EN 12824:1997
(Main)Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella (ISO 6579:1993 modified)
Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella (ISO 6579:1993 modified)
This European Standard specifies a horizontal method for the detection of Salmonella. Subject to the limitations discussed in the introduction, this European Standard is applicable to products intended for human consumption or feeding of animals. The incubation temperature (35°C or 37°C) is subject to agreement by the parties concerned and is specified in the test report.
Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum Nachweis von Salmonellen (ISO 6579:1993 modifiziert)
Diese Europäische Norm legt ein horizontales Verfahren zum Nachweis von Salmonellen fest. In Abhängigkeit von den in der Einleitung erörterten Einschränkungen gilt diese Europäische Norm für Produkte, die für den menschlichen Verzehr oder als Futtermittel bestimmt sind. Die Bebrütungstemperatur (35 °C oder 37 °C) wird zwischen den betreffenden Partnern vereinbart und im Untersuchungsbericht festgehalten.
Microbiologie des aliments - Méthode horizontale pour la recherche des Salmonella (ISO 6579:1993 modifiée)
La présente Norme européenne spécifie une méthode horizontale de recherche des Salmonella. Compte tenu des remarques signalées dans l'introduction, la présente norme européenne est applicable aux produits destinés à la consommation humaine ou à l'alimentation animale. La température d'incubation retenue (35°C ou 37°C) fait l'objet d'un accord entre les parties concernées et est notée dans le rapport d'essai.
Mikrobiologija živil in krmil - Horizontalna metoda za ugotavljanje prisotnosti salmonel (ISO 6579:1993, modificiran)
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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 salmonel (ISO 6579:1993, modificiran)Mikrobiologie von Lebensmitteln und Futtermitteln - Horizontales Verfahren zum Nachweis von Salmonellen (ISO 6579:1993 modifiziert)Microbiologie des aliments - Méthode horizontale pour la recherche des Salmonella (ISO 6579:1993 modifiée)Microbiology of food and animal feeding stuffs - Horizontal method for the detection of Salmonella (ISO 6579:1993 modified)07.100.30Mikrobiologija živilFood microbiologyICS:Ta slovenski standard je istoveten z:EN 12824:1997SIST EN 12824:1998en01-junij-1998SIST EN 12824:1998SLOVENSKI
STANDARD
SIST EN 12824:1998
SIST EN 12824:1998
SIST EN 12824:1998
SIST EN 12824:1998
SIST EN 12824:1998
SIST EN 12824:1998
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SIST EN 12824:1998
INTERNATIONAL ISO STANDARD 6579 Third edition 1993-09-01 Microbiology - General guidance on methods for the detection of Salmonella Microbiologie - Directives g&xSrales concemant /es methodes de recherche des Salmonella E!!! Reference number ISO 6579:1993(E) SIST EN 12824:1998
ISO 6579:1993(E) Contents Fage 1 2 3 4 5 6 7 a 9 10 11 12 Scope . 1 Normative references . 1 Definitions *.,.,. . . . . . . . . . . . . . . . . . . . . . . 1 Principle . 1 Culture media, reagents and sera . 2 Apparatus and glassware . 3 Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .*. 3 Preparation of the test Sample l . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 .> Expression of results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .*. 7 Test report . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Quality assurance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 Annexes A Diagram of procedure .,.,.,. 8 B Composition and preparation of culture media and reagents . 9 C Specification for brilliant green . . . . . . .I. 15 D Standard method of streaking agar plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16 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 ii SIST EN 12824:1998
ISO 6579:1993(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 mem.ber 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. I 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 6579 was prepared by Technical Committee ISO/TC 34, Agricultural food products, Sub-Committee SC 9, Microbiology. This third edition cancels and replaces the second edition (ISO 6579:1990), of which it constitutes a technical revision. Annexes A, B and C form an integral part of this International Standard. Annex D is for information only. SIST EN 12824:1998
ISO 6579:1993(E) Introduction This International Standard is intended to provide general guidance for the examination of products not dealt with by existing International Standards and to be taken into account by organizations preparing microbiological methods of test for application to foods or to animal feeding stuffs. Be- cause of the large variety of products within this field of application, these guidelines may not be appropriate in evety detail for certain products, and for other products it may be necessary to use different methods. Never- theless, it is hoped that in all cases every attempt will be made to apply the provided guidelines 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 these 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. SIST EN 12824:1998
INTERNATIONAL STANDARD ISO 6579:1993(E) Microbiology - General guidance on methods for the detection of SaImoneIla WARNING - In Order to safeguard the health of laboratory personnel, it is essential that tests for detecting Salmonella are only 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. 1 Scope This International Standard gives general guidance on methods for the detection of Salmonella. Subject to the limitations discussed in the Introduc- tion, this International Standard is applicable to pro- ducts intended for human consumption or feeding of animals. The incubation temperature (35 “C or 37 “C) shall be agreed by the Parties concerned and shall be specified in the test report. 2 Normative references The following Standards contain provisions which, through reference in this text, constitute provisions of this International Standard. At the time of publica- tion, 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 re- cent editions of the Standards indicated below. Members of IEC and ISO maintain registers of cur- rently valid International Standards. ISO 6887: 1983, Microbiology - General guidance for the preparation o f dilutions for microbiological exam- ina tion. ISO 72 18: 1985, Microbiology - General guidance for microbiological examina tioi 3 Definitions For the purposes of this following definitions apply. 1s. nternational Standard, the 3.1 Salmonella: Microorganisms which form typical colonies on solid selective media and which display the biochemical and serological characteristics de- scribed when tests are carried out in accordance with this International Standard. 3.2 detection of Salmonella: Determination of the presence or absence of these microorganisms, in a particular mass of product, when tests are carried out in accordance with this International Standard. 4 Principle The detection of Salmonella necessitates four suc- cessive stages (see also annex A). NOTE 1 Salmonelle may ‘be present in small numbers and are often accompanied by considerably larger numbers of other members of Entefobacteriacez? or of other families. Therefore, selective enrichment is necessary; furthermore, pre-enrichment is often necessary to permit detection of injured Salmonek 4.1 Pre-enrichment in non-selective liquid medium Inoculation of buffered peptone water (also used as diluent) with the test portion, and incubation at 35 “C or 37 “C (as agreed) for 16 h to 20 h. 4.2 Enrichment in selective liquid media Inoculation of magnesium chloride/malachite green medium and of a selenitelcystine medium with the culture obtained in 4.1. Incubation of the magnesium chloride/malachite green medium at 42 “C for 24 h and incubation of the 1 SIST EN 12824:1998
ISO 6579:1993(E) selenitelcystine medium at 35 “C or 37 “C (as agreed) for 24 h and a further 24 h. 4.3 Plating out and recognition From the cultures obtained in 4.2, inoculation of two selective solid media: - Phenol redlbrilliant green agar, unless the Inter- national Standard appropriate to the product to be examined, or other specific considerations (for ex- ample the isolation of lactose-positive Salmonella), require Substitution of some other medium as the one for obligatory use; - any other solid selective medium (see 5.2.4.2). Incubation at 35 “C or 37 “C (as agreed), and exam- ination after 24 h and, if necessary, after 48 h to check for the presence of colonies which, from their characteristics, are considered to be presumptive Salmonella. 4.4 Confirmation Subculturing of colonies of presumptive Salmonella, plated out as described in 4.3, and confirmation by means of appropriate biochemical and serological tests. 5 Culture media, reagents and sera 5.1 General For current laboratory practice, see ISO 7218. 5.2 Culture media and reagents NOTE 2 Because of the large number of culture media and reagents, it has been considered preferable, for the clarity of the text, to give their composition and preparation in annex B. 5.2.1 Non-selective pre-enrichment medium: Buffered peptone water See clause B.l. 5.2.2 First selective enrichment medium: Rappaport-Vassiliadis magnesium chloride/malachite green medi,um (RV medium) See clause B.2. 5.2.3 Second selective enrichment medium: Selenitelcystine medium See clause B.3. 5.2.4 Solid selective plating-out media 5.2.4.1 First medium: Phenol red/brilliant green agar (Edel and Kampelmacher) See clause B.4. This first medium is compulsory unless otherwise stated (sec 4.3). 5.2.4.2 Second medium The choice of the second medium is left to the dis- cretion of the testing laboratory, unless there is a specific International Standard relating to the product to be examined, which specifies the composition of this second medium. 5.2.5 Nutrient agar See clause B.5. 5.2.6 Triple sugar/iron agar (TSI agar) See clause B.6. 5.2.7 Urea agar (Christensen) See clause B.7. 5.2.8 L-Lysine decarboxylation medium See clause B.8. 5.2.9 Reagent for detection of fi-galactosidase (or prepared Paper discs, used in accordance with the manufacturer’s instructions) See clause B.9. 5.2.10 Reagents for Voges-Proskauer (VP reaction) See clause B.10. 5.2.10.1 VP medium 5.2.10.2 Creatine solution (ALamidinosarcosine) 5.2.10.3 1-Naphthol, ethanolic Solution 5.2.10.4 Potassium hydroxide Solution 5.2.11 Reagents for indole reaction See clause B.11. 2 SIST EN 12824:1998
ISO 6579:1993(E) 5.2.11 .l Tryptone-tryptophan medium 5.2.11.2 Kovacs reagent (N,N-dicyclohexyl- carbodiimide pentachlorophenol complex) 5.2.12 Semi-solid nutrient agar See clause B.12. 5.2.13 Saline Solution See clause B.13. 5.3 Sera Several types of agglutinant sera containing anti- bodies for one or several 0-antigens are available commercially, i.e. anti-sera containing one or more “0” groups (called monovalent or polyvalent anti-0 sera), anti-Vi sera, and anti-sera containing antibodies for one or several H-factors (called monovalent or polyvalent anti-H sera). Every attempt should be made to ensure that the anti-sera used are adequate to provide for the de- tection of all Salmonella serotypes. Assistance to- wards this objective may be obtained by using anti-sera prepared by a supplier recognized as com- Petent (for example, by an appropriate government agency). 6 Apparatus and glassware NOTE 3 Disposable apparatus is an acceptable alterna- tive to reusable glassware if it has suitable specifications. Usual microbiological laboratory equipment particular, the following. and, in 6.1 Apparatus for dry sterilization (oven sterilization (autoclave) See ISO 7218. ) or wet 6.2 Drying cabinet or oven, ventilated by con- vection, capable of operating between 37 “C + 1 “C and 55 “C + 1 “C. - 6.3 Incubator, capable of operating at 35 “C -f: 1 “C or 37 “C + 1 “C, depending on the temperature agreed. 6.4 Water bath, capable of operating at 42,0 “C k 1 “C or incubator, capable of operating at 42,0 “C + 0,5 "C. 6.5 Water baths, capable of operating at 45 “C + 1 “C, 55 “C + 1 “C and 70 “C k 1 “C. 6.6 Water bath, capable of operating at 35 “C + 1 “C or 37 “C + 1 “C, depending on the tem- perature agreed. 6.7 Loops, made of platinum/iridium or nickel/ chro- mium, of diameter approximately 3 mm. 6.8 pH-meter, having an accuracy of calibration of + 0,l pH unit at 25 “C. 6.9 Culture bottles or flasks NOTE 4 Bottles or flasks with non-toxic metallic or plastic screw-taps may be used. 6.10 Culture tubes, 8 mm in diameter and 160 mm in length. 6.11 Measuring cylinders 6.12 Graduated pipettes, of nominal capacities 10 ml and 1 ml, graduated respectively in 0,5 ml and 0,l ml divisions. 6.13 Petri dishes, of small size (diameter 90 mm to 100 mm) and/or large size (diameter 140 mm). 7 Sampling lt is important that the laboratory receive a Sample which is truly representative and has not been dam- aged or changed during transport or storage Sampling is not patt of the method specified in this International Standard. See the specific International Standard dealing with the product concerned. If there is no specific International Standard, it is rec- ommended that the Parties concerned come to an agreement on this subject. 8 Preparation of the test Sample Prepare the test Sample in accordance with the spe- cific International Standard dealing with the product concerned. If there is no specific International Stan- dard, it is recommended that the Parties concerned come to an agreement on this subject. 9 Procedure (See diagram in annex A.) 9.1 Test Portion and initia I Suspension 9.1.1 See ISO 6887 and the specific International Standard dealing with the product concerned. For preparation of the initial Suspension, use as di- lution fluid the p,re-enrichment medium specified in 5.2.1. 3 SIST EN 12824:1998
ISO 6579:1993(E) 9.1.2 In general, to prepare the initial Suspension, add a 25 g test portion to 225 ml of pre-enrichment medium (5.2.1), which is the ratio of test Portion to pre-enrichment medium specified in this method. If the prescribed test Portion is other than 25 g, use the necessary quantity of pre-enrichment medium to yield approximately a l/lO dilution (mass to volume). NOTES 5 To reduce the examination workload when more than one 25 g test Portion from a specified Iot of food has to be examined, and when evidente is available that compositing (pooling the test portions) does not affect the result for that particular food, the test portions may be composited. For example, if 10 test portions of 25 g are to be examined, combine the 10 units to form a composite test Portion of 250 g and add 225 litres of pre-enrichment broth. Alterna- tively, the 0,l ml (RV medium) and 10 ml (selenitelcystine medium) portions of the pre-enrichment broths from the 10 separate test portions (9.3.1) may be composited for enrichment in 0,l litre and 1 litre respectively of selective enrichment medium. 6 Dried or powdered food products may need a special rehydration procedure . to enhance the recovery of Salmonelle. Two techniques may be used for this purpose, that of immersion and that of agitation. Refer for this pur- pose to the specific International Standard dealing with the product under examination. If such a Standard is not avail- able, it is recommended that the Parties concerned come to an agreement on this subject. 9.2 Non-selective pre-enrichment Incubate the initial Suspension at 35 “C or 37 “C (as agreed) for not less than 16 h and not more than 20 h. 9.3 Selective enrichment 9.3.1 Transfer 0,l ml of the culture obtained in 9.2 to a tube containing 10 ml of the RV medium (5.2.2); transfer 10 ml of the culture obtained in 9.2 to a flask containing 100 ml of selenitelcystine medium (5.2.3). 9.3.2 Incubate the two inoculated media (9.3.1) for 18 h to 24 h as follows: a) the inoculated RV medium at 42 “C for 24 h; b) the inoculated selenitelcystine medium at 35 “C or 37 “C (as agreed) for 24 h and a further 24 h. NOTE 7 For the selenitelcystine medium, it may, in some cases, be advantageous to raise the incubation temperature to 42 “C. This modification should be indicated in the test report. 9.4 Plating out and identification 9.4.1 Using the culture obtained in the RV medium, after incubation for 24 h, inoculate, by means of a loop (6.7), the surface of one large-size Petri dish (6.13) containing the first selective plating-out me- dium (generally the Phenol red/briIIiant green agar, see 5.2.4.1), so that weil-isolated colonies will be ob- tained. In the absence of large dishes, use two small dishes, one after the other, using the same loop (see note 8). Proceed in the same way with the second selective plating-out medium (5.2.4.2) using a new loop and Petri dishes of appropriate size. NOTE 8 The following method of streaking is rec- ommended when Phenol red/brilliant green agar is used. Use one loop (6.7) for two dishes. Take a droplet from the edge of the surface of the fluid. Inoculate both dishes ac- cording to the two diagrams in annex D . Use the whole dish; loop streaks should be spaced about 0,5 cm apart. (DO not flame the loop or recharge it after making the first streak, nor when passing to the second dish.) When only one large dish is used, the method of streaking should be as indicated for the first dish in annex D. 9.4.2 Using the culture obtained in the selenite/ cystine medium after incubation for 24 h, repeat the procedure described in 9.4.1 with the two selective plating-out media. 9.4.3 Invert the dishes (9.4.1) and (9.4.2) so that the bottom is uppermost, and place them in the incubator (6.3) set at 35 “C or 37 “C (as agreed). 9.4.4 After a total incubation period of 48 h of the selenitelcystine medium (see 9.3.2 and 9.4.3), repeat the procedure described in 9.4.2 and 9.4.3. 9.4.5 After incubation for 20 h to 24 h, examine the dishes (9.4.3 and 9.4.4) for the presence of typical colonies of Salmonella. Typical colonies of Salmonella grown on Phenol red/brilliant green agar Cause the colour of the medium to Change from pink to red. 9.4.6 If growth is slight or if no typical colonies of Salmonella are present, reincubate at 35 “C or at 37 “C (as agreed) for a further 18 h to 24 h. Re-exam ine the plates colonies of Salmonella. for the presence of typical NOTE 9 Any typical or suspect colony should be sub- jected to a confirmation (9.5); the recognition of colonies of Salmonella is to a large extent a matter of experience, and their appearance may vary somewhat, not only from species to species, but also from batch to batch of medium. In this respect, agglutination, at this Stage, of colonies with polyvalent Salmonella anti-serum may facilitate recognition of suspected colonies. 4 SIST EN 12824:1998
ISO 6579:1993(E) , 9.4.7 Identification kits currently available commer- cially and permitting the identification of Salmonelle may be used. Typical Salmonelle cultures show alkaline (r-ed) slants with gas formation and acid (yellow) butts, with (in about 90 % of the cases) formation of hydrogen sul- fide (blackening of the agar). 9.5 Confirmation When a lactose-positive Salmonelle is isolated (see 4.3), the TSI slant is yellow. Thus, preliminaty confir- mation of Salmonella cultures shall not be based on the results of the TSI agar test only (see 9.5.3). 9.5.1 Selection of colonies for confirmation For confirmation, take from each dish of each selec- tive medium (see 9.4.5 and 9.4.6), five colonies con- sidered to be typical or suspect. 9.5.2.2 Urea agar (52.7) Streak the agar slope surface. If on one dish there are fewer than five typical or suspect colonies, take for confirmation all the typical or suspect colonies. Incubate at 35 “C or 37 “C (as agreed) for 24 h and examine at intervals. Streak the selected colonies onto the surface of pre- dried nutrient agar plates (5.2.5), in a manner which will allow weil-isolated
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