INTERNATIONAL STANDARD ISO 6579-1 First edition 2017-02 Microbiology of the food chain — Horizontal method for the detection, enumeration and serotyping of Salmonella — Part 1: Detection of Salmonella spp Microbiologie de la chne alimentaire — Méthode horizontale pour la recherche, le dénombrement et le sérotypage des Salmonella — Partie 1: Recherche des Salmonella spp Reference number ISO 6579-1:2017(E) © ISO 2017 ISO 6579-1:2017(E) COPYRIGHT PROTECTED DOCUMENT © ISO 2017, Published in Switzerland All rights reserved Unless otherwise specified, no part o f this publication may be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on the internet or an intranet, without prior written permission Permission can be requested from either ISO at the address below or ISO’s member body in the country o f the requester ISO copyright o ffice Ch de Blandonnet • CP 401 CH-1214 Vernier, Geneva, Switzerland Tel +41 22 749 01 11 Fax +41 22 749 09 47 copyright@iso.org www.iso.org ii © ISO 2017 – All rights reserved ISO 6579-1:2017(E) Contents Page Foreword v Introduction vii Scope Normative references Terms and definitions Principle 4.1 General 4.2 Pre-enrichment in non-selective liquid medium 4.3 Enrichment in/on selective media 4.4 Plating out on selective solid media Culture media, reagents, and antisera Equipment and consumables Sampling Preparation of test sample Procedure (see diagrams in Annex A) 9.1 Test portion and initial suspension 9.2 Non-selective pre-enrichment 9.3 Selective enrichment 9.3.1 General 9.3.2 Procedure for food, animal feed samples, and environmental samples from the food production area f f 9.4 Plating out 9.4.1 General 9.4.2 Procedure for food, animal feed samples, and environmental samples from the food production area f f 9.5.1 General f f 9.5.3 Biochemical testing 9.5.4 Serological testing 11 9.5.5 Interpretation of biochemical and serological reactions 11 12 10 Expression of results 12 11 Performance characteristics of the method 12 12 12 12 11.4 LOD 50 12 12 Test report 13 Annex A (normative) Diagrams of the procedures 14 Annex B (normative) Culture media and reagents 17 Annex C (informative) Method validation studies and performance characteristics 32 Annex D (normative) Detection of Salmonella enterica subspecies enterica serovars Typhi and Paratyphi 38 9.5 C o nfirmatio n 9.3 Pro cedure o r s amp les ro m the p rimary p ro ductio n s tage Pro cedure o r s amp les ro m the p rimary p ro ductio n s tage C o nfirmatio n 9.5 S electio n o 9.5 S ero typ ing co lo nies 1 I nterlab o rato ry s tudies 1 S ens itivity 1 S p ecificity © ISO 2017 – All rights reserved o r co nfirmatio n iii ISO 65 79 -1 : 01 7(E) (informative) 43 Bibliography 48 Annex E iv Examples of selective plating- out media © ISO 2017 – All rights reserved ISO 6579-1:2017(E) Foreword ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies (ISO member bodies) The work o f preparing International Standards is normally carried out through ISO 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 liaison with ISO, also take part in the work ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters o f electrotechnical standardization The procedures used to develop this document and those intended for its further maintenance are described in the ISO/IEC Directives, Part In particular the different approval criteria needed for the di fferent types o f ISO documents should be noted This document was dra fted in accordance with the editorial rules of the ISO/IEC Directives, Part (see www.iso org/directives) Attention is drawn to the possibility that some o f the elements o f this document may be the subject o f patent rights ISO shall not be held responsible for identi fying any or all such patent rights Details o f any patent rights identified during the development o f the document will be in the Introduction and/or on the ISO list of patent declarations received (see www.iso org/patents) Any trade name used in this document is in formation given for the convenience o f users and does not constitute an endorsement For an explanation on the meaning o f ISO specific terms and expressions related to formity assessment, as well as information about ISO’s adherence to the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following URL: www.iso org/iso/foreword.html This document was prepared by the European Committee for Standardization (CEN), Technical Committee CEN/TC 275, Food analysis — Horizontal methods, in collaboration with ISO Technical Committee TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the agreement on technical cooperation between ISO and CEN (Vienna Agreement) This first edition o f ISO 6579-1 cancels and replaces ISO 6579:2002 and ISO 6785:2001, which have been technically revised It also incorporates ISO 6579:2002/Amd 1:2007 and ISO 6579:2002/Cor 1:2004 The main changes, compared to ISO 6579:2002, are the following — ISO 6785 has been incorporated in this document — Samples from the primary production stage have been added to the scope — Detection of Salmonella Typhi and Salmonella Paratyphi is described in Annex D — Descriptions of preparations of initial suspensions have been removed and references made to relevant parts of ISO 6887, whenever possible — The temperature range for incubation of non-selective media has been extended from 37 °C ± °C to 34 °C to 38 °C without further tolerance — For selective enrichment, there is a choice between using the broth or the semi-solid agar of Rappaport Vassiliadis medium (RVS or MSRV) for food, animal feed samples, and for environmental samples from the food production area — The inoculation o f the isolation medium has become less prescriptive; the objective is to obtain well-isolated colonies after incubation — For confirmation, it is acceptable to per form the tests on only one suspect colony (instead o f one suspect colony o f each medium combination) I f this isolate tests negative for Salmonella, four more suspect isolates from different media combinations shall be tested © ISO 2017 – All rights reserved v ISO 65 79-1 : 01 7(E) — I t i s p erm itte d to p er form the bio chem ic a l fi rmation d i re c tly on a s u s p e c t, wel l-i s olate d colony from the s ele c tive pl ati ng me d iu m T he pu rity che ck on the non- s ele c tive aga r me d iu m ca n then b e performed in parallel — Two fi rmation te s ts have b e come op tiona l ( ß-galactosidase test and indole reaction) and one fi rmation te s t h as b e en dele te d ( Vo ge s -P ro s kauer re ac tion) — In th i s c u ment, s erolo gic a l fi rmation (to s ero group level) is de s crib e d For gu idance on s ero typi ng (to s erovar level) , re ference i s made to I S O/ T R 65 79 -3 — Table has been improved — Per forma nce te s ti ng for the qua l ity as s u nce o f the c u ltu re me d i s b e en adde d to — Performance characteristics of MSRV have been added to Annex C A list of all parts in the ISO 6579 series can be found on the ISO website vi Annex B © ISO 2017 – All rights reserved ISO 65 79-1 : 01 7(E) Introduction This document describes a horizontal method for the detection of Salmonella spp in food (including m i l k and m i l k pro duc ts , origi na l ly de s crib e d i n I S O 678 ) , i n an i ma l envi ron menta l s ample s from fe e d, i n an i ma l fae ce s , a nd i n the pri ma r y pro duc tion s tage (the latter two were origi na l ly de s crib e d i n ISO 6579:2002/Amd 1:2007) The main changes, listed in the foreword, introduced in this document compared to ISO 6579:2002, are considered as minor (see ISO 17468 [37] ) A procedure for the enumeration of Salmonella spp is described in ISO/TS 6579-2 [3] f f Salmonella spp is described in ISO/TR 6579-3 [24] Gu id ance or s ero typi ng o © ISO 2017 – All rights reserved vii INTERNATIONAL STANDARD ISO 6579-1:2017(E) Microbiology of the food chain — Horizontal method for the detection, enumeration and serotyping of Salmonella — Part 1: Detection of Salmonella spp 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 Persons using this document should be familiar with normal laboratory practice This document does not purport to address all of the safety aspects, if any, associated with its use It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any national regulatory conditions Scope f f Salmonella It is applicable to the following: — products intended for human consumption and the feeding of animals; — environmental samples in the area of food production and food handling; T h i s c u ment s p e ci fie s a hori z onta l me tho d — s a mple s from or the de te c tion o the pri ma r y pro duc tion s tage s uch a s an i ma l fae ce s , du s t, a nd s wab s With this horizontal method, most of the Salmonella serovars are intended to be detected For the Salmonella Annex D Salmonella de te c tion o f s ome s p e c i fic s erovars , add itiona l c u ltu re s tep s may b e ne e de d For Typh i a nd Paratyph i , the pro ce du re i s des c rib e d i n for the detection of motile Salmonella and is not appropriate for the detection of non-motile Salmonella strains T he s ele c tive enrich ment me d ium mo d i fie d s em i- s ol id Rapp ap or t-Va s s i l i ad i s (M S RV ) agar i s i ntende d Normative references T he fol lowi ng c u ments are re ferre d to i n the te xt i n s uch a way that s ome or a l l o f thei r content s titute s re qu i rements o f th i s c u ment For date d re ference s , on ly the e d ition cite d appl ie s For u ndate d re ference s , the l ate s t e d ition o f the re ference d c u ment (i nclud i ng any amend ments) appl ie s ISO 6887 (all parts), Microbiology offood and animal feed — Preparation oftest samples, initial suspension and decimal dilutions for microbiological examination ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for microbiological examinations ISO 11133:2014, Microbiology of food, animal feed and water — Preparation, production, storage and performance testing of culture media Terms and definitions For the pu rp o s e s o f th i s c u ment, the © ISO 2017 – All rights reserved fol lowi ng term s and defi nition s apply ISO 65 79 -1 : 01 7(E) ISO and IEC maintain terminological databases for use in standardization at the following addresses: — IEC Electropedia: available at http://www.electropedia org/ — ISO Online browsing platform: available at http://www.iso org/obp Salmonella m ic ro orga n i s m wh ich form s typic a l or le s s typic a l colon ie s on s ol id s ele c tive me d ia a nd wh ich d i s plays the ch arac teri s tics de s c rib e d when fi rmation te s ts a re c arrie d out i n accord ance with th i s c ument 3.2 detection of Salmonella determination of (3.1 f (e.g boot socks), when tests are carried out in accordance with this document Salm on ella 4.1 ) , i n a p ar tic u la r mas s or volu me o pro duc t or s u r face are a or obj e c t Principle General The detection of NOTE of other numbers of Salm on ella re qui re s fou r s ucce s s ive s tage s a s s p e c i fie d i n Annex A Salm on ella c a n b e pre s ent i n s m a l l nu mb ers a nd i s o ften acco mp a n ie d b y s iderab l y l a rger nu mb ers En terobacteriaceae 4.2 or bacteria of other families Pre-enrichment is used to permit the detection of low Salm on ella o r i nj u re d Salm on ella Pre-enrichment in non-selective liquid medium Buffered peptone water at ambient temperature is inoculated with the test portion, then incubated between 34 °C and 38 °C for 18 h For large quantities (e.g l or more), it is recommended to pre-warm the BPW to 34 °C to 38 °C before mixing it with the test portion 4.3 Enrichment in/on selective media Rapp ap or t-Va s s i l iad i s me d iu m with s oya (RVS bro th) or M o d i fie d S em i- s ol id Rapp ap or t-Va s s i l iad i s (MSRV) agar and Muller-Kauffmann tetrathionate-novobiocin broth (MKTTn broth) are inoculated with the culture obtained in 4.2 The RVS broth or the MSRV agar is incubated at 41,5 °C for 24 h and the MKTTn broth at 37 °C for 24 h For s ome pro duc ts , it may b e ne ce s s ar y to i nc ub ate the s ele c tive en rich ment me d iu m/me d i a additional 24 h NOTE MSRV agar is intended for the detection of motile detection of non-motile strains Salm on ella for an strains and is not appropriate for the Salm on ella 4.4 Plating out on selective solid media From the cultures obtained in 4.3, the following two selective solid media are inoculated: — Xylo s e Lys i ne D e ox ycholate aga r ( XL D agar) ; Annex E) The XLD agar is incubated at 37 °C and examined after 24 h The second selective agar is incubated according to the manufacturer’s instructions — any o ther s ol id s ele c tive me d ium complementar y to XL D agar (for example s , s e e © ISO 2017 – All rights reserved ISO 65 79 -1 : 01 7(E) Annex D (normative) Detection of Salmonella enterica subspecies enterica serovars Typhi and Paratyphi D.1 General Certain serovars of Salm on ella a re no t s i s tently de te c te d b y the me tho d de s crib e d i n th i s c u ment T h i s a n nex s p e ci fie s add itiona l s tep s to b e ta ken when the de te c tion o f Salm on ella enterica enterica s erovars Typh i and Pa ratyph i i s o f s p e ci fic concern T he shall also be performed fu l l subspecies me tho d de s crib e d i n th i s c ument S tra i n s o f s ero va r Ga l l i n a r u m ( b iova rs ga l l i n a r u m a nd pu l lor u m) a re o f no s ign i fic a nce to hu m a n NO TE health and so their isolation is not included in this document (see Reference [19]) However, if these biovars are described in this annex s p e c i fic a l l y s o ught, S elen ite C ys ti ne me d iu m (S C ) c a n b e u s e d i n add itio n to RVS b ro th a nd M KT Tn b ro th a s D.2 Detection of D.2 Salmonella Typhi and Salmonella Paratyphi Principle WARNING Salmonella — Typhi Salmonella and Paratyphi are Hazard Group organisms Appropriate containment facilities should be used when handling these strains D.2 1 General See 4.1 D.2 Pre-enrichment in non-selective liquid medium See 4.2 D.2 Enrichment in selective liquid media See 4.3 inoculation of RVS broth and MKTTn broth The SC medium is incubated at 37 °C (6.3) for 24 h and 48 h S elen ite c ys ti ne me d iu m (S C ) D P l a t i n g o u t a n d i d e n t i f i c a t i o i s i no c u late d with the c u ltu re ob tai ne d i n 4.2 in addition to n See 4.4 Bismuth sulphite agar (BS) is inoculated with the cultures obtained in 4.3 and D.2.1.3 in addition to XLD agar The BS agar is incubated at 37 °C (6.3 f f f ) and e xam i ne d a ter h , a nd agai n, i D C o n f i r m a t i o n o f i d e n t i ne ce s s a r y, a ter h t y See 4.5 38 © ISO 2017 – All rights reserved ISO 65 79-1 : 01 7(E) D.3 Culture media D.3 Selenite cystine medium (SC) WARNING — Sodium hydrogen selenite is potentially teratogenic and may harm the unborn child Appropriate protective precautions should be taken when preparing and handling this medium D.3 1 Base medium D.3 1 Composition Peptone a Lactose 5,0 g 4,0 g 10,0 g Water 4,0 g 000 ml Disodium hydrogen phosphate dodecahydrate (Na2 HPO 4.12H O) Sodium hydrogen selenite a For example, enzymatic digest o f casein D.3 1 Preparation Dissolve the sodium hydrogen selenite in the water, then add the remaining ingredients Heat to boiling to dissolve D.3 D.3 L-Cystine solution Composition L-Cystine Sodium hydroxide solution, c (NaOH) = mol/l Sterile water D.3 2 0,1 g 15 ml Approx 85 ml Preparation Add the components to a sterile 100 ml one-mark volumetric flask ( D.4.2) sterile water Do not sterilize D.3 D.3 Complete medium Composition Base (D.3.1.1) L-Cystine solution ( D.3.1.2) D.3 Dilute to the mark with 000 ml 100 ml Preparation Add the L-cystine solution aseptically to the base Adjust the pH, i f necessary, so that the final pH will be 7,0 ± 0,2 at 25 °C Dispense the medium aseptically into sterile tubes or flasks (6.12) to achieve a depth o f at least cm Sterilize by steaming for 15 Do not autoclave Store the poured tubes at °C (6.8) The medium may be used until a red precipitate occurs © ISO 2017 – All rights reserved 39 ISO 65 79 -1 : 01 7(E) D.3.2 Bismuth sulfite agar (BS) D.3 Composition Enzymatic digest o f animal tissues Meat extract Dextrose Disodium hydrogen phosphate (anhydrous) (Na HPO 4) Ferrous sul fate (anhydrous) Bismuth sulfite (indicator) Brilliant green Agar Water D.3 2 10,0 g 5,0 g 5,0 g 4,0 g 0,3 g 8,0 g 0,025 g 20,0 g 000 ml Preparation Add the components to the water and heat with frequent agitation until boiling Continue to boil gently for 30 s to 60 s to dissolve the agar and obtain a uniform suspension (precipitate will not dissolve) Cool to 47 °C to 50 °C, then gently agitate to suspend the precipitate Adjust the pH, i f necessary, so that it is 7,7 ± 0,2 at 25 °C Pour 20 ml to 25 ml into Petri dishes (6.14) and allow to set Correctly prepared plates will be a pale straw colour with a smooth cream-like opacity Prepare the plates the day be fore use and store in the dark at ambient temperature Dry the plates, i f necessary, be fore use Do not overdry D.3 Performance testing The definition o f selectivity and productivity is specified in ISO 11133 The inoculum volume should be the same as that used in the method for that medium and should contain the number of target or non- target organisms specified in ISO 11133:2014, 5.4 40 © ISO 2017 – All rights reserved ISO 6579-1:2017(E) Table D.1 — Performance testing for the quality assurance of the culture media Medium Function Selenite Incubation Typhimurium c,d Salm on ella Enteritidis c,d Productivity cystine Control strains Salm on ella 24 h ± h/ 37 °C ± °C Selectivity + + d Esch erich ia coli Pseudom on a s aerugin osa d Esch erich ia coli En terococcu s faecalis Bismuth sulphite agar d Typhimuriumc,d Salm on ella Enteritidis c,d Productivity Salm on ella 2x(24 h ± h)/ 37 °C ± °C Selectivity/ specificity Selectivity d Esch erich ia coli En terococcu s faecalis d WDCM Criteriae numbers a 00031 >10 characteristic colonies on XLD agar or 00030 other medium of choice 00012 or 00013 00025 00012 or Partial inhibition 00013 ≤100 colonies on TSA 00009 or