SIST EN ISO 11290-1:1997
(Main)Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of Listeria monocytogenes - Part 1: Detection method (ISO 11290-1:1996)
Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of Listeria monocytogenes - Part 1: Detection method (ISO 11290-1:1996)
Specifies a horizontal method for the detection of Listeria monocytogenes. Applicable to products intended for human consumption or as animal feeding stuffs.
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren für den Nachweis und die Zählung von Listeria monocytogenes - Teil 1: Nachweisverfahren (ISO 11290-1:1996)
Dieser Teil von ISO 11290 legt ein horizontales Verfahren für den Nachweis von Listeria monocytogenes fest. Vorbehaltlich der in der Einleitung angegebenen Einschränkungen, ist dieser Teil von ISO 11290 auf Lebensmittel und Futtermittel anwendbar.
Microbiologie des aliments - Méthode horizontale pour la recherche et le dénombrement de Listeria monocytogenes - Partie 1: Méthode de recherche (ISO 11290-1:1996)
La présente partie de l'ISO 11290 prescrit une méthode horizontale pour la recherche de Listeria monocytogenes. La présente partie de l'ISO 11290 est applicable aux produits destinés à la consommation humaine ou à l'alimentation animale, avec les quelques restrictions signalées dans l'introduction.
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje prisotnosti in števila Listeria monocytogenes - 1. del: Metoda za ugotavljanje prisotnosti (ISO 11290-1:1996)
General Information
Relations
Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 11290-1:1997
01-december-1997
Mikrobiologija živil in krme - Horizontalna metoda za ugotavljanje prisotnosti in
števila Listeria monocytogenes - 1. del: Metoda za ugotavljanje prisotnosti (ISO
11290-1:1996)
Microbiology of food and animal feeding stuffs - Horizontal method for the detection and
enumeration of Listeria monocytogenes - Part 1: Detection method (ISO 11290-1:1996)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren für den
Nachweis und die Zählung von Listeria monocytogenes - Teil 1: Nachweisverfahren (ISO
11290-1:1996)
Microbiologie des aliments - Méthode horizontale pour la recherche et le dénombrement
de Listeria monocytogenes - Partie 1: Méthode de recherche (ISO 11290-1:1996)
Ta slovenski standard je istoveten z: EN ISO 11290-1:1996
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 11290-1:1997 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 11290-1:1997
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SIST EN ISO 11290-1:1997
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SIST EN ISO 11290-1:1997
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SIST EN ISO 11290-1:1997
INTERNATIONAL
IS0
STANDARD 11290-I
First edition
1996-l 2-15
Microbiology of food and animal feeding
stuffs - Horizontal method for the
detection and enumeration of Listeria
monocytogenes -
Part 1:
Detection method
Microbiologic des aliments - Mkthode horizontale pour la recherche et le
dknombrement de Listeria monocytogenes -
Partie I: Mkthode de recherche
Reference number
IS0 11290-1:1996(E)
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SIST EN ISO 11290-1:1997
IS011290=1:1996(E)
Page
Contents
1
1 Scope .
1
2 Normative references .
1
3 Definitions .
2
4 Principle .
2
5 Culture media and reagents .
.............................................................. 3
6 Apparatus and glassware
3
7 Sampling .
3
8 Preparation of test sample .
3
......................................................................................
9 Procedure
7
.....................................................................
10 Expression of results
7
11 Test report .
Annexes
8
....................................................................
A Diagram of procedure
9
........
B Composition and preparation of culture media and reagents
16
Henry illumination test .
C
0 IS0 1996
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
Case Postale 56 l CH-1211 Geneve 20 l Switzerland
Printed in Switzerland
ii
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SIST EN ISO 11290-1:1997
@ IS0 ISO11290-1:1996(E)
Foreword
IS0 (the lnternational Organization for Standardization) is a worldwide fed-
eration of national standards bodies (IS0 member bodies). The work of
preparing International Standards is normally carried out through IS0
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 liason with ISO, also take part in the work. IS0 collab-
orates closely with the International Electrotechnical Commission (IEC) on
all matters of electrotechnical standardization.
Draft International Standards adopted by the technical committees are cir-
culated 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 IS0 11290-I was prepared by Technical Committee
ISO/TC 34, Agricultural food products, Subcommittee SC 9, Microbiology.
IS0 11290 consists of the following parts, under the general title Micro-
biology of food and animal feeding stuffs - Horizontal method for the de-
tection and enumeration of Listeria monocytogenes:
- Part I: Detection method
- Part 2: Enumeration method
Annexes A and B form an integral part of this part of IS0 11290. Annex C
is for information only.
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SIST EN ISO 11290-1:1997
IS011290=1:1996(E) 0 IS0
Introduction
Because of the large variety of food and feed products, this horizontal
method may not be appropriate in every detail for certain products for
which it may be necessary to use different or specific methods. Neverthe-
less, in all cases, every attempt should be made to apply this horizontal
method as far as possible and that deviations from this will only be made if
absolutely necessary for justified technical reasons.
When this part of IS0 11290 is next reviewed, account will be taken of all
information then available regarding the extent to which this horizontal
method has been followed and the reasons for deviations from it 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 this horizontal method. It is hoped that
when such standards are reviewed they will be changed to comply with this
part of IS0 11290 so that eventually the only remaining departures from
this horizontal method will be those necessary for well-established techni-
cal reasons.
IV
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SIST EN ISO 11290-1:1997
IS0 11290-I : 1996(E)
INTERNATIONAL STANDARD @ IS0
Microbiology of food and animal feeding stuffs - Horizontal
method for the detection and enumeration of Listeria
monocytogenes -
Part 1:
Detection method
In order to safeguard the health of laboratory personnel, it is strongly recommended that
WARNING -
tests for detecting Listeria monocytogenes are undertaken in properly equipped laboratories, under the
control of a skilled microbiologist, and that great care is taken in the disposal of all incubated materials. In
particular, it is strongly recommended that pregnant personnel do not manipulate cultures of L. monocyto-
genes.
IS0 7218: 1996, Microbiology of food and animal feed-
1 Scope
ing stuffs - General rules for microbiological exam-
ina tions.
This part of IS0 11290 specifies a horizontal method
for the detection of Listeria monocytogenes.
Subject to the limitations discussed in the introduction,
this part of IS0 11290 is applicable to products in-
tended for human consumption or animal feeding.
3 Definitions
For the purposes of this part of IS0 11290, the follow-
2 Normative references
ing definitions apply.
The following standards contain provisions which,
through reference in this text, constitute provisions of
3.1 Lis teria monocytogenes: Microorganisms
this part of IS0 11290. At the time of publication, the
which form typical colonies on solid selective media
editions indicated were valid. All standards are subject
and which display the morphological, physiological
to revision, and parties to agreements based on this
and biochemical characteristics described when tests
part of IS0 11290 are encouraged to investigate the
are carried out in accordance with this part of
possibility of applying the most recent editions of the
IS0 11290.
standards indicated below. Members of IEC and IS0
maintain registers of currently valid International Stan-
dards.
3.2 detection of Listeria monocytogenes: Deter-
mination of the presence or absence of these micro-
organisms, in a given mass or volume of product,
IS0 6887:1983, Microbiology - General guidance for
when tests are carried out in accordance with this part
the preparation of dilutions for microbiological exam-
of IS0 11290.
ina tion.
1
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SIST EN ISO 11290-1:1997
IS0 11290-l :1996(E)
5 Culture media and reagents
4 Principle
Within the limits of this part of IS0 11290, the detec-
5.1 General
tion of L. monocytogenes necessitates four successive
stages (see annex A for a flowchart).
For current laboratory practice, see IS0 7218.
NOTE 1 Listeria spp. may be present in small numbers
NOTE 2 Because of the large number of culture media and
and are often accompanied by considerably larger numbers
reagents, it has been considered preferable, for clarity of
of other genera, therefore selective enrichment is neces-
the text, to give their composition and preparation in
sary. It is also necessary to detect injured Listeria spp. and
annex B.
the primary selective enrichment medium, with reduced in-
hibitor concentration, fulfils at least part of this function.
5.2 Selective primary enrichment medium:
Fraser broth with reduced concentration of
4.1 Primary enrichment in a selective liquid
selective agents (half Fraser broth)
enrichment medium with reduced
concentration of selective agents (half Fraser
See clause B.l .
broth)
5.3 Selective secondary enrichment medium
inoculation of a selective primary enrichment medium
with full concentration of selective agents
containing one volume of lithium chloride and half a
(Fraser broth)
volume of both acriflavine and nalidixic acid (half
Fraser broth), which is also used as a dilution fluid for
See clause B.2.
the test portion (9.1).
Incubation of the test portion at 30 “C for 24 h.
5.4 Selective solid plating-out media
5.4.1 First medium: Oxford agar
4.2 Secondary enrichment with a selective
liquid enrichment medium with full
See clause B.3.
concentration of selective agents (Fraser
broth)
5.4.2 Second medium: PALCAM agar
Inoculation of full-strength secondary liquid enrichment
medium (Fraser broth) with a culture obtained from
See clause B.4.
4.1.
5.5 Solid culture medium: Tryptone soya
Incubation of the Fraser broth at 35 OC or 37 OC for
yeast extract agar (TSYEA)
4% h.
See clause B.5.
4.3 Plating out and identification
5.6 Liquid culture medium: Tryptone soya
From the cultures obtained in 4.1 and in 4.2, plating
yeast extract broth (TSYEB)
out on the two selective solid media:
a) Oxford agar; See clause B.6.
b) PALCAM agar.
5.7 Sheep blood agar
Incubation at 30 OC, 35 OC or 37 “C and examination
after 24 h and, if necessary, after 48 h to check for the See clause B.7.
presence of characteristic colonies which are pre-
sumed to be L. monocyfogenes.
5.8 Carbohydrate utilization broth
(rhamnose and xylose)
4.4 Confirmation
See clause B.8.
Subculturing of the colonies of presumptive L. mono-
cytogenes, plated out as described in 4.3, and con- 5.9 Motility agar (optional)
firmation by means of appropriate morphological,
See clause B.9.
physiological and biochemical tests.
2
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SIST EN ISO 11290-1:1997
IS0 11290-l : 1996(E)
@ IS0
6.9 Total-delivery graduated pipettes, of nominal
5.10 CAMP (Christie, Atkins, Munch-
capacities 10 ml and 1 ml, graduated respectively in
Petersen) medium and test strains
05 ml and 0,l ml divisions.
See clause B.10.
6.10 Petri dishes, of diameter 90 mm to 100 mm.
5.11 Hydrogen peroxide solution
6.11 Jars, suitable for microaerobic incubation
(opt ion al).
See clause B.11.
6.12 Gas mixture (optional), of specified compo-
sition for microaerobic incubation:
5.12 Phosphate-buffered saline (PBS)
5%to12%COZ,5%to15%0,,and75%N,.
See clause B.12.
6.13 Equipment for the Henry illumination test
(optional).
6 Apparatus and glassware
See annex C.
Usual microbiological equipment (see IS0 7218) and,
in particular, the following. 6.14 Microscope, preferably with phase-contrast,
and with slides and coverslips.
6.1 Apparatus for dry sterilization (oven) or wet
sterilization (autoclave)
7 Sampling
See IS0 7218.
It is important that the laboratory receive a sample
6.2 Drying cabinet or incubator, capable of being
which is truly representative and has not been dam-
maintained at between 25 OC & 1 OC and 50 OC + 1 OC.
aged or changed during transport or storage.
6.3 Incubators, for maintaining the inoculated me- Sampling is not part of the method specified in this
dia, plates and tubes within the following temperature part of IS0 11290. If there is no specific International
ranges: Standard dealing with sampling of the product con-
cerned, it is recommended that the parties concerned
a) 25 OC + 1 “C;
come to an agreement on this subject.
b) 30 “C & 1 “C; and
c) 35”Cf.l oCor37°C&1 OC.
8 Preparation of test sample
6.4 Water bath, capable of being maintained at
47 “C I!I 2 “C.
Prepare the test sample in accordance with the
specific International Standard appropriate to the
6.5 Loops, of platinum/iridium or nickel/chromium,
product concerned. If there is no specific International
approximately 3 mm in diameter, and wires of the
Standard, it is recommended that the parties con-
same material, or hockey-stick-shaped glass rods
cerned come to an agreement on this subject.
or single-use loops.
6.6 pH-meter, capable of being read to the nearest
0,Ol pH unit at 25 OC, enabling measurements to be
9 Procedure
made which are accurate to + 0,l pH unit.
9.1 Test portion and initial suspension
6.7 Test tubes or flasks, of appropriate capacity, for
sterilization and storage of *culture media and incu-
See IS0 6887 and any specific International Standard
bation of liquid media.
appropriate to the product concerned.
6.8 Measuring cylinders, of capacity 50 ml to
For preparation of the initial suspension, use as di-
1 000 ml, for preparation of dilutions and complete
lution fluid the selective primary enrichment medium
media.
specified in 5.2.
3
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SIST EN ISO 11290-1:1997
IS0 11290-l :1996(E) 0 IS0
35 “C or 37 “C because Listeria spp. tend to develop at the
In general, to prepare the initial suspension, add a test
same time as the supplementary flora. In all cases, the in-
portion of x g or x ml to 9x ml or 9x g of the selective
cubation temperature of the agar should be agreed upon
primary enrichment medium (5.2), to obtain a ratio of
between the parties concerned and recorded in the test re-
test portion to selective primary enrichment medium of
port.
l/IO (mass to volume or volume to volume).
9.4.4 After incubation for 24 h and for an additional
18 h to 24 h (if growth is slight or if no colonies are ob-
9.2 Primary enrichment
served after 24 h of incubation), examine the dishes
(9.4.3) for the presence of colonies presumed to be
Incubate [6.3 b)] the initial suspension, prepared in ac-
Listeaia spp.
cordance with 9.1, at 30 OC for 24 h + 2 h.
9.4.4.1 Oxford agar: Typical colonies of Listeria spp.
coloration may develop during the incu-
NOTE 3 A black
bation. grown on Oxford agar for 24 h are small (1 mm) grey-
ish colonies surrounded by black halos. After 48 h
colonies become darker, with a possible greenish
9.3 Secondary enrichment
sheen, and are about 2 mm in diameter, with black
halos and sunken centres.
9.3.1 After incubation of the initial suspension
(primary enrichment) for 24 h & 2 h (9.2), transfer
9.4.4.2 PALCAM agar: For plates incubated micro-
0,l ml of the culture obtained in 9.2 (regardless of its
aerobically, after incubation expose the PALCAM agar
colour) to a tube (6.7) containing 10 ml of secor
dary
plates to air for 1 h to allow the medium to regain its
enrichment medium (Fraser broth) (5.3).
pink to purple colour. After 24 h Listeria spp. grow as
small or very small greyish green or olive green colo-
9.3.2 Incubate the inoculated medium (9.3.1 for
nies, I,5 mm to 2 mm in diameter, sometimes with
48 h + 2 h at 35 OC or 37 “C.
black centres, but always with black halos. After 48 h
Listeria spp. appear in the form of green colonies
NOTE 4 The temperature of the inoculated medium s lould
about I,5 mm to 2 mm in diameter, with a central de-
between the parties concerned and re-
be agreed upon
pression and surrounded by a black halo.
corded in the test report.
9.4 Plating out and identification
9.5 Confirmation of LiSferia spp.
9.4.1 From the primary enrichment culture incubated
9.5.1 Selection of colonies for confirmation
for 24 h rfI 2 h at 30 OC (9.2), take, by means of a loop
or glass rod (6.5), a portion of the culture and inocu-
9.5.1.1 For confirmation, take from each plate of
late the surface of the first selective plating medium
each selective medium (see 9.4.4.1 and 9.4.4.2), five
(Oxford agar) (5.4.1) so that well-separated colonies
colonies presumed to be Listeria spp.
are obtained.
If on one plate there are fewer than five presumed
Proceed in the same way with the second selective
colonies, take for confirmation all of them.
plating-out medium (PALCAM agar) (5.4.2).
9.5.1.2 Streak the selected colonies onto the surface
NOTE 5 This plating out is carried out regardless of the
of pre-dried plates of tryptone soya yeast extract agar
colour of the medium.
(TSYEA) (5.5) in a manner which will allow well-
separated colonies to develop.
9.4.2 From the secondary enrichment medium incu-
bated for 48 h + 2 h at 35 OC or 37 OC (9.3.2), repeat
Place the plates in the incubator 16.3 c)] set at 35 OC
the procedure described in 9.4.1 with the two selective
or 37 “C for 18 h to 24 h or until growth is satisfactory.
plating-out media.
NOTE 7 The temperature of the inoculated medium should
9.4.3 Invert the dishes obtained in 9.4.1 and 9.4.2
be agreed upon between the parties concerned and re-
and place them in an incubator (6.3) set at 30 OC,
corded in the test report.
35 OC or 37 OC. PALCAM agar plates are incubated
either microaerobically in a jar (6.11) containing the
Typical colonies are 1 mm to 2 mm in diameter, con-
gas mixture (6.12) or aerobically.
vex, colourless and opaque with an entire edge. If the
colonies are not well separated, pick a typical Listeria
NOTE 6 Incubation of the Oxford agar at 30 “C is suitable
spp. colony onto another TSYEA plate. Carry out the
for foodstuffs only lightly contaminated by a supplementary
following tests from colonies of a pure culture on the
flora. For products heavily contaminated by a supplemen-
tary flora, it is preferable to incubate the Oxford agar at TSYEA.
4
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SIST EN ISO 11290-1:1997
IS0 11290-l :1996(E)
@ IS0
The Henry illumination test (see annex C) may be inoculate the sheep blood agar plates (5.7) to deter-
NOTE 8
conducted, if necessary. For this test, it is important that the mine the haemolytic reaction.
agar medium is thin (15 ml/plate).
Dry the agar surface well before use. Take a colony
9.5.2 Catalase reaction
separated in 9.5.1.2 and plate and stab one space for
each culture, using a wire (6.5). Simultaneously stab
Take an isolated colony obtained in 9.5.1.2 and sus-
positive (L. monocytogenes) and negative control cul-
pend it in a drop of hydrogen peroxide solution (5.11)
tures (L. innocua).
on a slide. The immediate formation of gas bubbles
indicates a positive reaction.
After incubation at 35 OC or 37 OC for 24 h + 2 h, ex-
amine the test strains and controls. L. monocytogenes
9.5.3 Gram staining
show narrow, clear, light zones (P-haemolysis*); see
figure 1); L. innocua show no clear zone around the
Perform the Gram stain on a colony separated in
stab. L. seeligeri show a weak zone of haemolysis.
951.2. Listeria spp. are revealed as Gram-positive
L. ivanovii usually show wide, clearly delineated zones
slim, short rods.
of P-haemolysis. Examine the plates in a bright light to
compare test cultures with controls.
9.5.4 Motility test (if necessary)‘)
NOTE 9 The haemolytic reaction may also be carried out
Take an isolated colony obtained in 9.5.1.2 and sus- using red blood corpuscles. Disperse the colony in 150 ~1 of
pend it in a tube containing TSYEB (5.6). TSYEB (5.6); incubate at 35 OC or 37 OC for 2 h. Add 150 ~1
of sheep red blood corpuscles in a 2 %. solution of PBS
(5.12). Incubate at 35 “C or 37 “C for between 15 min and
Incubate in the incubator [6.3 a)] set at 25 OC for 8 h to
60 min, then refrigerate at 3 OC + 2 OC for about 2 h. Then
24 h until a cloudy
...
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