MICROBIOLOGICAL EXAMINATION METHODS OF FOOD AND WATER MICROBIOLOGICAL EXAMINATION METHODS OF FOOD AND WATER A Laboratory Manual S E NEUSELY DA SILVA, MARTA HIROMI TANIWAKI, VALÉRIA CHRISTINA AMSTALDEN JUNQUEIRA, NELIANE FERRAZ DE ARRUDA SILVEIRA, MARGARETE MIDORI OKAZAKI & RENATO ABEILAR ROMEIRO GOMES Institute of Food Technology – ITAL, Campinas, SP, Brazil Originally published in Portuguese as: ‘Manual de Metodos de Análise Microbiológica de Alimentos e Água ©2010, Livraria Varela, Sao Paulo, Brazil E nglish edition ‘Microbiological Examination Methods of Food and Water: A Laboratory Manual’, CRC Press/Balkema, Taylor & Francis Group, an informa business ©2013 Taylor & Francis Group, London, UK Translation to English: Paul van Dender† CRC Press/Balkema is an imprint of the Taylor & Francis Group, an informa business © 2019 Taylor & Francis Group, London, UK Typeset by Apex CoVantage, LLC All rights reserved. No part of this publication or the information contained herein may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, by photocopying, recording or otherwise, without written prior permission from the publisher. Although all care is taken to ensure integrity and the quality of this publication and the information herein, no responsibility is assumed by the publishers nor the author for any damage to the property or persons as a result of operation or use of this publication and/or the information contained herein. Published by: CRC Press/Balkema Schipholweg 107c, 2316 XC Leiden, Th e Netherlands e-mail: [email protected] www.crcpress.com – www.taylorandfrancis.com Library of Congress Cataloging-in-Publication Data Names: Silva, Neusely da., author. Title: Microbiological examination methods of food and water : a laboratory manual / Neusely da Silva, Marta Hiromi Taniwaki, Valeria Christina Amstalden Junqueira, Neliane Ferraz de Arruda Silveira, Margarete Midori Okazaki & Renato Abeilar Romeiro Gomes. Other titles: Manual de metodos de analise microbiologica de alimentos e agua. English Description: Second edition. | Leiden, Th e Netherlands ; Boca Raton : CRC Press/Balkema, [2018] | Includes bibliographical references and index. Identifi ers: LCCN 2018034220 (print) | LCCN 2018035352 (ebook) | ISBN 9781315165011 (ebook) | ISBN 9781138091887 (hardcover : alk. paper) | ISBN 9781138057111 (pbk. : alk. paper) Subjects: MESH: Bacteriological Techniques—methods | Food Microbiology—methods | Water Microbiology | Laboratory Manuals Classifi cation: LCC QR115 (ebook) | LCC QR115 (print) | NLM QW 25.5.B2 | DDC 579/.16—dc23 LC record available at https://lccn.loc.gov/2018034220 ISBN: 978-1-138-05711-1 (Pbk) ISBN: 978-1-138-09188-7 (Hbk) ISBN: 978-1-315-16501-1 (eBook) Table of contents About the authors XXVII Preface XXIX List of tables XXXI List of fi gures XXXV 1 Sampling, transport and storage of samples for analysis 1 Revision history 1 1.1 Introduction 1 1.1.1 Lot 1 1.1.2 Lot sample and sample unit 1 1.1.3 Lot sampling plans 2 1.1.4 Analytical unit 2 1.2 Collecting samples for analysis 2 1.2.1 Selection and preparation of containers for the sampling of foods contained in non-individual packages 3 1.2.2 Procedures for the sampling of foods contained in non-individual packages 3 1.2.3 Sampling of foods involved in foodborne diseases 4 1.2.4 Sampling of water 4 1.3 Transportation and storage of samples until analysis 5 1.3.1 Foods with low water activity 5 1.3.2 Frozen foods 5 1.3.3 Refrigerated foods 5 1.3.4 Commercially sterile foods in sealed packages 6 1.3.5 Water samples 6 1.4 References 7 2 Preparation of samples for analysis 9 Revision history 9 2.1 Introduction 9 2.2 Homogenization of samples and withdrawal of the analytical unit 10 2.2.1 Procedure for homogenization and withdrawal of analytical units from liquid products 11 2.2.2 Procedure for homogenization and withdrawal of analytical units from solid or concentrated liquid products 11 2.2.3 Procedure for withdrawing the analytical unit using the surface swabbing technique 12 2.2.3.1 Swab sampling 12 2.2.3.2 Sponge swab sampling 13 2.2.4 Procedure for withdrawing the analytical unit using the surface washing technique 13 2.2.4.1 Procedure for washing poultry carcasses 13 2.2.4.2 Procedure for washing other foods 13 2.2.4.3 Procedure for washing packages 14 2.2.5 Keeping of counter-samples 14 2.3 Preparation of the first dilution of the analytical unit 14 VI Table of contents 2.3.1 Diluents for presence/absence tests 14 2.3.2 Diluents for tests requiring differentiated handling of the sample 14 2.3.3 Diluents for general quantification tests 14 2.3.4 How to prepare an initial 1:10 (10− 1 ) dilution 15 2.3.5 How to prepare an initial dilution different from 1:10 16 2.3.6 Procedure for the preparation of the first dilution of liquid samples 16 2.3.7 Procedure for the preparation of the first dilution of solid or concentrated liquid samples 16 2.3.8 Procedure for the preparation of the first dilution of samples obtained by surface swabbing or surface washing 16 2.4 Serial decimal dilution of the sample 16 2.5 References 17 Annex 2.1 – Procedures for homogenizing the content and withdrawal of the analytical unit of different types of foods 18 a) Powdered products 18 b) Pasty or ground products 18 c) Yogurts with fruit pieces 18 d) Cheeses 18 e) Very hard food products 18 f) Pieces of solid foods 18 g) Eggs in the shell 18 h) Meat cuts for analysis of non-surface contamination 19 i) Bivalves 19 j) Gastropods 19 k) Whole and sliced cephalopods 19 l) Whole crustaceans such as crabs 19 m) Sea urchins 19 n) Whole fresh fish 19 Annex 2.2 – Special cases in which there are variations in the analytical unit and/or dilution and/or diluents recommended for the preparation of the first dilution of samples of different types of foods 19 a) Liquids with low levels of contamination 19 b) Fatty foods 20 c) Lump-forming powders 20 d) Thickeners or products containing natural antimicrobial compounds 20 e) Gelatin 20 f) Acid products 20 g) Fine flours or meals, cereal grains, animal feed 21 h) Cocoa and chocolate 21 i) Egg white 21 j) Fermented products containing live microorganisms intended for the quantification of the contaminating microflora (except probiotics) 21 k) Powdered dairy products (dried milk, dried sweet whey, dried acid whey, dried buttermilk, lactose) 21 l) Butter 21 m) Frozen dairy products and ice cream 21 n) Cheeses 22 o) Fermented dairy products 22 p) Casein and caseinates 22 q) Rennet casein difficult to dissolve 22 Table of contents VII r) Powdered milk-based infant foods 22 s) Custard, desserts and sweet cream (pH > 5) 23 t) Mollusks (bivalves and gastropods) and sea urchins 23 u) Sea cucumbers ( Holothuroidea ) and tunicates 23 3 Basic plate count techniques for enumeration of microorganisms 25 Revision history 25 3.1 Introduction 25 3.2 Pour plate technique 26 3.2.1 Material required for the analyses 26 3.2.2 Procedure 26 3.3 Spread plate technique 27 3.3.1 Material required for the analyses 28 3.3.2 Procedure 28 3.4 Drop plate technique 29 3.4.1 Material required for the analyses 29 3.4.2 Procedure 29 3.5 Membrane filtration 30 3.5.1 Material required for the analyses 30 3.5.2 Procedure 30 3.6 Counting colonies and calculating results according to APHA 31 3.6.1 Pour plate calculations 31 3.6.1.1 Rules for calculating the pour plate results in the standard situation 32 3.6.1.2 Rules for calculating the pour plate results in unusual situations 33 3.6.1.3 Calculating the pour plates results for samples prepared by the surface swabbing technique (swabs or sponges) 35 3.6.1.4 Calculating the pour plate results of samples prepared by the surface washing technique 36 3.6.2 Spread plate calculations 36 3.6.3 Drop plate calculations 36 3.6.4 Membrane filtration calculations 36 3.7 (revised) Counting colonies and calculating results according to ISO 7218:2007/ Amd .1:2013 37 3.7.1 (new) General requirements for the calculation of results 37 a) Number of Petri dishes per dilution 37 b) Maximum and minimum acceptable number of colonies on counting plates 37 c) Decimal dilution of the number of colonies 38 d) Acceptable variation between counts of the pair of plates of a duplicate 38 e) Presentation of the result 38 3.7.2 General rules for the calculation of results 38 3.7.3 Rules for calculation in unusual situations 41 3.8 References 43 Annex 3.1 – (new) Limits of agreement for sums of colony counts of two parallel Petri dishes or colony counts from one Petri dish per dilution step over two 10-fold dilution steps (with a probability of 99% per comparison) (ISO 14461-2:2005) 44 Annex 3.2 – (new) Limits of agreement for colony counts of two parallel Petri dishes (with a probability of 99% per comparison) (ISO 14461-2:2005) 45 4 Basic techniques for microbial enumeration by the most probable number (MPN) method 47 Revision history 47 4.1 Introduction 47 VIII Table of contents 4.2 Multiple dilution test 48 4.2.1 Material required for the analyses 49 4.2.2 Procedure 49 4.3 Single dilution test 50 4.3.1 Material required for the analyses 50 4.3.2 Procedure 50 4.4 Calculation of the results 51 4.4.1 Calculating the results of the multiple dilution test 51 4.4.1.1 Calculation using the MPN tables (for decimal dilutions) 51 4.4.1.2 Calculating using the Thomas formula (for non-decimal dilutions) 52 4.4.1.3 Calculating the results of the samples prepared by the surface swabbing or surface washing techniques 52 4.4.2 Calculating the results of the single dilution test 53 4.4.2.1 Rules for calculations performed using Table MPN-3 53 4.4.2.2 Calculation for samples prepared by the surface swabbing or surface washing techniques 53 4.5 References 53 Annex 4.1 – MPN tables 54 5 Basic techniques for the detection of the presence/absence of microorganisms 57 Revision history 57 5.1 Introduction 57 5.1.1 Enrichment 57 5.1.1.1 Pre-enrichment 58 5.1.1.2 Selective enrichment 58 5.1.2 Isolation in solid media (selective differential plating) 58 5.1.3 Confirmation 59 5.1.3.1 Catalase test 59 5.1.3.2 Citrate test 59 5.1.3.3 Amino acid decarboxylation tests 59 5.1.3.4 Phenylalanine deaminase test 59 5.1.3.5 Carbohydrate fermentation tests 59 5.1.3.6 Indole test 60 5.1.3.7 Malonate test 60 5.1.3.8 Oxidation/fermentation (O/F) test 60 5.1.3.9 Oxidase test 61 5.1.3.10 Nitrate reduction test 61 5.1.3.11 Urease test 61 5.1.3.12 Methyl red (MR) test 61 5.1.3.13 Voges-Proskauer (VP) test 62 5.2 Material required for the analyses 62 5.3 Procedure 62 5.3.1 Pre-enrichment 62 5.3.2 Selective enrichment 62 5.3.3 Differential plating 62 5.3.3.1 Streak plating technique for obtaining pure cultures 62 5.3.4 Selection of colonies and subculturing of cultures for confirmation 63 5.3.4.1 Technique for the subculturing of pure cultures starting from colonies isolated from plates 63 Table of contents IX 5.3.5 Confirmation tests 63 5.3.5.1 Gram staining (Hucker’s method) 63 5.3.5.2 (new) KOH test for confirmation of doubtful Gram staining (Gregersen, 1978) 64 5.3.5.3 Spore staining (Schaeffer-Fulton’s method) 64 5.3.5.4 Spore staining (Ashby’s method) 64 5.3.5.5 Wet mounts for direct (fresh) microscopic observation 64 5.4 References 64 6 Aerobic plate count 65 Revision history 65 6.1 Introduction 65 6.1.1 The importance and significance of the total aerobic mesophilic count 65 6.1.2 Definition of psychrotrophs 66 6.1.3 Comments on methods of analysis 67 6.2 ( revised ) Plate count method APHA 8:2015 for aerobic mesophilic bacteria in foods and water 69 6.2.1 Material required for analysis 69 6.2.2 Procedure 70 6.2.2.1 Pour plate technique 70 6.2.2.2 Spread plate technique 71 6.2.2.3 Membrane filtration technique 71 6.3 ( revised ) Petrifilm ™ AOAC official methods for aerobic mesophilic bacteria in foods 73 6.3.1 Material required for analysis 73 6.3.2 Procedure 73 6.4 ( revised ) Plate count method APHA 13.61:2015 for aerobic psychrotrophic bacteria in foods 73 6.4.1 Material required for analysis 74 6.4.2 Procedure 74 6.5 ( new ) Plate count methods ISO 4833-1:2013 and ISO 4833-2:2013/Corr.1:2014 for aerobic mesophilic bacteria in foods 75 6.5.1 Material required for analysis 75 6.5.2 Procedure 75 6.6 (new) Plate count method BAM/FDA:2001 for aerobic mesophilic bacteria in foods 77 6.6.1 Material required for analysis 77 6.6.2 Procedure 77 6.7 References 79 7 Yeasts and molds 81 Revision history 81 7.1 Introduction 81 7.1.1 Yeasts and molds in foods 81 7.1.2 Comments on methods of analysis for total yeast and mold counts 82 7.1.3 Psychrotrophic fungi 82 7.1.4 Heat-resistant molds 84 7.1.5 Preservative-resistant yeasts (PRY) 84 7.1.5.1 Zigosaccharomyces bailii 85 7.1.5.2 Zygosaccharomyces bisporus 85 7.1.5.3 Schizosaccharomyces pombe 85