ISO 5552:1997

Meat and meat products — Detection and enumeration of Enterobacteriaceae without resuscitation — MPN technique and colony-count technique

Viande et produits à base de viande — Recherche et dénombrement des Enterobacteriaceae sans ressuscitation — Technique de NPP et technique de comptage de colonies

General Information

• Status: Withdrawn
• Publication Date: 03-Dec-1997
• Withdrawal Date: 03-Dec-1997
• ICS: 07.100.30 - Food microbiology
• Technical Committee: ISO/TC 34/SC 6 - Meat, poultry, fish, eggs and their products
• Drafting Committee: ISO/TC 34/SC 6 - Meat, poultry, fish, eggs and their products
• Current Stage: 9599 - Withdrawal of International Standard
• Completion Date: 16-Aug-2004

Relations

• Revised: ISO 21528-1:2004 - Microbiology of food and animal feeding stuffs — Horizontal methods for the detection and enumeration of Enterobacteriaceae — Part 1: Detection and enumeration by MPN technique with pre-enrichment
• Effective Date: 15-Apr-2008
• Revised: ISO 21528-2:2004 - Microbiology of food and animal feeding stuffs — Horizontal methods for the detection and enumeration of Enterobacteriaceae — Part 2: Colony-count method
• Effective Date: 15-Apr-2008
• Revises: ISO 5552:1979 - Meat and meat products — Detection and enumeration of Enterobacteriaceae (Reference methods)
• Effective Date: 15-Apr-2008

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 5552
Second edition
1997-12-01
Meat and meat products — Detection and
enumeration of Enterobacteriaceae without
resuscitation — MPN technique and colony-
count technique
Viande et produits à base de viande — Recherche et dénombrement des
Enterobacteriaceae sans ressuscitation — Technique de NPP et technique
de comptage de colonies
AA
Reference number
ISO 5552:1997(E)

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ISO 5552:1997(E)
Contents
1 Scope .1
2 Normative references .1
3 Definitions .2
4 Principle.2
4.1 Preparation of dilutions .2
4.2 Detection of Enterobacteriaceae in a specified quantity of sample .2
4.3 Enumeration of Enterobacteriaceae .2
5 Diluent, culture media and reagent.3
5.1 General.3
5.2 Diluent.3
5.3 Culture media.3
5.4 Oxidase reagent.6
6 Apparatus and glassware .6
7 Sampling.7
8 Preparation of test sample.7
9 Procedure .7
9.1 Test portion .7
9.2 Detection.7
©  ISO 1997
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced
or utilized in any form or by any means, electronic or mechanical, including photocopying and
microfilm, without permission in writing from the publisher.
International Organization for Standardization
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Internet [e-mail]
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Printed in Switzerland
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© ISO
ISO 5552:1997(E)
9.3 MPN technique. 7
9.4 Colony-count technique . 8
9.5 Confirmation . 8
10 Expression of results . 9
10.1 Detection of Enterobacteriaceae in a specified quantity of sample. 9
10.2 Calculation of the most probable number (MPN). 9
10.3 Calculation of colony count . 9
10.4 Precision. 11
11 Test report . 11
Annex A (normative) Determination of most probable number . 12
Annex B (normative) Confidence limits for the estimation of small numbers of colonies. 14
Annex C (informative) Bibliography . 15
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© ISO
ISO 5552:1997(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO
member bodies). The work of preparing International Standards is normally carried out through ISO technical
committees. Each member body interested in a subject for which a technical committee has been established has
the right to be represented on that committee. International organizations, governmental and non-governmental, in
liaison with ISO, also take part in the work. ISO collaborates closely with the International Electrotechnical
Commission (IEC) on all matters of electrotechnical standardization.
Draft International Standards adopted by the technical committees are circulated to the member bodies for voting.
Publication as an International Standard requires approval by at least 75 % of the member bodies casting a vote.
This second edition cancels and replaces the first edition (ISO 5552:1979), which has been technically revised.
International Standard ISO 5552 was prepared by Technical Committee ISO/TC 34, Agricultural food products,
Subcommittee SC 6, Meat and meat products.
Annexes A and B form an integral part of this International Standard. Annex C is for information only.
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© ISO
ISO 5552:1997(E)
Introduction
During a meeting in March 1996 of Subcommittees SC 5, Milk and milk products, SC 6, Meat and meat products,
and SC 9, Microbiology, it was recommended to put forward one horizontal standard for the detection and
enumeration of Enterobacteriaceae. This needs a revision of the two horizontal standards ISO 7402 and ISO 8523
into one standard, divided into three parts.
ISO 5552 will be withdrawn as soon as the combined horizontal method is published.
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INTERNATIONAL STANDARD  © ISO ISO 5552:1997(E)
Meat and meat products — Detection and enumeration of
Enterobacteriaceae without resuscitation — MPN technique and
colony-count technique
1 Scope
This International Standard specifies a method for the detection and enumeration of Enterobacteriaceae present in
all kinds of meat and meat products, including poultry. Enumeration is carried out as follows:
– by calculation of the most probable number (MPN) after incubation at 35 °C or 37 °C in liquid medium; or
– by counting colonies in a solid medium after incubation at 35 °C or 37 °C.
The temperature used is to be the subject of agreement between the parties concerned, and is to be stated in the
test report.
NOTE In the case of frozen foods, an incubation temperature of 30 °C is preferred when the aim of the enumeration is
technological.
For low numbers, the MPN method is preferable, otherwise the colony-count method is preferred.
This International Standard does not include resuscitation procedures and the results should not, therefore, be
related to criteria or specifications based on the assumption that resuscitation has been carried out.
A limitation on the applicability of this International Standard is imposed by susceptibility of the methods to a large
degree of variability. The methods should be used and the results interpreted in the light of the information given in
10.3.
2 Normative references
The following standards contain provisions which, through reference in this text, constitute provisions of this
International Standard. At the time of publication, the editions indicated were valid. All standards are subject to
revision, and parties to agreements based on this International Standard are encouraged to investigate the
possibility of applying the most recent editions of the standards indicated below. Members of IEC and ISO maintain
registers of currently valid International Standards.
ISO 3100-2:1988, Meat and meat products — Sampling and preparation of test samples — Part 2: Preparation of
test samples for microbiological examination.
ISO 6887:1983, Microbiology of food and animal feeding stuffs — General guidance for the preparation of dilutions
for microbiological examination.
ISO 7218:1996, Microbiology of food and animal feeding stuffs — General rules for microbiological examinations.
ISO 7402:1993,
Microbiology — General guidance for the enumeration of Enterobacteriaceae without resuscitation
— MPN technique and colony-count technique.
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© ISO
ISO 5552:1997(E)
3 Definitions
For the purposes of this International Standard, the following definitions apply.
3.1 Enterobacteriaceae
Microorganisms which ferment glucose and show a negative oxidase reaction when the test is carried out according
to the method specified.
3.2 detection of Enterobacteriaceae
Determination of the presence or absence of these bacteria, in a particular mass of product, when tests are carried
out in accordance with this International Standard.
3.3 count of Enterobacteriaceae
Number of Enterobacteriaceae found per millilitre or per gram of the test sample when the test is carried out
according to the method specified.
4 Principle
4.1 Preparation of dilutions
Preparation of decimal dilutions from the test sample.
4.2 Detection of Enterobacteriaceae in a specified quantity of sample
Introduction of 1 ml of the test sample if the product is liquid or of the initial suspension in the case of other products
(or of decimal dilutions of these), into a tube containing a selective enrichment broth.
Incubation of the tubes at 35 °C or 37 °C for 24 h, followed by streaking of the cultures onto violet red bile glucose
agar. After incubation of the streaked agar plates at 35 °C or 37 °C for 24 h, subjection of suspected colonies to
biochemical confirmation tests.
4.3 Enumeration of Enterobacteriaceae
4.3.1 Most probable number (MPN) technique
NOTE This technique is recommended when the number sought is expected to be in the range 1 to 100 per millilitre or per
gram of the test sample.
Inoculation of three tubes of double-strength medium with a specified quantity of test sample if the product is liquid,
or with a specified quantity of the initial suspension in the case of other products.
Inoculation of three tubes of single-strength medium with a specified quantity of the test sample if the product is
liquid, or with a specified quantity of the initial suspension in the case of other products. Then under the same
conditions, inoculation of three tubes of single-strength medium with the first decimal dilution of the test sample or of
the initial suspension.
Incubation of the tubes at 35 °C or 37 °C (as agreed) for 24 h.
From the number of confirmed positive tubes, calculation of the most probable number of Enterobacteriaceae per
millilitre or per gram of the test sample using the MPN table (see annex A).
4.3.2 Colony-count technique
NOTE This technique is recommended when the number sought is expected to be greater than 100 per millilitre or per
gram of the test sample.
Inoculation of violet red bile glucose agar contained in two Petri dishes (poured-plate technique) with a specified
quantity of the test sample if the initial product is liquid, or with a specified quantity of the initial suspension in the
case of other products. Covering with an overlayer of the same medium.
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Preparation of other pairs of plates under the same conditions, using decimal dilutions of the test sample or of the
initial suspension.
Incubation of the dishes at 35 °C or 37 °C (as agreed) for 24 h ± 2 h.
Calculation of the number of Enterobacteriaceae per millilitre or per gram of the test sample from the number of
confirmed typical colonies per dish.
5 Diluent, culture media and reagent
5.1 General
For current laboratory practice, see ISO 7218.
5.2 Diluent
See ISO 6887.
5.3 Culture media
5.3.1 Buffered brilliant green bile glucose broth (EE broth)
5.3.1.1 Composition
a) Double-strength medium b) Single-strength medium
Peptone 20,0 g 10,0 g
Glucose 10,0 g 5,0 g
Disodium hydrogen phosphate 12,90 g 6,45 g
(NaHPO)
2 4
Potassium dihydrogen phosphate 4,0 g 2,0 g
(KH PO )
2 4
Ox bile, dehydrated 40,0 g 20,0 g
Brilliant green 0,027 g 0,0135 g
Water 1 000 ml 1 000 ml
5.3.1.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by boiling. Adjust the pH, if necessary,
so that after boiling it is 7,2 at 25 °C. Do not heat the medium for longer than 30 min. Cool the medium rapidly.
Aseptically transfer 10 ml portions to sterile tubes or bottles (6.8).
Do not autoclave the medium.
The medium may be stored for up to 1 week at between 0 °C and 5 °C.
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© ISO
ISO 5552:1997(E)
5.3.2 Violet red bile glucose agar (VRBG)
5.3.2.1 Composition
Peptone 7,0 g
Yeast extract 3,0 g
Bile salts 1,5 g
Glucose 10,0 g
Sodium chloride 5,0 g
Neutral red 0,03 g
Crystal violet 0,002 g
1)
Agar in powder or flake form 8 g to 18 g
Water 1 000 ml
1) Depending on the gel strength of the agar.
5.3.2.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by boiling. The medium must not be
boiled for more than 2 min.
Adjust the pH, if necessary, so that after boiling it is 7,4 at 25 °C.
Transfer the culture medium to sterile tubes, flasks or bottles (6.8) of capacity not more than 500 ml.
Do not autoclave the medium.
Prepare this medium just before use (see 9.3.2 and 9.4.1).
5.3.2.3 Preparation of agar plates (required for detection and MPN technique, see 9.3.2)
Dispense immediately approximately 15 ml of the culture medium, cooled to approximately 47 °C in the water bath
(6.5), into Petri dishes (6.6) and allow to solidify.
Immediately before use, dry the plates, preferably with the lids off and the agar surface downwards, in the oven
(6.3) until the agar surface is dry.
If prepared in advance, the undried plates shall not be kept for longer than 4 days at between 0 °C and 5 °C.
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5.3.3 Glucose agar
5.3.3.1 Composition
Tryptone 10,0 g
Yeast extract 1,5 g
Glucose 10,0 g
Sodium chloride 5,0 g
Bromocresol purple 0,015 g
1)
Agar in powder or flake form 8 g to 18 g
Water 1 000 ml
1) Depending on the gel strength of the agar.
5.3.3.2 Preparation
Dissolve the components or the dehydrated complete medium in the water by heating, if necessary.
Adjust the pH, if necessary, so that after sterilization it is 7,0 at 25 °C.
Dispense the culture medium in quantities of 15 ml into tubes or flasks (6.8).
Sterilize for 15 min in an autoclave (6.1) set at 121 °C.
Leave the tubes or flasks in a vertical position.
The medium may be stored for up to 1 week at between 0 °C and 5 °C.
Just before use, heat in boiling water or flowing steam for 15 min, then cool rapidly to the incubation temperature.
5.3.4 Nutrient agar
5.3.4.1 Composition
Beef extract 3,0 g
Peptone 5,0 g
1)
Agar in powder or flake form 8 g to 18 g
Water 1 000 ml
1) Depending on the gel strength of the agar.
5.3.4.2 Preparation
Dissolve the components or dehydrated complete medium in the water by heating, if necessary.
Adjust the pH, if necessary, so that after sterilization it is 7,0 at 25 °C.
Transfer the culture medium to tubes, bottles or flasks (6.8) of capacity not more than 500 ml.
Sterilize for 15 min in the autoclave (6.1) set at 121 °C.
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5.3.4.3 Preparation of agar plates (see 9.4.1)
Transfer immediately about 15 ml of the culture medium, melted and cooled to approximately 47 °C, to Petri dishes
(6.6) and allow to solidify.
Just before use, dry the plates, preferably with the lids off and the agar surface downwards, in the oven (6.3), until
the agar surface is dry.
If prepared in advance, the undried plates may be stored for up to 2 weeks at between 0 °C and 5 °C.
5.4 Oxidase reagent
5.4.1 Composition
1,0 g
N,N,N ¢,N ¢-Tetramethyl-p-phenylenediamine
dihydrochloride
Water 100 ml
5.4.2 Preparation
Dissolve the reagent in the cold water. The reagent shall be prepared immediately prior to use.
6 Apparatus and glassware
Usual micr
...