SIST EN ISO 7932:1998

SLOVENSKI STANDARD
SIST EN ISO 7932:1998
01-junij-1998
Mikrobiologija - Splošno navodilo za ugotavljanje števila Bacillus cereus - Štetje
kolonij pri 30 °C (ISO 7932:1993)
Microbiology - General guidance for the enumeration of Bacillus cereus - Colony-count
technique at 30 °C (ISO 7932:1993, including Technical Corrigendum 1:1997)
Mikrobiologie - Allgemeine Anleitung zur Zählung von Bacillus cereus -
Koloniezählverfahren bei 30 °C (ISO 7932:1993, einschließlich Technische Korrektur
1:1997)
Microbiologie - Directives générales pour le dénombrement de Bacillus cereus - Méthode
par comptage des colonies a 30 °C (ISO 7932:1993, Rectificatif Technique 1:1997
inclus)
Ta slovenski standard je istoveten z: EN ISO 7932:1997
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 7932:1998 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 7932:1998

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SIST EN ISO 7932:1998

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SIST EN ISO 7932:1998

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SIST EN ISO 7932:1998
ISO
INTERNATIONAL
7932
STANDARD
Second edition
1993-1 O-l 5
- General guidance for the
Microbiology
enumeration of Bacillus cereus -
Colony-count technique at 30 “C
Microbiologie - Directives g&&ales pour Ie denombrement de Bacillus
- Methode par camptage des colonies 6 30 “C
cereus
Reference number
ISO 7932:1993(E)

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SIST EN ISO 7932:1998
ISO 7932:1993(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. Esch member body interested in a subject for
which a technical committee has been established has the iight 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.
International Standard ISO 7932 was prepared by Technical Committee
ISODC 34, Agricultural food products, Sub-Committee SC 9,
Microbiology.
This second edition cancels and replaces the first edition
(ISO 7932:1987), which has been technically revised.
Annex A forms an integral part of this International Standard. Annex B is
for information only.
0 ISO 1993
All rights reserved. No part of this publication may be reproduced or utilized in any form or
by any means, electronie or mechanical, including photocopying and microfilm, without per-
mission in writing from the publisher.
International Organization for Standardization
Case Postale 56 l CH-l 211 Geneve 20 l Switzerland
Printed in Switzerland

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SIST EN ISO 7932:1998
Introduction
0.1 This International Standard is intended to provide general guidance
for the microbiological examination of food products not dealt with by ex-
isting International Standards and to be taken into account by organizations
preparing microbiological methods of test for application to foods or to
animal feeding stuffs. Because of the large variety of products within this
field of application, these guidelines may not be appropriate in every detail
for certain products and for some other products it may be necessary to
use different methods. Nevertheless, it is hoped that in all cases every
attempt will be made to apply the guidelines provided as far as possible
and that deviations from them will only be made if absolutely necessary
for technical reasons.
When this International Standard is next reviewed, account will be taken
of all information then available regarding the extent to which the guide-
lines have been followed and the reasons for deviation from them in the
case of particular products. ’
The harmonization of test methods cannot be immediate and, for certain
groups of products, International Standards and/or national Standards may
already exist that do not comply with the guidelines. In cases where
International Standards already exist for the product to be tested, they
should be followed, but it is hoped that when such Standards are reviewed
they will be changed to comply with this International Standard so that
eventually the only remaining departures from these guidelines will be
those necessary for weil-established technical reasons.
0.2 For the purposes of a practicable test method, the definition of
Bacillus cereus given in clause 3 and used as a basis for the procedure
does not exclusively describe strains of 5. cereus. In particular, the con-
firmatory tests are inadequate to distinguish between B. cereus and other
closely related but less commonly encountered bacillus species such as
B. anthracis, 6. thuringiensis, 6. mycoides, etc.
0.3 lt appears that the spores of many, if not most, strains of B. cereus
germinate readily on the surface of culture media employed for enumer-
ation. In most cases there does not seem to be a need for heat shock
treatment to provoke germination. Sometimes a heat shock procedure is
desirable, for example for Spore counts or to inhibit growth of vegetative
bacterial cells. In such cases, treatment of 15 min. at 70 “C is rec-
ommended.
. . .
Ill

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SIST EN ISO 7932:1998
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SIST EN ISO 7932:1998
ISO 7932:1993(E)
INTERNATIONAL STANDARD
- General guidance for the enumeration
Microbiology
of Bacillus cereus - Colony-count technique at 30 “C
4 Principle
1 Scope
This International Standard gives general guidance for
Surface plating, on a solid selective culture me-
4.1
the enumeration of viable Bacillus cereus in products
dium contained in Petri dishes, of a specified quantity
intended for human consumption or animal feeding
of the test Sample if the initial product is liquid, or of
stuffs by means of the colony-count technique at
a specified quantity of an initial Suspension in the case
30 “C.
of other products.
Preparation of other plates, under the Same con-
2 Normative references
ditions, using decimal dilutions of the test Sample or
of the initial Suspension.
The following Standards contain provisions which,
through reference in this text, constitute provisions
of this International Standard. At the time of publi-
4.2 Aerobic incubation of the plates at 30 “C for
cation, the editions indicated were valid. All Standards
18hto48h.
are subject to revision, and Parties to agreements
based on this International Standard are encouraged
Calculation of the number of 5. cereus per gram
4.3
to investigate the possibility of applying the most re-
or per millilitre of Sample from the number of con-
cent editions of the Standards indicated below.
firmed colonies obtained on plates at dilution levels
Members of IEC and ISO maintain registers of cur-
chosen so as to give a significant result, and confir-
rently valid International Standards.
mation according to the tests specified.
ISO 6887: 1983, Microbiology - General guidance for
the preparation of dilutions for microbiological exam-
5 Diktion fluid, culture media and
ina tion.
reagents
ISO 72 18: 1985, Microbiology - General guidance for
microbiological examina tions. 5.1 General
For current laboratoty practice, see ISO 7218.
3 Definition
Commercially prepared ready-to-use reagents
NOTE 1
For the purposes of this International Standard, the
may be used.
following definition applies.
5.2 Dilution fluid
3.1 Bacillus cereus: A microorganism that forms
colonies on the surface of a selective culture medium
See ISO 6887 and any specific Standard dealing with
and which gives positive confirmation reactions under
the product to be examined.
the conditions specified in this International Standard.
1

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SIST EN ISO 7932:1998
ISO 7932:1993(E)
Shell to the other. Put the yolks into a sterile measur-
5.3 Agar medium (see reference [l] in
ing cylinder and add four Parts by volume of sterile
annex B)
water. Transfer aseptically into a sterile flask (6.7) and
mix vigorously.
5.3.1 Base medium
Heat the mixture for 2 h in a water bath (6.4) set at
45 “C. Then leave for 18 h to 24 h at 0 “C to 5 “C to
5.3.1 .l Composition
allow a precipitate to form.
Collect the supernatant emulsion aseptically.
Beef extract 1,o g
The emulsion may be stored at 0 “C to 5 “C for not
Peptone
no g
longer than 72 h.
D-Mannit01
no g
Sodium chloride (NaCI)
no g
5.3.4 Complete medium (MYP agar)
Phenol red 0,025 g
Agar 12to18g’)
5.3.4.1 Composition
Water 900 ml
90 ml
Base medium (5.3.1)
1) Depending on the gel strength of the agar.
1,O ml
Polymyxin B Solution (5.3.2)
IO,0 ml
Egg yolk emulsion (5.3.3)
5.3.1.2 Preparation
Dissolve the components or the dehydrated complete
5.3.4.2 Preparation
medium in the water, by heating if necessary.
Adjust the pH, if necessary, so that after sterilization
Melt the base medium and cool it in a water bath
it is 7,2 at 25 “C.
(6.4) set at 50 “C.
Dispense the medium in quantities of 90 ml into
Add the other liquids, mixing weil after each addition.
flasks (6.7) of appropriate capacity.
Cool the complete medium in a water bath (6.4) at
Sterilize for 15 min in an autoclave (6.1) set at
45 “C.
121 “C.
5.3.5 Preparation of agar plates for enumeration
5.3.2 Polymyxin B solution
Pour 15 ml to 20 ml portions of the completeImedium
(5.3.4) into sterile Petri dishes (6.8) and allow to
5.3.2.1 Composition
solidify.
The plates may be stored Prior to drying at between
106 I.U.
0 “C and 5 “C for up to 4 days.
100 ml
Immediately before use, dry the plates, preferably
with the lids off and the agar surface downwards, in
a drying cabinet or incubator (6.2) set between 37 “C
and 55 “C until the agar surface is dry.
5.3.2.2 Preparation
Dissolve the polymyxin B sulfate in the water. Sterilize
Preparation of agar plates for isolation
5.3.6
by f iltration.
Pour portions of about 15 ml of the base medium
5.3.3 Egg yolk emulsion
(5.3.1), previously melted, cooled and kept in a water
bath (6.4) at 45 “C, into sterile Petri dishes (6.8) and
Use fresh hens’ eggs with their shells intact. Wash
allow to solidify.
the eggs, using a brush, in liquid detergent. Rinse
Immediately before use, dry the plates, preferably
under running water, dip in 95 % (WV) ethanol for
with the lids off and the agar surface downwards, in
30 s and dry. Using aseptic procedures, break each
a drying cabinet or incubator (6.2) set between 37 “C
egg and separate the yolk from the white by repeat-
and 55 “C until the agar surface is dry.
edly transferring the yolk from one half of the egg
2

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SIST EN ISO 7932:1998
ISO 7932:1993(E)
5.4 Glucose agar Sterilize for 15 min in an autoclave (6.1) set at
121 “C.
5.4.1 Composition
5.6 Voges-Proskauer (VP) test reagents
Tryptone
IO,0 g
5.6.1 a-Naphthol Solution
Yeast extract
1,5 g
5.6.1 .l Composition
Glucose
IO,0 g
Sodium chloride (NaCI)
5,O g
Bromocresol purple 0,015 g a-Naphthol
5,O g
Agar
12to18g’) 100 ml
Ethanol, 95 % (VW)
Water 1 000 ml
5.6.1.2 Preparation
1) Depending on the gel strength of the agar.
Dissolve the a-naphthol in the ethanol.
5.4.2 Preparation
Store at between 0 “C and 5 “C in a hermetically
sealed brown culture flask.
Dissolve the components or the dehydrated complete
medium in the water, by heating if necessary.
5.6.2 Potassium hydroxide Solution
Adjust the pH, if necessary, so that after sterilization
5.6.2.1 Composition
it is 7,0 at 25 “C.
Dispense the culture medium in quantities of 15 ml
into test tubes (6.7).
Potassium hydroxide (KOH)
40 g
Water 100 ml
Sterilize for 15 min in an autoclave (6.1) set at
121 “C.
Immediately before use, melt the medium in a boiling
5.6.2.2 Preparation
water bath or flowing steam for IO min, then cool
rapidly to about 30 “C, keeping the tubes in a vertical
Dissolve the potassium hydroxide slowly in the water.
Position.
5.6.3 Creatine, crystalline.
5.5 Voges-Proskauer (VP) medium
5.7 Nitrate medium
5.5.1 Composition
5.7.1 Composition
Peptone
7,o g
Glucose
5,o g
Peptone
5,o g
Dipotassium hydrogen
5,O g
Beef extract
3,o g
Phosphate (K,HPO,)
Potassium nitrate (KNO,)
w g
Sodium chloride (NaCI)
50 g
Water 1 000 ml
Water 1 000 ml
5.5.2 Preparation 5.7.2 Preparation
Dissolve the components or the dehydrated complete Dissolve the components or the dehydrated complete
medium in the water. medium in the water.
Adjust the pH, if necessary, so that after sterilization
Adjust the pH, if necessary, so that after sterilization
it is 7,0 at 25 “C.
it is 7,0 at 25 “C.
Dispense the medium in quantities of 5 ml into test Dispense the medium in quantities of 5 ml into test
tubes (6.7). tubes (6.7).

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SIST EN ISO 7932:1998
ISO 7932:1993(E)
Sterilize for 15 min in an autoclave (6.1) set at
Discard immediately any unused complete reagent.
121 “C.
5.9 Zinc dust
5.8 Nitrite reagent
6 Apparatus and glassware
5.8.1 5-Amino-2-naphthalenesulfonic acid
(5-2 ANSA) Solution
NOTE 3 Disposable apparatus is an acceptable alterna-
tive to reusable glassware if it has similar specifications.
5.8.1 .l Composition
Usual microbiological laboratory equipment and, in
particular, the following.
5-2 ANSA
OJ g
Acetic acid (2,6 mol/l) 100 ml
6.1 Apparatus for dry sterilization (oven) or wet
sterilization (autoclave)
See ISO 7218.
5.8.1.2 Preparation
6.2 Drying cabinet or incubator, ventilated by con-
Dissolve the 5-2 ANSA in the acetic acid and filter
vection, for drying the agar plates, capable of operat-
through Paper?
ing between 37
...