SIST EN ISO 11290-2:1999
(Main)Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of Listeria monocytogenes - Part 2: Enumeration method (ISO 11290-2:1998)
Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of Listeria monocytogenes - Part 2: Enumeration method (ISO 11290-2:1998)
Migrated from Progress Sheet (TC Comment) (2000-07-10): Should be put on the VA list (see resolution TC 275/WG 6 - but wait for official ++ request from TC Secretary) - (NT/961023)
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren für den Nachweis und die Zählung von Listeria monocytogenes - Teil 2: Zählverfahren (ISO 11290-2:1998)
Dieser Teil von ISO 11290 legt ein horizontales Verfahren für die Zählung von Listeria monocytogenes fest. ANMERKUNG: Dieses Verfahren ermöglicht auch die Zählung von anderen Listeria Spezies, die gegebenenfalls als Indikatoren für den Hygienestatus von Lebensmitteln und Futtermitteln herangezogen werden. 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 2: Méthode de dénombrement (ISO 11290-2:1998)
Mikrobiologija živil in krmil - Horizontalna metoda za ugotavljanje prisotnosti in števila Listeria monocytogenes - 2. del: Metoda štetja (ISO 11290-2:1998)
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2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Mikrobiologija živil in krmil - Horizontalna metoda za ugotavljanje prisotnosti in števila Listeria monocytogenes - 2. del: Metoda štetja (ISO 11290-2:1998)Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren für den Nachweis und die Zählung von Listeria monocytogenes - Teil 2: Zählverfahren (ISO 11290-2:1998)Microbiologie des aliments - Méthode horizontale pour la recherche et le dénombrement de Listeria monocytogenes - Partie 2: Méthode de dénombrement (ISO 11290-2:1998)Microbiology of food and animal feeding stuffs - Horizontal method for the detection and enumeration of Listeria monocytogenes - Part 2: Enumeration method (ISO 11290-2:1998)07.100.30Mikrobiologija živilFood microbiologyICS:Ta slovenski standard je istoveten z:EN ISO 11290-2:1998SIST EN ISO 11290-2:1999en01-december-1999SIST EN ISO 11290-2:1999SLOVENSKI
STANDARD
SIST EN ISO 11290-2:1999
SIST EN ISO 11290-2:1999
SIST EN ISO 11290-2:1999
SIST EN ISO 11290-2:1999
SIST EN ISO 11290-2:1999
AReference numberISO 11290-2:1998(E)INTERNATIONALSTANDARDISO11290-2First edition1998-07-01Microbiology of food and animal feedingstuffs — Horizontal method for thedetection and enumeration of Listeriamonocytogenes —Part 2:Enumeration methodMicrobiologie des aliments — Méthode horizontale pour la recherche et ledénombrement de Listeria monocytogenes —Partie 2: Méthode de dénombrementSIST EN ISO 11290-2:1999
ISO 11290-2:1998(E)©
ISO 1998All rights reserved. Unless otherwise specified, no part of this publication may be reproducedor utilized in any form or by any means, electronic or mechanical, including photocopying andmicrofilm, without permission in writing from the publisher.International Organization for StandardizationCase postale 56 · CH-1211 Genève 20 · SwitzerlandInternetiso@iso.chPrinted in SwitzerlandiiContentsPage1Scope .12Normatives references .13Definitions .24Principle .25Culture media and reagents .26Apparatus and glassware .27Sampling .38Preparation of test sample .49Procedure .410Expression of results (see ISO 7218) .811Precision .912Quality control of culture media .1013Test report .10Annex A (normative) Diagram of procedure .11Annex B (normative) Composition and preparation of media andreagents .12Annex C (informative) Henry illumination test .20SIST EN ISO 11290-2:1999
© ISOISO 11290-2:1998(E)iiiForewordISO (the International Organization for Standardization) is a worldwidefederation of national standards bodies (ISO member bodies). The work ofpreparing International Standards is normally carried out through ISOtechnical committees. Each member body interested in a subject for whicha technical committee has been established has the right to be representedon that committee. International organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISOcollaborates closely with the International Electrotechnical Commission(IEC) on all matters of electrotechnical standardization.Draft International Standards adopted by the technical committees arecirculated to the member bodies for voting. Publication as an InternationalStandard requires approval by at least 75 % of the member bodies castinga vote.International Standard ISO 11290-2 was prepared by Technical CommitteeISO/TC 34, Agricultural food products, Subcommittee SC 9, Microbiology.ISO 11290 consists of the following parts, under the general titleMicrobiology of food and animal feeding stuffs — Horizontal method for thedetection and enumeration of Listeria monocytogenes:—Part 1: Detection method—Part 2: Enumeration methodAnnexes A and B form an integral part of this part of ISO 11290. Annex Cis for information only.SIST EN ISO 11290-2:1999
ISO 11290-2:1998(E)© ISOivIntroductionBecause of the large variety of food and feed products, this horizontalmethod may not be appropriate in every detail for certain products. In thiscase, different methods, which are specific to these products may be usedif absolutely necessary for justified technical reasons. Nevertheless, everyattempt should be made to apply this horizontal method as far as possible.When this part of ISO 11290 is next reviewed, account will be taken of allinformation then available regarding the extent to which this horizontalmethod has been followed and the reasons for deviations from this methodin the case of particular products.The harmonization of test methods cannot be immediate, and for certaingroups of products International Standards and/or national standards mayalready exist that do not comply with this horizontal method. It is hoped thatwhen such standards are reviewed they will be changed to comply with thispart of ISO 11290 so that eventually the only remaining departures fromthis horizontal method will be those necessary for well-establishedtechnical reasons.SIST EN ISO 11290-2:1999
INTERNATIONAL STANDARD
© ISOISO 11290-2:1998(E)1Microbiology of food and animal feeding stuffs — Horizontalmethod for the detection and enumeration of Listeriamonocytogenes —Part 2:Enumeration methodWARNING — In order to safeguard the health of laboratory personnel, it is strongly recommended thattests for detecting Listeria monocytogenes are undertaken in properly equipped laboratories, under thecontrol of a skilled microbiologist, and that great care is taken in the disposal of all contaminated materials.In particular, it is strongly recommended that female laboratory staff are made aware of the particular riskto the developing foetus presented by infection of the mother through exposure to Listeria mono-cytogenes. National legislation may involve more specific demands.1
ScopeThis part of ISO 11290 specifies a horizontal method for the enumeration of Listeria monocytogenes.NOTE —
The method also allows enumeration of other Listeria species which may be used as indicators of the hygienicquality of food or feed products.Subject to the limitations discussed in the introduction, this part of ISO 11290 is applicable to products intended forhuman consumption or animal foodstuffs.In general (see note in 9.2.1), the lower limit of enumeration of this method is 10 L. monocytogenes per millilitre ofsample for liquid products, or 100 L. monocytogenes per gram of sample for other products.2
Normative referencesThe following standards contain provisions which, through reference to the text, constitute provisions of thisInternational Standard. At the time of publication, the editions indicated were valid. All Standards are subject torevision, and parties to agreements based on this International Standard are encouraged to investigate thepossibility of applying the most recent editions of standards indicated below. Members of IEC and ISO maintainregisters of currently valid International Standards.ISO 6887-1:—1), Microbiology of food and animal feeding stuffs — Preparation of test samples, initial suspensionand decimal dilutions for microbiological examination — Part 1: General rules for the preparation of the initialsuspension and of decimal dilutions.ISO 7218:1996, Microbiology of food and animal feeding stuffs — General rules for microbiological examination.ISO 11290-1:1996, Microbiology of food and animal feeding stuffs — Horizontal method for the detection andenumeration of Listeria monocytogenes — Part 1: Detection method.
1)
To be published. (Revision of ISO 6887:1993)SIST EN ISO 11290-2:1999
ISO 11290-2:1998(E)© ISO23
DefinitionsFor the purposes of this part of ISO 11290, the following definitions apply.3.1Listeria monocytogenesmicroorganisms which form typical colonies on the solid selective medium described and which display themorphological, physiological and biochemical characteristics described when the analysis is carried out inaccordance with this part of ISO 112903.2enumeration of Listeria monocytogenesdetermination of the number of colony-forming units (CFU) of Listeria monocytogenes (see 3.1), in a given quantityof product, when the analysis is carried out in accordance with this part of ISO 112904
PrincipleWithin the limits of this part of ISO 11290, the enumeration of Listeria monocytogenes requires six successive steps(see annex A for a flowchart).4.1
Preparation of the initial suspension in one of the two diluents described, as necessary.4.2
Resuscitation for 1 h at 20 °C.4.3
Surface plating, on the solid selective culture medium contained in two Petri dishes, of a specified quantity ofthe test sample for liquid products or the initial suspension for other products.Preparation of other dishes, under the same conditions, using decimal dilutions of the test sample or initialsuspension.4.4
Incubation of the dishes at 35 °C or 37 °C and examination after 24 h and 48 h.4.5
Confirmation of presumptive colonies of Listeria monocytogenes with the tests described.4.6
From the number of confirmed colonies, calculation of the number of Listeria monocytogenes per gram or permillilitre of the test sample.5
Culture media and reagentsFor current laboratory practice, see ISO 7218.NOTE —
Because of the large number of culture media and reagents, it has been considered preferable, for clarity of the text,to describe them in annex B.6
Apparatus and glasswareUsual microbiological equipment (see ISO 7218) and, in particular, the following.6.1
Apparatus for dry sterilization (oven) or wet sterilization (autoclave)See ISO 7218.SIST EN ISO 11290-2:1999
© ISOISO 11290-2:1998(E)36.2
Drying cabinet or incubator, capable of being maintained between 25 °C ± 1 °C and 50 °C ± 1 °C.6.3
Incubators, for maintaining the inoculated media, plates and tubes within the following temperature ranges:a)20 °C ± 1 °C (optional);b)25 °C ± 1 °C (optional);c)35 °C ± 1 °C or 37 °C ± 1 °C.6.4
Water bath, capable of being maintained at 47 °C ± 2 °C.6.5
Loops and wires of platinum/iridium or nickel/chromium, or Pasteur pipettes or single-use loops.6.6
Glass or plastic spreaders, sterile.6.7
pH-meter, capable of being read to the nearest 0,01 pH unit at 25 °C, enabling measurements to be madewhich are accurate to ± 0,1 pH unit.6.8
Test tubes or flasks, of appropriate capacity, for sterilization and storage of culture media and incubation ofliquid media.6.9
Total-delivery graduated pipettes, of nominal capacities 1 ml and 10 ml, graduated respectively in 0,1 mland 0,5 ml divisions.6.10
Petri dishes, of diameter 90 mm and 140 mm.6.11
Jars, suitable for microaerobic incubation (optional).6.12
Gas mixture (optional), of specified composition for microaerobic incubation:5 % to 12 % CO2, 5 % to 15 % O2, and N2 up to 100 %.6.13
Equipment for the Henry illumination test (optional)See annex B.6.14
Microscope, preferably with phase-contrast.7
SamplingSampling is not part of the method specified in this part of ISO 11290. If there is no specific International Standarddealing with sampling of the product concerned, it is recommended that the parties concerned come to anagreement on this subject.It is important that the laboratory receive a sample which is truly representative and has not been damaged orchanged during transport or storage (see ISO 7218).SIST EN ISO 11290-2:1999
ISO 11290-2:1998(E)© ISO48
Preparation of test samplePrepare the test sample in accordance with the specific International Standard appropriate to the productconcerned. If there is no specific International Standard, it is recommended that the parties concerned come to anagreement on this subject.9
ProcedureWARNING — Whenever a choice is given between 35 °C or 37 °C for the incubation temperature, thistemperature shall be agreed between the parties concerned and recorded in the test report.9.1
Test portion, initial suspension and dilutionsSee ISO 6887-1 and any specific International Standard appropriate to the product concerned.Use as diluent for preparing the initial suspension either buffered peptone water (B.1), or half-Fraser broth basemedium (B.2).Half-Fraser broth base without the addition of selective agents may be used as a diluent for the food or feedsample when both the detection method (see ISO 11290-1) and this enumeration method are carried out on thesame test sample. This procedure is to avoid the need to prepare two initial suspensions; the selective agents areadded to the suspension once the test portion for enumeration has been used. Use of this procedure should benoted in the test report.Let the initial suspension stand for 1 h ± 5 min at 20 °C ± 2 °C [by using, if necessary, the incubator 6.3 a)], in orderto resuscitate the stressed microorganisms.If a dilution range is used, prepare it after resuscitation.9.2
Inoculation and incubation9.2.1
Transfer, by means of a sterile pipette (6.9), 0,1 ml of the initial suspension (9.1) to each of two dishes ofPALCAM agar (B.3), dried beforehand if necessary in the incubator (6.2).Repeat the procedure using further decimal dilutions if necessary.NOTE —
When, for certain products, it is necessary to estimate low numbers of Listeria monocytogenes, the limit ofenumeration can be lowered by a factor of 10 by examining 1,0 ml of the initial suspension. Distribute the 1 ml of inoculumeither on the surface of the agar medium in a large Petri dish (140 mm) or over the surface of the agar medium in three smalldishes (90 mm) using a sterile spreader (6.6). In both cases, prepare duplicates by using two large dishes or six small dishes.9.2.2
Carefully spread the inoculum as quickly as possible over the surface of the agar plate without touching thesides of the dish with the spreader. Use a fresh sterile spreader for each plate.2) Leave the plates closed for about15 min at ambient temperature for the inoculum to be absorbed into the agar.9.2.3
Invert the dishes prepared in 9.2.2 and place them in an incubator [6.3 c)] set at 35 °C or 37 °C. IncubatePALCAM agar dishes either microaerobically in a jar (6.11) containing the gas mixture (6.12) or aerobically.9.3
Enumeration of characteristic colonies9.3.1
After incubation for 24 h, and for an additional 18 h to 24 h if growth is slight or if no colonies are observedafter 24 h of incubation, examine the dishes (9.2.3) for the presence of colonies presumed to be Listeria spp.(see 9.3.3).
2)
It is possible to use the same spreader for a given sample, by beginning with the higher dilution.SIST EN ISO 11290-2:1999
© ISOISO 11290-2:1998(E)59.3.2
For dishes incubated microaerobically, after incubation leave the PALCAM agar dishes (9.3.1) to air for 1 h toallow the agar to regain its pink to purple colour.9.3.3
After 24 h the characteristic colonies of Listeria spp. grow as small or very small greyish green or olive greencolonies, sometimes with black centres, but always with black halos. After 48 h Listeria spp. appear in the form ofgreen colonies, about 1,5 mm to 2 mm in diameter, with a central depression and surrounded by a black halo.9.3.4
Count all the colonies presumed to be Listeria spp. (9.3.3) on each dish containing less than 150characteristic or non-characteristic colonies.9.4
Confirmation of Listeria spp.9.4.1
Selection of colonies for confirmation9.4.1.1
After the period of incubation (9.3.1), keep the dishes containing less than 150 presumptive Listeria spp.colonies, at all dilutions and, if possible, at two successive dilutions.Select five of the presumptive colonies on each plate retained. If there are fewer than five presumptive colonies on adish, select for confirmation all presumptive colonies.9.4.1.2
Streak the selected colonies onto the surface of predried plates of tryptone soya yeast extract agar(TSYEA) (B.4) in a manner which will allow well-separated colonies to develop.Place the plates in the incubator [6.3 c)] set at 35 °C or 37 °C for 18 h to 24 h or until growth is satisfactory.Typical colonies are 1 mm to 2 mm in diameter, convex, colourless and opaque with an entire edge. If the coloniesare not well separated, pick a typical Listeria spp. colony onto another TSYEA plate. Carry out the following testsfrom colonies of a pure culture on the TSYEA.NOTE —
The Henry illumination test may be conducted if necessary (see annex C and note in B.4.2). The colonies thenappear bluish with a granular surface.9.4.2
Catalase reactionTake a colony separated in 9.4.1.2 and suspend it in a drop of hydrogen peroxide solution (B.10) on a slide. Theimmediate formation of gas bubbles indicates a positive reaction (see ISO 7218).9.4.3
Gram stainingPerform the Gram stain on a colony separated in 9.4.1.2 (see ISO 7218). Listeria spp. are revealed asGram-positive slim, short rods (of approximately 0,4 mm to 0,5 mm diameter, and 1 mm to 2 mm length).9.4.4
Motility test (if necessary)3)Take a colony separated in 9.4.1.2 and suspend it in a tube containing TSYEB (B.5).Incubate in the incubator [6.3 b)] set at 25 °C for 8 h to 24 h until a cloudy medium is observed.Deposit a drop of the above culture using a loop (6.5) onto a clean glass microscope slide. Place a coverslip on topand examine it with the microscope (6.14). Listeria spp. appear as slim, short rods with tumbling motility.Cultures grown above 25 °C may fail to exhibit this motion. Always compare them to a known culture. Cocci, largerods, or rods with rapid swimming motility are not Listeria spp.
3)
This examination is not necessary in all cases if the analysis is carried out by a microbiologist who regularly works on thedetection of L. monocytogenes.SIST EN ISO 11290-2:1999
ISO 11290-2:1998(E)© ISO6As an alternative test for motility, using an inoculating needle (6.5), stab the motility agar (B.8) with a typical colonyon TSYEA (9.4.1.2). Incubate it for 48 h in the incubator [6.3 b)] set at 25 °C.Examine for growth around the stab. Listeria spp. are motile, giving a typical umbrella-like growth patternimmediately below the surface of the agar. If growth is not sufficient, incubate for up to an additional 5 days andexamine the culture during this time.9.5
Confirmation of L. monocytogenes9.5.1
Haemolysis test9.5.1.1
If the morphological and physiological characteristics and catalase reaction are indicative of Listeria spp.,determine the haemolytic reaction on sheep blood agar dishes (B.6).Before use, thoroughly dry the blood agar surface, then mark the agar into squares. For each culture, take a colonyseparated in 9.4.1.2 and stab one labelled square using a wire (6.5). Also stab positive (L. monocytogenes)
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