INTERNATIONAL ISO

STANDARD 16654

First edition
2001-05-01

AMENDMENT 2
2023-01

Microbiology of food and animal

feeding stuffs — Horizontal method
for the detection of Escherichia coli
O157
AMENDMENT 2: Inclusion of
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STANDARD testing of all culture media
and reagents
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Microbiologie des aliments — Méthode horizontale pour la recherche
des Escherichia coli O157
ISO 16654:2001/Amd 2:2023
AMENDEMENT 2: Inclusion d'essais de performance de tous les
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milieux de culture et réactifs
16654-2001-amd-2-2023

Reference number
ISO 16654:2001/Amd.2:2023(E)

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Foreword
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the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
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www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee TC 34, Food products, Subcommittee SC 9,
Microbiology, in collaboration with the European Committee for Standardization (CEN) Technical
ISO 16654:2001/Amd 2:2023
Committee CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical
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cooperation between ISO and CEN (Vienna Agreement).
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Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

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 ISO 16654:2001/Amd.2:2023(E)

Microbiology of food and animal feeding stuffs —

Horizontal method for the detection of Escherichia coli
O157
AMENDMENT 2: Inclusion of performance testing of all culture
media and reagents

Clause 2
Add the following normative reference:
ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and
performance testing of culture media

Clause 5, first sentence

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Replace the text with the following:

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For current laboratory practice, see ISO 7218 and ISO 11133.
The general specifications of ISO 11133 are applicable to the preparation and performance testing of
the culture media and reagents ISO described in this clause.
16654:2001/Amd If culture media or reagents are prepared
2:2023
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dehydrated complete media/reagents or if ready-to-use media/reagents are used, follow the
manufacturer’s instructions regarding preparation, storage conditions, expiry date and use.
16654-2001-amd-2-2023
The shelf lives of the media and reagents indicated in this clause have been determined in previous
studies. The user shall verify these under their own storage conditions (in accordance with ISO 11133).
Performance testing of culture media and reagents is described in 5.11.

Clause 5, after 5.10

Add the following text as a new subclause.
5.11 Performance testing
The definitions of productivity, selectivity and specificity are specified in ISO 11133. For performance
testing of culture media, follow the procedures in accordance with ISO 11133 and Table 1.

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ISO 16654:2001/Amd.2:2023(E)

Table 1 — Performance testing for the quality assurance of the culture media and reagents
WDCM Method of
Medium Function Incubation Control strains Criteriab
numbersa control
00014 > 10 characteristic
(non- colonies on CT-SMAC or
Escherichia coli O157:H7
toxigenic other medium of choice
6 h/
Productivity strain) Qualitative (characteristic
mTSB + (41,5 ± 1) °C
appearance of the
Nc + Staphylococcus 00032 colonies should refer to
aureus subsp. aureuse or 00034 the different media used)
(21 ± 3) h/ Staphylococcus 00032
Selectivity Qualitative Total inhibition (0) on TSA
(41,5 ± 1) °C aureus subsp. aureuse or 00034
Good growth (2) of
00014
transparent colonies
(non-
Productivity Escherichia coli O157:H7 Qualitative with a pale yellowish-
toxigenic
brown appearance and
strain)
a diameter ~1 mm
(21 ± 3) h/
CT-SMACc
(37 ± 1) °C Staphylococcus 00032
Qualitative Total inhibition (0)
aureus subsp. aureusd or 00034
Selectivity Partial inhibition (1)
00012
Escherichia colid Qualitative with growth of some pink
or 00013
colonies
00012
or 00013
Nutrient (21 ± 3) h/ or 00014
agar
Productivity
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(37 ± 1) °C
Escherichia colid
(non-
toxigenic
Qualitative Good growth (2)

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strain)
(21 ± 3) h/ 00012
(37 ± 1) °C, 00013
then Kovac’s 00014
reagent is ISO 16654:2001/Amd (non-2:2023 Positive reaction:
added and Escherichia colid toxigenic Qualitative Formation of a red ring
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allow to strain) within 10 min
Tryptone stand at room 16654-2001-amd-2-2023
00090
/ tryp- temperature 00179
tophan Detection of for 10 min
medium indole
Klebsiella aerogenes
with formation
(formerly Enterobacter 00175
indole from
aerogenes)d
reagent tryptophan
(Kovac’s Citrobacter freundiid 00006 Negative reaction:
reagent) (21 ± 3) h/ Salmonella enterica Qualitative Yellow/brown ring within
(37 ± 1) °C serovar 00031
Typhimuriumd,e 10 min

Salmonella enterica
serovar 00030
Enteritidisd,e
a Refer to the reference strain catalogue on http://​w ww​.wfcc​.info for information on culture collection strain numbers and contact
details; WDCM: World Data Centre for Microorganisms.
b Growth is categorized as 0: no growth; 1: weak growth (partial inhibition); 2: good growth (see ISO 11133).
c mTSB +N: Modified Tryptone soya broth supplemented with 20 mg/l of novobiocin; CT-SMAC: Cefixime tellurite sorbitol MacConkey
agar.
d Strain free of choice; one of the strains has to be used as a minimum. For the confirmation medium and reagent, the user may choose
any of the strains cited for positive and negative reactions (see ISO 11133).
e Some national restrictions and directions require the use of a different serovar. Make reference to national requirements relating to
the choice of Salmonella serovar(s).

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