTC 74 did not grow well in tetrathionate broth. Although this is me only selective broth

IM'V1UNOLOGICAL DETECTION

or

SALMONELLAS

263

used in the 1- 2 Test it did not seem to affect the detection of Sa1m. typhimurium. This may be due to the differences in the 1-2 Test tetrathionate broth formulation or the relatively short time the salmonellas are in the broth prior to entering the non-selective motility medium. The TECRA and Bio-Enzabead ELISA kits would probably be best suited to the routine screening of large numbers of samples, although the centrifugation step for high-moisture foods in the latter kit may pose a problem in some laboratories. The Kirkegaard and Perry ELISA requires more manipulations and development of the enrichment procedure by the user. This, however, does provide the kit with more flexibility and could allow more rapid detection. The Salmonella 1- 2 Test would perhaps be more suitable for testing small numbers of samples and in laboratories that are not equipped to perform ELISAs.

References IBKi\HIM, G.F. & FLEET, G.I I.

1985. Detection of salmonellae using accelerated methods. 1lltematillllal]oumal oI Food ;l1iaobiolob.'Y 2, 259~ 272. WOOD, ].M. & GUlBS, P.A. 1982. New Developments in the rapid estimation of microbial populations in foods. In Dt"-JdoplIIC1ltS ill Food /vliembiolo&,v - 1, ed. Davies, R. London & New Jersey: Applied Science Publishers.

Immunoassay Kits for the Detection of Toxins Associated with Foodborne Illness SALLY

A. ROSE, N. P. PATEL, A. O. SCOTT AND

M. F. STRINGER Campden Food Preseroatioll Research Association, Chipping Campden, Gloucestershire GL55 6LD, UK

There are a number of toxins and toxic substances of bacterial (Cliver & Cochrane 1986), fungal (Salunkhe & Wu 1977), small organism (Turnbull & Gilbert 1982), and plant origin (Salunkhe & Wu 1977; Page 1984), which have been associated with human foodborne illness. Intoxications of bacterial origin can be due to either the consumption of pre-formed toxins in food (e.g. staphylococcal enterotoxins and Clostridium botulinum neurotoxins) or the consumption of food containing large numbers of organisms which subsequently produce toxins within the gastro-intestinal tract (e.g. Clostridium perfringens and Salmonella). Mycotoxins (e.g. aflatoxins and ochratoxin A) are fungal metabolites generally pre-formed in food, although metabolism of these toxins by the host may occur to produce secondary metabolites (e.g. aflatoxin M} in milk). Toxic substances may also occur as normal constituents of some plants (e.g. glycoalkaloids in potatoes and lectins in kidney beans) and in addition, high levels of toxic elements (e.g. arsenic and lead) may be found in some foods. Occurrence of these types of food contaminants represents a continual hazard and necessitates regular monitoring of the quality of a wide variety of food products. The increasing awareness of toxins as agents of human foodbome illness has prompted their study and has led to the requirement for inexpensive, rapid techniques to detect and quantify toxic contaminants in food products. Until recently, methods of toxin isolation and quantification have either been unavailable or required lengthy and sophisticated procedures, in addition to specialized facilities and expertise. Examples of these procedures are the in vivo kitten emetic test (Dolman & Wilson 1940), the slide immunodiffusion methods for measuring staphylococcal enterotoxins (Casman et al. 1969), Cop)'riKhl Rapid Microbiological Methods for Foods, Beverages and PharmJccutiea!s

© /989 by AI/ riKhls

Ihe Solid)' .liJY Applied Baaeri"lo!!J'

"f "prodl/(I;"1/ ;11 all)' form reserved 0-632-02629-"

265

266

S. A. ROSE ET AL.

and chromatographic methods such as thin-layer and high-pressure liquid chromatography for mycotoxin measurement (lIunt et al. 1978; Patterson & Roberts 1979). Recent innovations in clinical immunodiagnostics, however, have produced simple, rapid techniques, suitable for routine assay, which can now be employed in the monitoring of food production. These immunological techniques rely on the highly specific interaction between an antibody and its antigen (the analyte) in the food sample. Various methods are used to reveal the antibody- antigen complex, such as immunoprecipitation (Casman et al. 1969), activity of conjugated enzymes (Maggio 1980) and agglutination (Silverman et at. 1968). Assays for the identification and quantification of some toxins (indicated in Table 1) during food quality control are now conunercially available in kit form and will be discussed in this chapter. The object of this study was to look at the practical aspects of the use of these kits rather than to verify the assays (this has been carried out elsewhere: Anon. 1987; Barrett 1987; Berry et al. 1987; Wieneke & Gilbert 1987). In view of this, little detail and discussion of the methods (which are available with the kits) is included in this chapter.

Materials and Methods Eleven commercially available kits, for the detection of toxins that causefoodbome illness, were investigated. Each kit was used in strict accordance with the manufacturer's instructions unless otherwise stated. In all microtitre plate Enzyme-Linked Immunosorbent Assay (ELISA) kits, sample replicates were randomly distributed throughout the whole plate and plate washing was carried out by pipetting the stated volume of wash solution into the wells. The wash solution was removed by aspiration to reduce the possibility of crosscontamination of reagents between wells and to optimize containment of toxic solutions for safety reasons. Aflatoxin assays

Purified lyophilized toxins, aflatoxins Bl, BZ, Gl and GZ, ochratoxin A, patulin and T2 toxin (Sigma), were reconstituted in dimethyl sulphoxide to give 1 mg/m! working stocks solutions which were stored at -18°C. These were then diluted in phosphate butTered saline (PBS) to appropriate levels and each assayed by all the aflatoxin kits to determine specificity and sensitivity. An initial sample of 1 kg of food was taken as required by the AOAC official method for aflatoxin analysis (Stoloff & Scott 1984). This sample was then either milled (Retsch disc mill, I-mm grating), grated to form a fine powder or blended (Kenwood Gourmet or Moulinex Magimix using shredder and blade attachments). Prepared samples of peanuts, peanut butter, and

TOXIN KITS

267

maize were spiked with the minimum volume (20-100 ~l) of diluted aflatoxin Bl prior to extraction, at various levels appropriate to the test kit. In all cases, kits were used in the quoted sensitivity range except for the Microtest kit where the sensitivity was matched to the level of toxin being tested. The Aflatest kit was used in the semi-quantitative mode only (using the florisil tips), as this was considered to be the mode which would be most frequentIy used. Staphylococcal enterotoxin assays Purified staphylococcal enterotoxins (SETs), types A and 0, were kindly provided by Prof. M. Bergdoll (Food Research Institute, University of Wisconsin/Madison) and stored at - IS o C or obtained from the Oxoid SETRPLA (reverse passive latex agglutination) kit (SETA, SETB, SETC, SETD). l'oxins were diluted in PBS to appropriate levels and were assayed by both kits to determine specificity and sensitivity. Samples (100 g) of canned salmon, cooked minced beef and cheeses (Cheddar and Stilton) were each inoculated with 2.0 ml SETA solution prior to extraction, to give concentrations of 0.1, 0.5 and 1.0 ng g ~ I of food. Samples were extracted by stomacher with 100 ml of a 0.2 moll-I NaCl solution containing 5.0% (v Iv) normal rabbit serum and 0.5% (v/v) Tween-20, for 5 min, and the resulting slurry was centrifuged at 10000 g for 20 min at 5°C. The supernatant liquid was retained for assay. Clostridium perfringens enterotoxin assll.Y Extracts of faeces from patients suffering from food poisoning were prepared by blending 2 g of specimen with 2 ml of PBS, centrifuging at 15000 g for 30 min at 4°C followed by membrane filtration (0.22 ~m) of the supernatant fluid. EX1racts were assayed according to the kit instructions. Results and Discussion Comparisons of the extraction procedures, technical information, and practical aspects of the different toxin kits used in this study are shown in Tables 1- 3, respectively. This information is presented as a guide for sectors of the food industry interested in pursuing these types of assay for routine quality control procedures. Aflatoxin assll.Y All aflatoxins are potentially hazardous to health and have to be handled with great care. Decontamination of all the apparatus after use is necessary. All the kits used in this study emphasize this and recommend the use of sodium

TABLE

1. Comparison of extraaion procedures jOr the toxin assay kits

--

Type of kit Total Aflatoxin Assay-Biokits

Aflasure

Quantitox

Transia

Chemafla

Ez-Screen

Antigen

Aflatoxin

Aflatoxin

Aflatoxin

Aflatoxin

Aflatoxin

Aflatoxin

Equipment required

General*'

General;*' centrifuge; separating funnel; rotary evaporator

General'*'

General'*'

General;*' nitrogen stream apparatus

General'*'

50 g

109 (foods)

20 g (foods)

5g (foods)

50 g or 3 g (foods)

55% (v/v) ,Methanol: H 2 O

80% (v/v) Methanol: H 2 O

90°,{} (v/v) l\fethanol: H 2 O

80% (v/v) l\1ethanol: IIzO

25 m1

100 m1 or 6 ml

Weight of sample required

Choice (foods)

Extraction chemicals

(foods)

50% (v/v) Acetonitrile: H 2O

55% (v/v) Methanol: 1120, Hexane, NaCl, Chloroform

Total extraction volume

5 ml/g

350 ml

50 m1

60 m1

Blending time

2 min

I min

3 min

3 min

-

Shaking time Total extraction time per sample Quoted extraction efficiency

I min 3 min

I min

6 min

45 min

7 min

10 min

30 min

6 min

NQ

90%

NQ

NQ

NQ

NQ

U

Type of kit Atlatest

Microtest

SET-RPLA

SET-EIA

PET-RPLA

VET-RPLA

Antigen

Atlatoxin

Atlatoxin

Staphylococcal enterotoxins

Staphylococcal enterotoxins

C. perfnngens enterotoxin

V. cholerae + E. coli (LT) enterotoxins

Equipment required

General*

General*

Blender; centrifuge; membrane filter

Blender; centrifuge

Blender; centrifuge

Centrifuge

50 g (foods)

109 (foods)

10 gt (foods)

choice H (toods)

choice t (faeces)

0.25 ml (culture supernatant)

60'}'o (v/v) :YIethanol: H 2O

60% (v/v) Methanol: H 2 O

0.85% NaCI

DW/PBS

0.85% NaCl

NA

Total extraction volume

250 ml

20 ml

100 m]

I ml/g

10:1 v/v

NA

Blending time

1 min

I min

c. I min

c. ] min

c. I min

NA

NA

NA

NA

NA

Weight of sample required Extraction chemicals

Shaking time Total extraction time per sample

5 min

5 min

30 min

30-90 min~ + 30 min

25 min

25 min

Quoted e"traction"* efficiency

NQ

NQ

NQ

NQ

NA

NA

* General = automatic blender, filter paper, associated glassware. t Can also be used for examination of culture supernatants. §

Extraction procedure variable dependent on product (additional steps of acid precipitation, centrifugation and fat removaliCHCl3 extraction may be indicated).

~ Incubation of extract with normal rabbit serum to absorb protein A which may cause non-specific reactions.

"* NO, not quoted; NA, not applicable.

TABLE 2. Technical information fOr the toxin assay kits Type of kit Total aflatoxin assay

Aflasure B

Quantitox B

Aflatoxin test

Chemafla

Ez-Screen

Supplier

Biokits Ltd.

Cambridge Life Sciences

May & Baker Diagnostics Ltd

Transia

Chemunex

Analytes

Aflatoxins (BI,B2,G I,G2)

Aflatoxins (Bl,B2)

Aflatoxin (Bl)

Aflatoxins (BI,B2,Gl,G2)

Aflatoxin (Bl)

Aflatoxins (Bl,B2,Gl,M 1)

Foods for assay

Edible nuts and nut products

Groundnuts, groundnutmeal, peanut butter, other foods and raw materials (with appropriate extraction methods)

Peanuts, peanut products, maize, palm kernel

Cereals, oilseeds, and foods and feedstuffs derived from these

Grains, cereals, peanuts, etc.

Com, feeds, raw and roasted peanuts, peanut butter, grains, cereal

Assay principle

Competitive double-antibody plate ELISA

Competitive double-antibody plate ELISA

Competitive plate ELISA

Competitive plate ELISA

Competitive double -antibody plate ELISA

Card ELISA colour change

Environmental Diagnostics Inc.

*.

End point Reading

MI4

A450

M50

A405

M50/650

(,isual)

+

+

+

+

+

±

Assay sensitivity

2 ng/ml

0.28 ng/ml

2 ng/ml

I ng/ml

0.25 ng/ml

10 ng/ml

Working range (ng/ml)

2-200§

0.30-50

2-30§

1-6§

0.25-2.5§

10-100

£160

£142 (1422 FF)

£I 60 (1600 FF)

£10

62~

36~

40~

2

£3.44

£3.94

£4.00

£5.00

Quantitative"

Cost of kit§ § No. assays in kit Cost per assay Provided standards Standard curve Positive control Negative control

£190 37~

Not currendy available

£5.14

+ (toxins) +

+ (toxins)

+ (graph) -

+ (toxins)

+ (toxins)

-

As in sc

As in sc

Sample

-

Sample

+

+

TABLE 2. Contd. Type of kit

Supplier

Aflatest P/IO

Microtest Aflatoxin assay

SET-RPLA

May & Baker

Microtest

Oxoid Ltd

SET-EIA (i) Labor:

PET-RPLA

VET-RPLAtt

Oxoid Ltd

Oxoid Ltd

Dr W. Bonuneli (ii) Biosure: UK distributor Analytes

Aflatoxins P(B I,B2,l\11,G I, G2) IO(BI,B2,MI)

Aflatoxins (Bl,B2,GI,G2)

Staphylococcal emerotoxins (A,B,C,D)

Staphylococcal enterotoxins (A,B,D,C)

Clostridium peifringens enterotoxin

Vibrio eholerae + E. coli (LT) enterotoxins

Foods for assay

Groundnut, nut, nut products maize, soya, cotton seed, palm kernel

J\'1any matrices including: sunflower seed, palm kernel, maize, cotton seed, milk, wheat

Various foods, culture supernatants

Various foods, culture supernatants

Faeces

Culture supernatants

Assay principle

Immunoaffinity column/LTV

Immunoaffinity column/VV

Reverse passive latex agglutination

Sandwich bead ELISA

Reverse passive latex agglutination

Reverse passive latex agglutination

Fluorescence visual! fluroimeter)

Fluorescence (visual)

Agglutination (visual)

A405/visual

Agglutination (visual)

Agglutination (-visual)

±

+/-

±

±

Endpoint Reading Quantitative*

±/+

±

Assay sensithity Working range (ng/ml) Cost of lat§§ No. assays in Idt Cost per assay Provided standards Standard curve Positive control Negative control

2.0 ng/ml

I ng/ml

NA

NA

2.0 ng/ml t ~

Sensiti\ity

0.1 ng/mJ ~

Sensitivity 0.1-2.0

2.0 ng/ml ~

Sensitivity

1.0 ng/ml ~

Sensitivity

£100

£98.21

(i) 150 SwFR/£61.54 (ii) £100

£55.09

£55.69

25

20

10

20

20

£2.78

£2.78

£6.40

£4.60

£4.91

(i) £0.15 (ii) £10.00

+(non toxic comparitor)

-

Control latex

Control bead

+ Control latex

"+ Quantitative assays enable accurate determination of the concentration of toxin samples by interpolation from a standard curve. ± Semi-quantitative assays enable the concentration of toxin in samples to be estimated: (i) by comparison with provided positive standards (Aflatest) or negative controls (SET-Eli\); (ii) presence/absence at the level of sensitivity of the assay (Microtest + RPLAs). t Experimental evidence suggest an actual sensjtivity for the SET-RPLA or 0.25 ng/in!. § The range of assays may be extended by assaying diluted extracts. ~ Number of duplicated assays possible per lat. "" Available from Biosure (UK). tt This lat has not been evaluated in our laboratory. §§ Costs of lats as September 1987.

+ Control latex

TABLE

3. Comparison

0/ practical aspeas 0/ the toxin

assay kits

Type of kit T otaI aflatoxin (Biokits)

Instructions (a) Clarity/ease of use (b) Information on reagents (c) hazards warning & decontamination

2 Approx. time (11) to

Aflatoxin test (Transia)

Chemafla

Ez-Screen

Concise but brief Poor

Clear

Poor

Comprehensive but confusing Good

+

+

+

+

1.25

2

1.50

3

0.25

None Easy

Minimal Easy

Minimal Easy

Complex Difficult

None

None

Good (strip plate)

Good (strip plate)

None

None

None

Good

Plate reader, plate shaker

Plate reader

Plate reader

Plate reader

None

Aflasure

Quantitox B

Clear but brief

V. good

Clear and comprehensive Poor

+

+

4

Good but verbose

Poor

perfOrm assay (exd. extraction) 3

Interpretation 0/ results (a) Calculation (b) Ease of interpretation

4 Flexibility (Le. ability to choose no. samples to assay)

Ea~)'

5 Additional

equipment Suggested, considered necessary

0 not

Plate reader, plate shaker (plate washer)

Type of kit Aflatest

Instructions (a) Clarity I ease of use (b) Information on reagents (c) hazards waming & decontamination 2

Approx. time (h) to perfimn assay (excl. extraction)

Microtest aflatoxin assay

SET-RPLA

SET-EIA

PET-RPLA

VET-RPLA*

Clear

Clear

Clear

Clear

NA

Good

Clear and comprehensive Good

Good

Good

0.25

0.75

16

24

18

18

None Easy but subjective

Easy but subjective

Minimal Easy but subjective

Minimal Easy

Minimal Easy but subjective

Minimal Easy but subjective

Good

Good

Good

Good

Good

Good

UV light box, clamp stand

UV light box, syringes

Microtitre plate (moisture box) (plate shaker)

Plate reader, plate shaker (microtitre plate), tubes, aspirator

Clear but brief NA

+

3 Interpretation oI results (a) Calculation (b) Ease of interpretation 4

Flexibili~y

(Le. ability to choose no. samples to assay)

5 AdditiOtlal equipment Suggested, () not considered necessary

* This kit has not been evaluated in our laboratory. NA, not applicable.

As

As

SET-RPLA

SET-RPLA

276

S. A. ROSE ET AL.

hypochlorite solution (Chloros) for decontamination. None of the assay kit instructions cautions the operator of the corrosive nature of this chemical which prevents its use with apparatus containing metal parts, e.g. a blender. In this situation we suggest that it may be more appropriate to use acetone which is, however, not as powerful as sodium hypochlorite for decontamination purposes. Another general point concerns the type of water used to prepare solutions. Some kits prescribe the use of de-ionized water, others distilled water. A high-grade water supply may not be readily available in all laboratories and therefore we feel that advice on the suitability or otherwise of alternative water types, including tap water if possible, would be helpful. Several methods of grinding food samples (I kg) to give a fine powder before sub-sampling were investigated. A disc mill was found suitable for maize kernels. A food processor with the shredder blade produced a good powder of peanuts, whilst the ordinary blade also produced a slurry of peanut butter suitable for extraction purposes. The extraction procedure of the Quantitox kit was easily performed. A standard curve is provided and the results are calculated by comparison to a non-toxic reference standard which gives maximum colour development. Whilst the provision of a standard curve in graphical form obviates the handling of toxic standards, the assay requires absolute compliance with the stipulated protocol to achieve the optimum results and is heavily dependent on the reference standard being non-contaminated. In this study the kit performed as the instructions indicated. Cross-reactions were observed with all the aflatoxins tested and the kit therefore does not appear to be specific for BIas claimed. Cross-reactivity with the other mycotoxins tested was not observed. Good agreement was found between levels of toxin recovered from spiked samples and the amount of toxin added. The instructions were clear and easy to follow. During extraction for the Transia kit the necessity to discard the first 1 ml of filtrate was difficult to accommodate when assaying a large number of samples. Toxin standards are provided in the kit and arc assayed to produce the standard curve, thereby allowing for certain flexibility in the assay protocol without compromising the accuracy of interpolated results. The need to dilute these standards, however, although allowing for accurate quantitation, introduces additional hazards and may be an additional source of operator error. In this study the results produced a flat standard curve thereby reducing the working range and increasing inaccuracies during interpolation. We have no evidence to suggest that this is an inherent fault of the kit; but we suggest that the short antibody incubation time may contribute to such a problem. Results confirmed that the assay was capable of detecting aflatoxins B1, B2, Gland G2, but a low level of cross-reactivity with the other mycotoxins was also

TOXIN KITS

277

observed. Good replication between randomly distributed replicates was observed. The kit has the in-built flexibility of separate strip wells enabling the numbers of samples to be varied, although care is needed to prevent the strips falling out of the holder. The instructions for calculating the results were not easy to follow. The extraction procedure for the Biokits assay was easy to perform. In this study the blender option was used for comparison with other kits although an alternative, simpler procedure is given in the instructions which does not require the use of a blender. Useful additional information on the storage of extracts (overnight refrigeration) is given in these instructions. The standard curve in this kit was constructed using pre-diluted standards thus minimizing the handling of toxins. Generally the assay performed as the instructions indicated. In our experience the assay was capable of detecting aflatoxins B1 and Gland showed a very low level of cross-reactivity with G2 but none with B2. No cross-reactivity with other mycotoxins was found. The instructions are very detailed and whilst this may be a little daunting at first, all the information contained was found to be usefuL The extraction procedure for the Aflasure kit requires both a large food sample (50 g) and solvent volume (350 ml) which may cause handling problems and be expensive for large numbers of samples. Also the several steps involved in the extraction procedure require additional handling and equipment. This makes the overall procedure tedious and time-consuming and also results in the need to decontaminate large amounts of glassware. Pre-diluted aflatoxin standards are provided and are separately contained within a metal container, a safety aspect followed by this kit alone. The assay (in strip well format) was easy to carry out and produced a good standard curve and replication. We found the assay sensitive to 0.28 ng/ml aflatoxin B} which was the highest sensitivity of the kits studied (although a sensitivity of 0.25 ng/ml is claimed for the Chemafla assay). Cross-reactivity was observed with aflatoxin Bz but not with G} or G z as stated in the instructions, and no cross-reactivity was found with the other mycotoxins. The instructions for the assay are well presented and are easy to follow. The extraction procedure given in the Chemafla kit was simple to perform, and did not require the use of a blender. The subsequent evaporation of samples under a nitrogen stream was very time-consuming, however, and this facility may not be readily available in all laboratories. The ELISA was easy to perform although the preparation and dilution of toxin for the standard curve was tedious and therefore a possible source of operator error and additional hazard. \Ve can make no comment on the assay performance as meaningful results were not obtained in our study of this kit. The extraction method for the Microtest kit was easy to perform. The sensitivity of the assay is dependent on dilution factors within the extraction

278

S. A. ROSE ET AL.

step and therefore has to be pre-determined. \Vhilst this presents no problems where samples are to be screened on a pass/fail basis, estimation of the toxin in unknown samples requires multiple analysis at different sensitivities. A considerable variation in times for elution of samples from columns was observed. All the aflatoxins tested were detected as claimed by the instructions and no cross-reactivity with other mycotoxins was found. The exclusion of extraneous light when observing fluorescence ofcolumns is absolutely necessary to achieve accurate and reproducible interpretation of results. The assay was generally easy and safe to handle and performed as the instructions suggested. The AfIatest instructions were clear and the procedure simple. This assay has the advantages of requiring little additional equipment and the inclusion of a card with spots of different fluorescent intensity with which results are compared, thereby avoiding the necessity for handling toxic standards. Crossreactivity with all the aflatoxins was observed and a low level of interaction with ochratoxin A was suggested. No cross-reaction with either T2 toxin or patulin was observed. Discrimination between 0 and 10 mg/l (or mg/kg) aflatoxin B1 in buffers and in foods proved difficult, resulting in some false positives and a few false negatives. Results at levels of inoculation above 10 mg/ml were in general agreement with the comparator card although the brightness of the comparator fluorescence tended to depress the results slightly. It should be noted, however, that considerable variation in the interpretation of results was observed between individuals. The E z screen extraction procedure was very simple and may be readily carried out with the minimum of facilities. The assay procedure was very simple involving only the spotting of extracts and reagents on to a card. The end point is determined by. a colour change and, in our experience, the colour of some extracts resulted in confusion of end-point interpretation. In conclusion, we suggest that for a routine screening of samples, the Aflatest and l\1icrotest are both good semi-quantitative assays. These column assays are relatively quick, can be used in laboratories where minimal facilities are available, and may be performed by technicians with a minimum of training. By contrast the titre plate enzyme immunoassays are fully quantitative but require a greater degree of expertise than the column assay for optimum use. Although technicians, after training, may perform titre plate assays it would be advisable for a senior operator, cognizant of the problems of immunoassay, to view and interpret the results. Finally it would be useful for the kit manufacturers to include specific details of the principal components of each kit to enable greater flexibility of use and to aid identification of problems should they arise. It is important to note that our experiences suggest that immunoassay kits for analysis of aflatoxins in foods should be used only for products specified in the manufacturers' instructions (see Table 2). Where the need for analysis of

279

TOXIN KITS

other products arises, kits should be used only after they have been successfully validated for these products by comparison with official AOAC methods (Stoloff & Scott 1984).

Staphylococcal enterotoxin

ana~ysis

The SET-EIA is capable of detecting 0.1 ng SETlg of food and no crossreactivity between the different types of SET (SETA, B, C and D) was observed. The instructions are clear and give useful additional information about reagents and data interpretation. The choice of extraction method is at the user's discretion and some of the protocols given are time-consuming and exacting. The protocol given in the methods here, however, is relatively simple and has been found adequate for many types of foods. The assay procedure involves lengthy incubation periods (overnight and 6 h) and some difficulty may be encountered in completing the latter stages of the assay within a normal working day, particularly if large numbers of samples are to be investigated. No positive standards are included in the kit, but if available can be used to construct a standard curve enabling the assay to be used quantitatively. The e;\1:raction and assay procedures for the SET-RPLA are relatively simple and quick to perform and require no additional equipment. In our CA'Perience the assay has been found to be sensitive to 0.25 ng SET Iml in buffer rather than the 2.0 nglml claimed, although the dilution factor of the CA1:ract (I: 10) reduces the detection limit in foods to c. 2.5 nglg. This is sufficiendy sensitive for detection of levels of toxin usually associated with illness. Cheeses have been found to give non-specific reactions which may be interpreted as false positives by inCA'Perienced users, making the kit unsuitable for assay of these products. This problem may be overcome, however, by addition of 10 mmol/l sodium hexametaphosphate to dle diluent provided in the kit (Rose et al. 1989). Determination of the end-point was found to be subjective. Concentration of toxin present in samples can be estimated by multiplying dle sensitivity of the assay by the dilution factor of the sample in the last positive well. Positive standards, which aid interpretation of results, are included in dle kit. Neidler kit contains a test for the detection of staphylococcal enterotoxin E. Clostridium perfringens enterotoxin assay The extraction and assay procedures of the PET-RPLA are simple, quick and easy to perform requiring minimal specialized equipment. The assay was sensitive to 4 ng toxin I g faeces. Non-specific reactions are not uncommon in the first 2- 3 wells of the dilution series and it is advised that a sample is only designated positive if agglutination in the sensitized row exceeds that in the

280

S. A. ROSE ET AL.

control row by two or more wells. The presence of these reactions reduces the sensitivity of the assay, but is only likely to result in false negatives in a small percentage (c. 5) of samples. Vibrio cholerae enterotoxin analysis This RPLA assay has not been studied by ourselves, but adopts the same principles as the other RPLAs. The assay is known to cross-react with the heat-labile enterotoxin of Escherichia coli (but not the heat-stable toxin) and can therefore be used for assay of both toxins. Interpretation of end points and estimation of toxin concentrations are as for the other RPLA assays.

Concluding Remarks \Vhilst these kits represent significant advances in the monitoring of toxins associated with foodborne illness, there is still a need for further development in this area. Kits are not as yet available for several toxins associated with particularly severe or common symptoms. In addition, most of the kits still require specialized facilities and eApertise before they can be used and can often take more than a day to obtain results. Further developments, to produce more rapid tests which are easier to perform, are required. Such advances have been made in the field of clinical diagnostics, for example dipsticks, which can offer rapid screening of numerous samples. This type of test would be of particular benefit for routine quality control monitoring in the food industry. It is important to note that this study was carried out before 1 September 1987 and that some amendnlents to the instructions and format of the assay kits may have been made subsequently.

Acknowledgements We wish to thank the kit manufacturers for providing kits free of charge and for their co-operation throughout this study.

References Aflatoxin: The New Analytical /Welhods. Agtest Ltd, 150 Brompton Road, London. G.M. 1987. Two immunoassay kits (or detennining aflatoxin Bl in wheat and other

ANON. 1937. BARRE1T,

commodities: an evaluation. FMBRA Bulletin No.6, 252-257. Chorlcywood, Hertfordshire: F~1BRA.

P.R., \VIENEKE, A.A., RODHOUSE, J.c. & GILBERT, R.J. 1987. Use of commercial kits for the detection of Clostn'dium perfringens and Stap/~ylococcus aureus enterotoxins. In Immunological Ted111iljUeS in Microbiology, ed. Grange, ].M., Fox, A. & Morgan, N.L SAB Technical Series

BERRY,

TOXIN KITS

281

No. 24. pp. 245-254. Oxford: Blackwell Scientific Publications. CASMAN, E.P., BENNET, RW., DORSEY, A.C. & STONE,].C. 1969. The microslide gel double diffusion test for the detection of staphylococcal enterotoxin. Health Laboratory Science 6,

1&5-19&. CUVER, D.O. & COCHRANE, B.A. (eds) 1986. Prugress in Foods Safety. Food Research Institute. Madison, USA: University of Wisconsin/Madison. DOI.MAN, C. E. & WILSON, R.]. 1940. The kitten test for staphylococcus enterotoxin. Canadian

Journal of Public Health 31, 68-71. HUNT, D.C., BOURDON, AT., WILD, 1'.]. & CROSBY, NT. 1978. Use of high perfonnance liquid chromatography combined ",ith fluorescence detection for the identification and estimation of aflatoxins and ochratoxin in food. Journal uf the Scimce of Food and Agriculture 29,

234-238. l'vL~GGIO,

E.T. 1980. Enzymes as immunochemical labels. In Enzyme-Immunoassay ed. Maggio, E.T., pp. 53-70. Florida: CRC Press Inc. PAGE, S.W. 1984. Report on plant toxins. Journal of the Associatiun uf Official Analytical Chemists

67, 371. PATTERSON, 0.5.1'. & ROBERTS, B.A. 1979. Mycotoxins in animal feedstuffs: sensitive thin layer chromatographic detection of aflatoxin, ochratoxin A, sterigmatocystin, zearalenone, and 1'- 2 toxin. Journal uf the Association of OjJidal Analytical Chemists 63, 1265 -1267. ROSE, 5.A., BANKS, P. & STRINGER, M.F. 1989. Detection of staphylococcal enterotoxins in dairy products by the reversed passive latex agglutination (SET -RPLA) kit. International Journal of

Food iHicrobiology 8, 65-72. SALLNKHE, O.K. & Wu, M.T. 1977. Toxicants in plants and plant products. Critical Reviews in

Food Scienu and Nutrition 9, 265--324. SILVERMAN, SJ., KNOTT, A.R. & HOWARD, M. 1968. Rapid, sensitive assay for staphylococcal enterotoxin and a comparison of serological methods. Applied Microbiology 16, 1019-1023. STOLOFF, L. & SCOTT, P.M. 1984. Natural poisons. In Official Method ofAnalysis ofthe Association of Official Analytical Chemists, ed. Williams, S. pp. 477 -494. USA: Association of Official Analytical Chemists, Inc. TURNBULL, P.c.B. & GII.BERT, R.]. 1982. Fish and shellfish poisoning in Britain. In Adverse Ajfi:cts ofFouds, ed. Jelliffe, E.F.P. & Jelliffe, 0.13. pp. 297-306. New York: Plenum Press. WIENEKE, A.A. & GILBERT, R.]. 1987. Comparison of four methods for the detection of staphylococcal enterotoxin in foods from outbreaks of food poisoning. International Journal of

Fuod Microbiology 4, 135-143.

Rapid Detection of Viruses in Water and the Water Environment H. MERRETT AND C. E. STACKHOUSE Virology Unit, Welsh Water, PLC Engineering and Environment Ltd, Bidgend Office and Laboratory, Tremains House, Tremains Court, Bidgend, Mid Glamorgan, CF31 2AR Wales, UK

Rotaviruses, along with other similar small viruses, were discovered and characterized in the 1970s. They are 70 nm in diameter with a doublestranded RNA genome, and are classified with reoviruses and orbiviruses within the family Reoviridae (Matthews 1979). Rotaviral infections occur throughout the world, and in temperate climates are thought to be responsible for around 50% of all clinical cases of acute gastroenteritis in children under the age of two (Blacklow & Cukor 1981). Although the infection is seen predominantly in children, rotavirus can also cause severe diarrhoeal disease in adults, and in infected individuals viruses are excreted at the rate of 10 10 -lOll particles/g stool (Flewett 1982). There is unequivocal evidence that pathogenic human viruses may be transmitted via the water cycle and retrospective epidemiological studies indicate that contamination of water supplies by sewage was responsible for at least five outbreaks of rotaviral gastroenteritis (Gerba etal. 1985). There is therefore a real risk of infection from sewage-contaminated water supplies and a possible risk of infection from recreational bathing waters where sewage is discharged directly into the sea. A joint research project involving Welsh Water and the Water Research Centre was initiated in 1985 to study the distribution and survival of rotaviruses in the marine environment. Although rotaviruses are present initially in very high concentrations in sewage contaminated with stools from infected individuals, the sewage treatment process itself, and the subsequent dilution of sewage effluent in waters into which it is discharged, ensure that the final concentration of rotavirus in the aquatic environment is considerably less than the initial concentration in faeces. The detection of rotavirus in environmental samples thus involves Copyright Rapid 'Vlicrobiological Methods for Foods, [leverages and Pharmaceuticals

© 1989 by the Society jiJr App!it'd Baturio!o!,.')' All rights of reproduction in any fOrm rmrved 0-632-02629

283

284

H. MERRETT AND C. E. STACKHOUSE

concentration of a large volume (10-20 1) of water into a sOlall workable volume « 10 ml) which can then be assayed for the presence of virus by an appropriate assay for viral particles or antigens. Unlike some of the pathogenic enteroviruses, e.g. Coxsackie virus A and B, echovirus and poliovirus, mammalian rotavirus cannot be cultivated directly in vitro by current organ or cell culture techniques. Where growth has occurred it has been inconsistent and inadequate for viral diagnostic and characterization studies (Wyatt & James 1980). However, if the virus is centrifuged at low speed on to a pre-formed monolayer of cells, the cells become more susceptible to infection (Smith & Gerba 1982) and in the presence of trypsin and absence of serum, the virus undergoes an incomplete replicative cycle producing viral antigens in the cell. Although the infection is abortive and yields little or no infectious virus (Bryden et al. 1977; Thouless et al. 1977), the viral antigens that are produced can be detected using immunofluorescent-labelled antibodies. The immunofluorescence technique is based on the antibody-antigen reaction in which the antibody-antigen complex is made visible by incorporating a fluorochrome in the antibody molecule. Fluorescence is then detected by dark-ground illumination using ultraviolet light or visible blue light. This technique is virus-specific and very sensitive, detecting the presence of virus within 24 h. In addition, the method differentiates between infectious and non-infectious particles (McNulty 1978) which is of considerable relevance to this study, since the presence of non-infectious rotavirus particles in the environment is of little public health significance.

Materials and Methods

Concentration of sample In aqueous solution viruses behave as amphoteric, hydrophilic colloids and the net charge is a function of pI-I, ionic composition and ionic strength of the solution (l\tlorris & Waite 1981). These properties are exploited in the concentration of viruses from large volumes of water. At low pH in the presence of cations, viruses adsorb, by virtue of their surface charge, to a variety of media, including cellulose nitrate and glass fibre. Elution from this initial adsorptive phase is achieved by using a solution of an organic material, e.g. skimmed milk or beef ex1ract in glycine buffer or water, at high pI-I, resulting in a primary eluate of more manageable volume (400-500 rot). Further concentration ofviruses is achieved by a secondary concentration step. This procedure, known as organic flocculation (Katzenelson et al. 1976), utilizes the property of organic materials to precipitate or flocculate when the pH of the solution is lowered to near the isoelectric point of the material: viruses are effectively adsorbed onto this precipitate. The precipitate and associated viruses are sub-

DETECTION OF VIRUSES IN WATER

285

Water sample (10 I) pH 3.5 Addition of 1 molll AICI 3 to a final concentration of 0.0005 molll Adsorption

filtered under pressure Cellulose nitrate membrane (0.45 lim pore)

.

I

Viruses adsorbed to membrane (40 - 70% efficiency) --_.Filtrate discarded Elution

I

Viruses eluted with 400 ml 0.2% skimmed milk in 0.05 molll Glycine pH 9.5

I

Addition of 1 M glycine (pH 2.0) to final pH 4.5 and floc formation 30 min at 4°C

I

Flocculation

Centrifugation (2800 g 20 min)

I Pellet • Supernatant discarded Resuspended in 10 ml 0.15 molll Na2HP04

, Add 10 ml dithizone in chloroform (0.01 gil) , Rotamix

I

Removal of toxic products

Centrifugation 11500 9 15 min) - - - _ . Chloroform layer , discarded Aqueous layer exposed to current sterile air for 30 min to remove excess chloroform

I

Store at Assay FIG.

70 a e (if required)

•

Assay for viruses

1. Isolation of viruses fwm large volumes of water.

sequently collected by low-speed centrifugation. Viruses are then recovered for assay by dissolving the precipitate in a suitably small volume of moderately alkaline buffer. The entire procedure is summarized in Fig. I and described in detail below.

Adsorption Samples (10 I) of water were collected aseptically in sterile pots at the mean low water mark from six sample points along the designated beach and

286

H. MERRETT AND C. E. STACKHOUSE

transported to the Virology laboratory within 48 h ofsampling. All manipulations were carried out aseptically and all solutions were sterile throughout sample processing. The sample was acidified to pH 3.5 with concentrated Hel, then 1.0 mol/l aluminium chloride to a final concentration of 0.0005 molll was added to enhance virus adsorption (Goyal & Gerba 1982). The sample was then filtered through a 293-mm-diameter cellulose nitrate membrane, pore size 0.45 !lm (Sartorius, UK Ltd) at a pressure of 0.5 kg cm 2 compressed air or nitrogen. Because of the presence of sand and fine silt, a 257-mmdiameter fibre glass pre-filter (Sartorius, UK Ltd) was routinely used in series to prevent the pores of the membrane filter from becoming clogged.

Elution Adsorbed virus was then eluted from both the pre-filter and the membrane by passing 400 ml 0.2% (w/v) skimmed milk in 0.05 molll glycine (adjusted to pH 9.5 by addition of 1 moIl1 NaOH) through the filter at a pressure of 0.5 kg cm 2 (Fig. 1): 400 ml skimmed milk was sufficient to elute viruses retained from a 10-1 sample of water.

Flocculation Glycine (1 mol/I; pII 2.0) was added dropwise to the filter eluate until a fine white precipitate began to fonn. At pH = 4.5, the isoelectric point of casein, which generally coincided with the formation of a dense white precipitate, the eluate was transferred to the refrigerator at 4°C. After 30 min the precipitate took on a flaky appearance, forming a floc. This floc was centrifuged at 2800 g for 20 min and the resultant pellet re-suspended in 10 ml 0.15 molll Na zl-IP0 4 buffer.

Removal of toxic products Toxic products sometimes associated with marine samples were removed where necessary by addition of 10 ml dithizone in chloroform (0.01 gil) to the concentrated sample. After rotamixing for 1 min and centrifuging at 1500 g for 10 min, the chloroform layer was discarded. The aqueous layer was transferred to a sterile pot and the residual chloroform removed by exposure to a current of sterile air for 30 min. The resultant concentrated sample was then either transferred to the tissue culture laboratory for immediate assay or to the freezer for long-term storage at - 70°C.

Assay jor rotavirus LLC-MK2 cells (Flow Laboratories Ltd) derived from Rhesus monkey kidney, were grown routinely in flasks as monolayers. Growth medium was Eagle's

DETECTION OF VIRUSES IN WATER

287

minimal essential medium (MEM) modified with Hanks salts (Morton 1970) (Flow Laboratories Ltd) and supplemented with: Leibovitz LIS medium, 50%; foetal bovine serum, 5%" glutamine, 2 mmolll; vitamins, 1%; nonessential amino acids, 1
Immunofluorescence assll.Y In the immunofluorescence assay for rotavirus, LLC-MK2 cells were removed from flasks by trypsinization with 0.05 % trypsin - EDTA solution. After addition of growth medium, the resultant cell suspension was centrifuged at 800 g for 5 min. The supernatant was discarded and the cell pellet was re-suspended in serum-free medium (SFM). SFM was Eagle's minimal essential medium modified with Hanks salts containing sodium bicarbonate, 0.04%; streptomycin, 200 ~lg/ml; penicillin, 200 units; amphotericin B, 1.75 f1g/ml adjusted to pH 7.2 with NaOH, 0.1 molll and containing 0.5 f1g/ml trypsin. The protocol for the immunofluorescence assay is summarized in Fig. 2. After counting in an Improved Neubauer haemocytometer, cells were seeded in 96-well microtitration plates at a rate of 5 X 10 4 cells/IOO f11 SFmoliweli. The plates were incubated for 1 hat 3rC in 10% COz/air atmosphere and then for a further 1.5 h in 5% COz/air concentration: 100 f11 of sample concentrate (total 10 ml) was added to each well. In addition, 100-f11 control rotavirus concentrate derived from stools of infected individuals was added to 3-4 wells. The plates were centrifuged at 1400 g for 60 min (Fig. 2) and then incubated at 37°C for 1 h when the sample was removed and replaced \vith 150 f11 SFM (without trypsin). The plates were then incubated overnight at 37°C in 5% COz/air atmosphere.

Staining After overnight incubation, the medium was removed and each well was washed once with phosphate buffered saline (PBS). The cells were then fixed by adding 100 ~l ice-cold methanol and incubating at 4°C for 10 min. The methanol was removed and replaced with 100 f11 PBS, followed by incubation at room temperature for 10 min. The plates were then air-dried. After 10 min, 100 f11 rabbit- anti-rotavirus antiserum (Dako Ltd) (l:40 dilution in PBS) was added to the cells in each well and, after shaking for 5 min, the plates were incubated for 1 h at 37°e. Each well was then washed three times with PBS, and 100 ~l FITC (Fluorescein Isothiocyanate Isomer 1) conjugated goat-anti-rabbit antiserum (1:40 dilution in PBS) was added to each well. After shaking for 5 min, the plates were incubated for 1.5 h at 37°C. Each well was washed three times with PBS and 50 ~I of a 1% solution of amido

288

H. MERRETT AND C. E. STACKHOUSE

Cell culture LLC-MK2 cells

I

96 well microtitration plate (5 x 104 cells/well) in 100 JAI SFM with 0.5 JAg/ml trypsin

I

Incubate for 1 h (high CO 2 concentration) 37°C

I

Incubate for a further 1.5 h (low CO 2 concentration) 37°C

I

Add sample concentrate (total 10 ml) 100 JAI/well

I

Centrifuge 1400 g, 60 min

I

Incubate 1 h 37°C

I

•

Remove sample and replace with SFM Incubate overnight, 37°C, 5% CO 2 /Air §.!aininQ Remove medium Wash x 1 with PBS r

Add 100 iii/well ice cold methanol 4°C for 10 min

Fixation

Remove methanol Add 100 JAI/well PBS room temperature for 10 min

Rehydration

I

I

Air dry

I

Add 100 ~I rabbit anti-rotavirus antiserum (1 :40 dilution) and shake for 5 min

Addition of primary antibody

I

Incubate 1 hat 37°C

I

Wash x 3 PBS

I Add 100 ~{I FITC conjugated to goat-antirabbit-antiserum (1 :40) and shake 5 min

Addition of secondary antibody

I

Incubate 1.5 h at 37°C I Add 50 ~l Amido black ( 1 %)

I

Shake 10 min at room temperature

I

Wash x 3 PBS with shaking

I Air dry . Examine for fluorescent foci

..

SFM - serum free medium FIG.

2. The immunofluorescence assay.

Counterstain

DETECTION OF VIRUSES IN WATER

289

black was added to each well. After shaking for 10 min at room temperature, each well was washed three times with PBS, and then the plates were airdried and examined for fluorescent foci using a Nikon Diaphot inverted microscope at an excitation wavelength of 495 nm and magnification x200. Results

Incidence of rotavirus It is evident from the data collated throughout 1987, that rotavirus is present in marine waters in the designated area in South Wales. Out of a total of 46 water samples analysed during the sampling programme, 36 (78.3%) were positive for rotavirus. If one fluorescing cell is considered to be a consequence of infection by one rotavirus particle, then the numbers of rotavirus particles in 'positive' water samples ranged from 1 to 44 per 10 1.

Appearance of rotavirus-positive cells The fluorescing cells produced as a result of infection by rotavirus isolated directly from stools (controls), were well-defined and readily identified as bright green fluorescing foci on a black background. Viral antigen, made visible by fluorescent antibodies, was almost always detected in the cytoplasm as bright green fluorescing foci, either concentrated in granules or as fibrils throughout the cytoplasm (Bryden et al. 1977). In contrast, although rotaviruspositive cells resulting from infection by rotavirus isolated from environmental samples occasionally had similar appearance to those resulting from infection by control preparations, invariably they appeared as rounded, uniformly fluorescent, opaque entities on the black background. Discussion The immunofluorescence test is a highly specific assay that detects incomplete rotavirus replication in cell culture. To date, it is the only practical, specific method for measuring rotavirus infectivity (Smith & Gerba 1982) which is important in studies relating to the environment, when the presence or absence of viruses may be used to make predictions as to risk factors associated with recreational activities. Rotaviruses possess group, sub-group and serotype antigens and the majority of human and animal rotavirus strains possess the same group antigen. This group specificity is located on the outer surface of the inner (rough) capsid layer. Antibody to it forms tlle basis of a number of diagnostic tests including immunofluorescence. Thus, although the test used in this study could theoreti-

290

H. MERRETT AND C. E. STACKHOUSE

cally detect both human and a number of other rotaviruses including bovine and porcine rotavirus, it is thought that this is unlikely since we have to date failed to identify a possible source. After infection ofhost cells, rotavirus replication takes place in the cytoplasm. If cells are inoculated with high numbers of trypsin-activated virus, the replication cycle is completed in 12 h at 37°C. Viral protein synthesis is maximal at 3-5 h post-infection, which is consistent with viral antigens being first detectable by the fluorescent antibody technique by 4-6 h post-infection (Estes et al. 1983). Viral antigens detected in the cytoplasm are often seen as discrete perinuclear granules. Late in the infection, diffuse antigen is generally present throughout the entire cytoplasm and in addition, nuclear changes may be apparent. However, it is not known whether the fastidious human rotavirus strains such as are isolated from environmental samples exhibit the typical replication pattern as described above. Human enteric viruses cannot multiply in the environment and in this cell-free state are exposed to a variety of adverse factors of physico-chemical or biological nature (Bitton 1978). Thus, the decline of the rotavirus population in environmental waters follows firstorder kinetics, but may deviate from the exponential due to protective mechanisms in the suspending medium, aggregation of virus particles or adsorption to surfaces. Exposure to these conditions could conceivably select for an indigenous rotavirus strain whose replication cycle is different from the one predominantly present and frequently isolated directly from stools. For example, the opaque fluorescence associated with cells infected by environmentally derived rotavirus as opposed to the discrete cytoplasmic fluorescence observed after infection with rotavirus derived from stools, could simply represent a shortening of the overall replication cycle for indigenous rotavirus. In this instance, the observed opaque appearance could be attributed to the presence of diffuse antigen associated with events late in the infection process.

References BITTON~

G. 1978. Survival of enteric '\riruses. Water Pollution }r1icrobiology 2~ 273-299. N.R. & CUKOR, G.e. 1981. Viral gastroenteritis. New England Joumal ojMedicine

BLACKLOW,

304, 397 -406. BRYDEN, A.S.~ DAVIES, H.A.~ THOULESS, M.E. & FLEWETT, T.H. 1977. Diagnosis of rotavirus

infection by cell culture. Joumal of j\1edical Microbiology 10, 121-125. & OBIJESKI, J. 1983. Rotavirus: A Review. Current topics in Microbiological Immunity 105, 123-184. FLEWETT, T.H. 1982. Clinical features of rota,~rus infections. In Virus In/i:ctions oJtfte Gastrointestinal Trad eds Tyrell, D.A. & Kapikian, A.Z. New York: Marcel Dekker. GERBA, c.P., ROSE, ].B. & SINGH, S.M. 1985. Waterborne gastroenteritis and viral hepatitis. ESTES, M.K.~ PALMER, E.L.

eRe Critical Reviews ;11 Etlviromnental ComrolIS, 213-236. GoYAL, S.l\r!. & GERBA, c.P. 1982. Concentration of viruses from water by membrane filters. In

Methods in Enviromnetltal Virology, cds Gerba, C.I). & Goyal, S.M. pp. 59-109. New York: Marcel Dekker.

DETECTION OF VIRUSES IN WATER

291

KATZENELSON, E., FATTAL, B. & HOSTOVESKY, 1'. 1976. Organic flocculation: an efficient second step concentration method for the detection of viruses in tap water. Applied and Environmental Microbiology 32, 638-639. MeNuLTY, M.S. 1978. Rotaviruses. Journal of Gmeral Virology 40, 1-18. l'viATTHEWS, R.E.F. 1979. The classification of nomenclature of viruses. Summary of results and of the International Committee on Taxonomy of Viruses. bllen>irology II, 133 -135. MORRIS, R. & WAITE, W.M. 1981. Environmental virology and its problems. Journal of the Institution of Water Engineers and Scimtists 35, 232-245. MORTON, H.]. 1970. A survey of commercially available tissue culture media. In vitro 6, 89-108. SMITH, E.M. & GERllA, c.P. 1982. Development of a method of detection of human rotavirus in water and sewage. Applied and Ellvironmmtal Microbiology 43, 1440~ 1450. THOULESS, M.E., BRYDEN, A.S. & FLEWETT, T.H. 1977. Rotavirus neutralisation by human milk. British Medical Journal 2, 1390-1393. WYATT, R.G. & JAMES, W.o. 1980. Human rota,irus Type 2: cultivation ill vitro. Sdmce 207, 189-191.

Index

Acetohacter spp., 104 Acinetobacter, 237 Adenosine triphosphate, see ATP bioluminescence Aerobic plate count, 42, 90 Aeromonas spp., 128 Aeromonas hydrophila, 127 Affinity techniques for removal of bacteria from milk, 16, 19, 23 Aflasure kit, 277 Aflatest kit, 207, 277, 278 Aflatoxins, 265 assays for, 265, 267, 276-278 Amperometric biomass sensing, 214- 224 AMS image analysers, 74, 75, 77, 80 Antibody immobilization, 170, 171 APC, see Aerobic plate count Apyrase enzyme, 13, 15 ATP bioluminescence, for analysis of beer, I - 10 for analysis of milk, 13-32 reliability of, 9 Automated counting, for DEFT, 34, 52, 56 Automated electrometric methods, optimization of, 87-98

147, 149 for testing water samples, 120-125 protocol, cost for, 200 BEC, see Bioelectrochemical cell Beer, amperometric testing of, 225 detection of contaminants in, IOI-II7 PET bottled, analysis of, I-II use of DEFT with, 2 Benzalkonium chloride, as selective inhibitor,

36,37 Benzoquinone, 216 Beverages, use of DEFT on, 35 BlOCHECK, 213-225 Bioelectrochemical cell, 214 - 225 Bio-Enzabead Salmonella Screen kit, 169, 175,

179-181,249-254,262-263 Biokit, 279 Bioluminescent techniques for milk, 13-29 Breed smear, sensiti~ity of, 33 Brewery bacteria, 106-108 Brewing industry, use of electrometric techniques in, 10 1- II7 Brewing yeasts, 105, 107, 117 British Standard method, for detecting salmonellas in milk powders, 156-164

Bacillus spp., 237 Bacillus pumilus, 67 Bacteriological Analytical Manual, Food and Drug Administration, 251, 254 Bacteriological examination of drinking water supplies, 119 Bacteriuria, 59-64, 67 Bactomatic method, for TVCs of powdered products, 144, 147, 148 Bactometer, 32, 132, 134, 156, 159, 160, 168, 213 for conductimetric detection, 186-189, 197, 199, 200 for enterococci detection, 203, 205, 206, 209-211 for screening powdered products, 143, 144,

Ca Z+ sequestrant, 13 Cadmium, in Bio-Enzabeads, 180 Calcium phosphate-citrate complex, 13-26 Calibration curves, of standard and electrometric tests, 90, 91, 98, 121,

122, 144, 147, 149, 152,209,210,225 Calibration factor, in image analysis, 75 Calibration of Malthus and Bactometer instruments, 209 - 211 Carbon materials, use as an electrode, 215 Casein micelle, 13-26 Celite, use of in removal of bacteria from milk,

16, 19,27,28 Chelating agents, use of in dissociating casein micelles, 26

293

294

INDEX

Chelating systems, in removal of free ATP, 17 Chemafla kit, 277 Chicken, DEFT counts of, 53 detection of salmonellas in, 255, 256, 259 Chilli-eon-carne, detection of salmonellas in, 255 ELISA method for detection of salmonellas, 258 Chocolate, salmonellas in, 185, 187 Chromatography, for mycotoxin measurement, 266 citrate, in removal of free ATP, 17 CitroblUter spp., 128 Citroba
Clostridium botulinum, 265 perfringens, 265, 267, 279, 280 Clumps, of cells. per microscope field, 51, 52 CM medium, 120, 121 Cocoa powder, detection of salmonellas in, 255, 258 ELISA method for detection of salmonellas, 260 Cod, electrometric assay of, 93 Coliform organisms, in milk powders, 144, 145, 149, 152 in water, 120-124, 127-130 Colony count, of Lancefie1d Group D cocci, 204, 207, 209 Coloured latex agglutination test, 171, 172,

178, 179, 181, 182 Comminuted meats, bacterial counting of, S3~55

Computer-controlled multichannel system,

217 Computer key for mould identification,

curves, 168) 169, 180, 181, 187,205-209 for detection of contaminants in beer,

101-117 measurement, 1, 87-98 for Salmonella detection, 145-147, 152,

156-164 screen, 131 - 140 signals) from Enterobacteriaceae, 137, 138 temperature for, 89 Confectionery, salmonellas in, 185 - 200 Contaminants, detection of, in beer. 101 - 117 Contaminated fluids, DEFT analysis of,

64-67 Contamination, bacterial, assessment of, 227 - 239 by yeasts, 2 Contamination level, fonnula for, 66 Contrast, in image analysis systems, 74 Conventional assays, compared with eJectrometric, 90, 91 Cornflour, electrical screening of, 147-149 Correlation coefficients) between DT and TVC, 92, 147, 149 for Malthus and Bactometer instruments,

210 Counting, of bacteria, in DEFT samples,

51-55 Cream cakes, Enterobacteriaceae in, 136, 137 Critical Control Point, 185 Cross-reactivity, in aflatoxins, 276, 277 Crystal violet, as selective inhibitor) 36, 37 Current output, relationship with plate count,

228 Current time profile, 216 Curve quality, 89, 90 Cycloheximide, 106, 108 Cytochrome c oxidase, activity in milk and cream, 231, 232, 236- 238

245-248 Computer program for image analysis, 75 Concanavalin A, in affinity techniques, 16, 19,

23,28 Concentrated cells, bacterial ATP estimation on, 16 Concentration method, for enumeration of Enterobacteriaceae, 136, 137 Conductance, change, source of, 95 - 97 comparison with IMET, 170 for confectionery products, 185 - 200

DEFT (direct epifluorescent filtration technique), cost of equipping a laboratory for, 34 daily sample throughout, 56 DEFT/APC procedure, 44 medical and phannaceutical applications of,

59-79 recent developments for foods and beverages, 33-45 simplified protocol, 60 for urine examination, 60-64

INDEX

use of in milk and beer, 1, 60 use of with meat and poultry, 47-56 Detection routine, evaluation of, 104 Detection time, 87-98, 102, 104, 107, 109, 110, 115, 144, 149, 181 effect of pH on, 206-209 impedance, 121, 122 Dichlorophenol indophenol, 219, 228 Dichotomous keys, 242 Direct epifluorescent filtration technique, see DEFT Dried milk products, 195 DT, see Detection time Dye reduction tests, 227, 233 Dynal beads, 170, 171, 175, 178, 180, 181 Easter-Gibson procedure, 156-164 EDTA (ethylenediaminetetraacetic acid), as chelating agent, 15 in removal of free ATP by sequestrating agents, 17, 26 EGTA (ethyleneglycolaminoctylether tetraacetic acid), as chelating agent, 15 in removal of free ATP by sequestrating agents, 17 Electricity from microbial fuel cells, 227 Electrochemical cell, computer control of, 217 Electrometric assay, 88 method for detection of enterococci, 203-211 methods, optimization of, 87-98 screening of powdered dairy products, 143-153, 155-164 testing of water quality, 119- 130 Electron transfer, detection of, 227-239 Electrostatic charge, for removal of bacteria from milk, 16 Electrostatic interaction fiJr removal of bacteria from miik, 19 EI.JSA, see Enzyme linked immunosorbent assays Enlerobacler cloacae, 102, 108, 127, 157, 160, 164, 168 Enterobacteriaceae, in foods, 131-140 selective broth, 132-134 Enterococci, instrumental detection of, 203-211

295

Elllerococclts [aecalis, 203, 205, 207 [aeaum, 203, 207 Enterotoxin assays, 267, 279-280 Enzyme linked immunosorbent assays, 165, 166, 175, 186-200, 249-259, 262, 266, 277 Epifluorescence microscopy, cost of, 34 Equation for shelf life of pasteurized milk, 23 ESBBH, see Enterobacteriaceae selective broth Escherichia coli, 27, 67, 88, 120, 124, 125, 127-130,131,132,197,220-225, 280 Ethylenediaminetetraacetic acid, see EDTA Ethyleneglycolaminoctylether tetraacetic acid, see EGTA Faecal indicator bacteria, in water samples, 120 Farm milk, use of DEFT for grading, 35 Fat-filled powder, electrical screening of, 149-151 Ferrocene derivatives, 219 Filters, efficiency of, 7 Filtration, of DEFT meat and poultry preparations, SO, 51 efficiencies, in beer spoilage tests, 6, 7, times, for PET bottles of beer, 6 Fish, electromagnetic assay of, 93, 95 shelf-life of, 88, 96 Flavobacterium od()ratum, 238 Flocculation, organic, 284- 286 Food, Enterobacteriaceae in, 131-140 immunological detection of salmonellas in, 249-261 mycology, 241-248 poisoning, due to Salmonella spp., 155-164 preservation, use of gamma irradiation in, 42 samples, amperometric testing of, 225 suspensions, preparation of for DEFT, 35 Food and Drug Administration's Bacteriouigical A"a{ytical Manual, 251-254 Foodborne illness, toxins associated with, 274-280 Forcing tests, for beer, 101 Free ATP, in milk, 13-26 Frozen meat/fish, DEFT count of, 35

296

INDEX

Gamma radiation in food preservation, 42 Gastroenteritis, rotaviral, 283 Gluconohacter spp., 104 Gram-negative brewery bacteria, 106 medium for, 114, 115 Gram-positive brewery bacteria, 107, 108 medium for, 115

HajfiJia alvei, 170 Hazard analysis and critical control point, 185 Heat-treated meat products, use of DEFT with, 55 Helix pomatia, in affinity techniques, 16 'Hole fill' option, 78 Image analyser, with DEFT automated counting, 34, 52 Image analysis, for MIC determination, 73-85 IMET, see Immunomodified electrical technique Immunoassay kits, for toxin detection, 265-280 Immunofluorescence techniques, for detection of viruses in water, 284- 290 Immunological detection of salmonellas, 249-261 Immunomodified electrical technique, 166, 169, 170-172, 174-181 Impedance method, for beer, 1 for powdered milk products, 144, 145, 149, 151, 152 Impedimetric estimation, of colifonns, 132 In-line sampling of milk, 24 Incubation, period, open circuit, 234 times, for beer samples, 101 Infusion fluids, DEFT analysis of, 64-67 Inhibitors, for brewery micro-organisms,

106-108 Inorganic constituents of media, 96-98 Intravenous fluids, DEFT analysis of, 64-67 Irradiated foods, screening of, 42 use of DEFT for, 42 Keeping quality, of milk, 36, 38 of pasteurized cream, 37, 38 of pasteurized milk, 236- 237

Kirkcgaard and Perry Salmonella ELISA, 256~ 259, 262, 263 Kits, toxin, 265 - 280 K(t see Keeping quality Lactobacilli, in beer, I Lactobacillus spp., 104 La<:tOf{JCCUS lactis, 229 Lactose, fennentation of, 120, 132 Lancefield Group D cocci, 203 - 211 Lecrins, in affinity techniques, 16, 19, 20 in raw milk testing, 28 Luciferase, 14, 27 Luciferin-Iuciferase complex, 13, 15, 19, 23 Lumac Biocounter, 2 Lumacult, in ATP analysis,S Luminometers, 15 Lumit PM, 15, 19 M-broths, 253, 254, 257 Magnetite, use of in removal of bacteria from

milk, 16, 23, 27 Malthus, Columbia broth, 134 detection times, 109, 110, 115 drinking water test, 119, 125-130 Growth Analyser, 92, 93, 102-106, 125 -130, 156, 159, 160, 168, 169, 186, 209-211 instrument, 143, 145,213 method, 11 0, 114- 116 use with brewery spoilage organisms, 116 1\1ean generation times, for DEFT samples, 66,67 Meat, cytochrome c oxidase tcst for, 236-239 electrical signals from samples of, 235, 236 Enterobacteriaceae screening, use of, 139 in fuel cells, 231 use of DEFT with, 47-56 Media, for brewery micro-organisms, 103 database for, 247 for detection of salmonellas, 186 inorganic constituents of, 96-98 for moulds, 245 pre-enrichment, 157, 159-161 for Salmonella immuno/conductance detection, 166, J67

INDEX

selective, for cocci, 203, 205 Mediators, in bioelectrochemical method, 214-216 Medical and pharmaceutical applications of DEFT, 59-70 Membrane filters, in ATP analysis, 3, 7, for meat and poultry samples, 47, 50 for urine analysis, 60, 61 in water testing, 120 Membrane Iluorescence counts, total, 67 MESB, see 1\10dified Enterobacteriaceae selective broth Methylene blue test, 236 MIC (minimum inhibitory concentration), of copper sulphate, 106 of crystal violet, 105, 106 determination of, 73 - 85 Microbial fuel cell, electricity from, 227-231, 233,234 Microcolony DEl'~r counts, selectivity of, 4 I, 42 sensitivity of; 40 Microcolony formation, by pure cultures, 40 Microscope factor, the, 52, 62, 66, 67 Microtest kit, 277, 279 Milk, amperometric testing of, 224 assessment of with bioluminescent techniques, 13-29, Bacteria Kit, 14, 16 bacteria associated with, 237 c)1ochrome c oxidase activity in, 231, 232, 236-238 detection of bacteria, in, 13 electrical signals from samples of, 235, 236 ELISA method for Salmonella, 260 hygienic quality of, 227 incubation of in fuel cells, 231 post-pasteurization contalnination ot~ 236 powders, electrical screening of, 143-153 pre-incubation of, 232 psychrotrophic coliforms in, 236- 239 raw, resazurin test for, 236 spoilage of, 228 UHT,257 usc of DEFT with, 60 Mixed flora powders, 161, 163, 164 MLD, see Modified lysine decarboxylase broth Modified Enterobacteriaceae selective broth,

297

133, 134, 137, 139 Modified lysine decarboxylase broth, 187, 189, 194, 197, 199 Monoclonal antibodies, 249 Moulds, computer-assisted identification of, 241-248 isolation of, 243-245 media for, 243 Multipoint inoculator, 78-85 Mycology, of food, 241-248 Mycotoxins, 267, 266-277 Neucleopore membrane filters, 38-40 Nisin, as a selective inhibitor, 37 NRB, for extraction of ATP, 2

Obesumbacterium proteus, in pitching yeast, 1 Ochratoxin A, 267 Organic Ilocculation, 286- 288 Osmotolerant yeasts, in confectionery products, 38 Overcounting, 53, 55 Parenteral Iluids, DEFT analysis of, 64 Pasteurized milk, dye reduction grading, 233 microbiology ot~ 36 testing, 16, 29 variation in processing of, 17 Pediococcus spp" 104 Penicillin, as a selective inhibitor, 37 Penicillium, three-stage branched species of, 244-256 Penicillium chrysogenum, 248 italicum, 248 roquejOrtii, 248- 250 PET bottle filtration device, 3-6 PET bottles, analysis of beer in, 1-11 pH, effect on detection time, 207-209 Phage test, for identification of salmonellas, 189-194, 199 Phenazine ethosulphate, 219 Phosphate, in media, 97, 98 Pin-pattern date, 80-82 Pixels, 73, 74 Plate colony count, for beer samples, 103 comparison \\~th DEFT, 35, 52-56, 67

298

INDEX

for monitoring beer, 101, 1l0, 114-116 relationship with current output, 228 Plate diffusion assay, 75, 77 Population generation time, 139, 140 Positivity threshold, for significant bacteriuria,

63 Post-incubation DEFT, 65-67 Post-pasteurization contamination, of milk,

236 Post-pasteurizer samples, moitoring of, 116 Potassium ferricyanide, 216, 219, 220 Potentiostat, 217 Poultry, use of DEFT with, 47-56 Powdered dairy products, electrical screening of, 143-153 Prawns, detection of salmonellas in, 255, 258 Pre-enrichment incubation time, 171, 172,

175, 176, 179 Pre-filtration, 48- 50 Pre-incubated storage tank milk, bacterial ATP content of, 24 Pre-incubation, procedure for testing shelf life of milk,

23-25 selective, for detection of spoilage bacteria, 36-38 temperatures, 37, 38

Proteus mirabilis, 157, 160, 164, ]68 vulgan>s, 197 Prototype biocheck model, 220- 224 Pseudomonas aeruginosa, 67, 197,219 jluoresccns, 27 putida, 238 stutzeri, 27, 237 Psychrotrophic count of milk, 14 Psychrotrophs, growth of in milk, 29, 36 Quadratic regression analysis, using Bactometer software, 122 Radiometric medlods, 132 Raw milk, bacterial ATP assay of, 14 selective enumeration of bacteria in, 39 testing, 26, 27 Raw milks, comparison of assay systems for 17,

19 Redox activity, 215, 216 Redox mediator reduction, 236

Relative Light Unit values, 2, 6 Removal of free ATP by use of sequestrants,

17 Replication cycle, of rotaviruses, 290 Rotaviruses, 283, 286, 287, 289, 290 assay for, 287 RSI data analysis system, 80

Satcharomyces cerevisiae, 10 Salad vegetables, 140 Salmonella spp., 88, 96 antigens, 249-261 conductance method of testing fOf,

155-164 in confectionery products, 185- 200 in foods, immunological detection of,

249-261 freeze-injured cultures, 169 in milk powders, 145-147, 149, 152,

155-164 rapid detection of, 165 - 182 strains, in immuno/conductance determinations, 167, 168 1-2 test, 259-261, 263

Salmonella anatum, 179 ertteritidus, 169-172, 181, 255, 261 guinea, 195 houten, 195 london, 197 napoli, 187 newport, 255 stanley, 255, 261 typhimurium, 138, 187,25],255,259,261 virchow, 255 Sample suspension preparation techniques, with DEFT, 48-52 Scottish Milk Marketing Board, 14 SC/T/D, see Selenite-cystine-trymethylamine oxide-dulcitol medium SDS, see Sodium dodecyl (lauryl) sulphate Selective enumeration ofbacteria, by DEFT, 39 Selective pre-incubation, 36 Selenite-cystine-trimethylamine oxide -dulcitol medium, 186, 187, 189, 194, 197, 199 Sewage, amperometric testing of, 224 rotaviruses in, 283 Shelf life, of chilled packaged fish, 88, 96 test for pasteurized milk, 23

INDEX

Sigma enzyme, 19 Skimmed milk powder, electrical screening of, 144, 149-151, 157, 163 in immuno/conductance determination, 167, 168 in immunological detection of salmonellas, 255, 258, 259 Sodium azide, effect on conductance profiles, 208,209 Sodium dodecyl (lauryl) sulphate medium, 120-123 Software, for microbial activity monitor, 217 Somatic cell ATP, 13,27 Somase, use of in milk assay, 16, 17 Spectrophotometric assay of TMPD oxidation rates, 232 Spiral plate maker, 160-172 Spoilage flora of fish, 95, 96 Spray drying, of Lancefield Group D cocci, 204 Standard line calculation, 78 Standard plate count, for cornflour, 148, 149 for milk powders, 144, 152 Staphylococcal enterotoxin assays, 267, 279 Staphylococcus aureus, 67 Steak, use of in fuel cells, 231 Streptococcus bovis, 204, 207 equinus, 204, 205, 207 faeca/is, 27, 137 Surfactant treatment, for DEFT preparations, 49, 50 Synoptic keys, 244 Synthetic wort medium, see SYWM System III Image Analyser, 74, 75, 77 SYWM, 103-108, 114-116 Taxonomic accuracy, of computer keys, 246, 247 TBC of milks, 14, 17, 23 TECRA Salmonella visual assay, 253 - 256, 258, 262, 263, 256, 258 Temperature, incubation, 91 Tetra Brik machine, contamination reduction, 25 Thallous acetate, 208, 209 Thionine-mediated fuel cell, 219, 228-231, 233 Three-electrode constant-potential system,

299

214-216 TMAO/TMA reaction, 95, 96, 98 Total bacterial count, see TBC of milks Total viable count, 88, 90-92, 96, 98 of powdered products, 144, 147, 148, 151 Toxins, immunoassay for, 267-282 Trichloracetic acid, in ATP extraction, 23 Trisodium citrate, as chelating agent, 15 Triticum vulgaris, in affinity techniques, 16, 20 Trypsin, 49, 50 TV image analyser system, cost of, 34 TVC, see Total viable count Two-electrode configuration, 217 Urine, amperometric testing of, 225 examination of, by DEFT, 59-64 Vibrio cholerae enterotoxin analysis, 280 Video cameras, Chalnicon, 85 for image analysis, 73-75 Plumbicon, 85 Viruses, detection of in water, 283-290 Water, quality testing, electrical methods for, 119-130 y;ruses in, 283-290 Whey powder, electrical screening of, 157, 164 in immuno/conductance detection, 167-168 Whole milk powder electrical screening of, 157, 163 in immuno/conductance detection, 167, 168 Wines and equipment, use of DEFT on, 35 WLN broth, 103 Yeasts, cause of contamination in, 2, 5 used in beer monitoring, 102, 103 w;ld, 105 wild, medium for, 115 YEPG (yeast extract-peptone-glucose) broth, 103, 104, 106, 110, 114-116 YM broth, 103, 105 Zeta plus filters, for removal of milk bacteria, 19,23, 27, 28 Zones of inhibition, 75, 77 Zymomonas spp., 104

THE SOCIETY FOR APPLIED BACTERIOLOGY TECHNICAL SERIES General Editor: F.A. Skinner Identification Methods for Microbiologists Part A 1966 Ediled ky B.M. Gibbs and F.A. Skinner Out o/prilll 2 Identification Methods for Microbiologists Part B 1968 Edited ky B.M. Gibbs and D.A. Shapton Out 0/prilll 3

Isolation Methods for Microbiologists 1969 Ediled by D.A. Shapton and G.W. Gould

4

Automation, Mechanization and Data Handling in Microbiology 1970 Edited by Ann Baillie and RJ. Gilbert

5 Isolation of Anaerobes 1971 Edited by D.A. Shapton and R.G. Board 6 Safety in Microbiology 1972 cilited ky D.A. Shapton and R.G. Board 7 Sampling - Microbiological Monitoring of Environments 1973 Edited by R.G. Board and D.W. Lovelock 8 Some Methods for Microbiological Assay 1975 Edited by R.G. Board and D.W. Lovelock 9 Microbial Aspects of the Deterioration of Materials 1975 Edited by R.]. Gilbert and D.W. Lovelock 10 l\1icrobial Ultrastructure: The Use of the Electron 1\1icroscope 1976 Edited ky R. Fuller and D.W. Lovelock 11

Techniques for the Study of Mixed Populations 1978 ciiited kY D.W. Lovelock and R. Davies

12

Plant Pathogens 1979 Edited by D.W. Lovelock

13

Cold Tolerant Microbes in Spoilage and the Environment 1979 Edited ky A.D. Russell and R. Fuller

14

Identification Methods for Microbiologists (2nd Edn) 1979 Edited by F.A. Skinner and D.W. Lovelock

15

Microbial Growth and Survival in Extremes of Environment 1980 Edited by GW. Gould and Janet E.L. Corry

16

Disinfectants: Their Use and Evaluation of EtTectiveness 1981 1:aited by e.H. Collins, M.e. Allwood, Sally F. Bloomfield and A. Fox

SAB TECHNICAL SERIES

17

Isolation and Identification Methods for Food Poisoning Organisms J982 Edited by Janet E.L. Corry, Diane Roberts and F.A. Skinner

18 Antibiotics: Assessment of Antimicrobial Activity and Resistance 1983 Edited by A.D. RusseU and L.B. Quesnel 19

Microbiological Methods for Environmental Biotechnology 1984 Edited by J.J. Grainger and lM. Lynch

20

Chemical Methods in Bacterial Systematics 1985 Edited by M. Goodfellow and D.E. Minnikin

21

Isolation and Identification of Micro-organisms of Medical and Veterinary Importance 1985 Edited by C.H. Collins and ].M. Grange

22

Preservatives in the Food, Phannaceutical.and Environmental Industries 1987 Edited by R.G. -Board, M.e. Allwood and J.G. Banks

23

Industrial Microbiological Testing 1987 Edited by J.W. Hopton and E.C. Hill

24

Immunological Techniques in Microbiology 1987 Edited by J.M. Grange, A. Fox and N.L. Morgan

25

Rapid Microbiological Methods for Foods, Beverages and Phannaceuticals 1989 Edited by C.j. Stannard, S.B. Petitt and F.A. Skinner

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