Tiêu chuẩn iso 06579 1 2017

— The inoculation of the isolation medium has be ome les pr sc iptive; the o je tive is t o o tain wel-isolat ed colonies aft er incub tion... — It is pe mitt ed t o pe form the biochemi

Micr obiolog y ofthe food chain —

Part 1:

Micro iolo ie de la chaîne alimentaire — Méth de h rizontale

p ur la r che che, le dé ombr ment et le séroty a e des

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F reword v

Introduction vii

1 Sc ope 1

2 Nor mati ve r eferenc es 1

3 Terms an definitions 1

4 Principle 2

4.1 General 2

4.2 Pr e-enrichment in non-sele tive lq id medium 2

4.3 Enrichment in/ n sele tive media 2

4.4 Plating out on sele tive sol d media 2

4.5 Confirmation 3

5 Cultur e media, r eag ents, and antisera 3

6 Equipment and c onsumables 3

7 S mpl ng 4

8 Preparatio of test sample 4

9 Proc ed re (se diag rams in A nne x A) 4

9.1 Tes p rtion an initial suspension 4

9.2 Non-sele tive pr e-enrichment 4

9.3 S le tive enrichment

5 9.3.1 General 5

9.3.2 Pr oc d re for fo d, animal fe d samples, an envir onmental samples fr om the fo d prod ction area 5

9.3.3 Pr oc d re for samples fr om the primary pr od ction s ag e 5

9.4 Plating out 6

9.4.1 General 6

9.4.2 Pr oc d re for fo d, animal fe d samples, an envir onmental samples fr om the fo d prod ction area 6

9.4.3 Pr oc d re for samples fr om the primary pr od ction s ag e 6

9.5 Confirmation 7

9.5.1 General 7

9.5.2 S le tion of colonies for confirmation 7

9.5.3 Biochemical tes ing 8

9.5.4 S rolog ical tes ing 1

9.5.5 Inte pr etation of biochemical an ser olog ical r eactions 1

9.5.6 S rotyping 1

10 Ex pres io of r esults 12

11 Perfor manc e character istic of the metho .12

1 1 Inter la or atory s u ies 1

1 2 S nsitivity 1

1 3 Spe ificity 1

1 4 LOD 5

1 12 Test repor t 13

A nne x A (normative) Diag rams of the pr oc edures 14

A nne x B (normative) C lture media and reag ents 17

A nne x C (informative) Meth d valdatio studies and perfor mance character istic .32

A nne x D (normative) Detectio of S lmone lla e nte rica subspecies e nte rica serovar sT yphi

and Paratyphi 38

A nne x E (informative) Examples of selecti ve plating -o t media 43

Biblog raph y 48

ISO (he Int ernational Org nization for Stan ardization) is a worldwide fede ation of national s an ards

b dies (ISO membe b dies) The work of pr p ring Int ernational Standards is normaly car ied out

through ISO t ech ical committ ees Each membe b dy int er st ed in a subje t for w hich a t ech ical

committ ee has be n es a l shed has the right t o be r pr sent ed on that committ ee Int ernational

org nizations, g ove nmental an non-g ove nmental, in laison with ISO, also take part in the work

ISO cola orat es closely with the Int ernational Ele trot ech ical C mmis ion (IEC) on al matt ers of

ele trot ech ical s an ardization

The proc d r s used t o develo this document an those int en ed for it furthe maint enanc ar

desc ibed in the ISO/IEC Dir ctives, Part 1 In p rticular the dife ent a pro al c it eria ne ded for the

dife ent ty es of ISO document should be not ed This document was draft ed in ac ordanc with the

edit orial rules of the ISO/IEC Dir ctives, Part 2 ( e www iso org dir ctives)

A tt ention is drawn t o the p s ibi ity that ome of the element of this document ma be the subje t of

p t ent right ISO shal not be held r sponsible for identifying any or al such pat ent right Detais of

any p t ent right identif ied d ring the develo ment of the document wi be in the Introd ction an / r

on the ISO ls of p t ent de larations r c ived ( e www iso org p t ent )

Any trade name used in this document is information given for the convenienc of use s an does not

cons itut e an en orsement

For an ex lanation on the meaning of ISO spe ific terms an ex r s ions r lated to conformity as es ment,

as wel as information a out ISO’s adhe enc to the Wor ld Trade Org nization (WTO) principles in the

Te h ical Bar ie s to Trade (TBT) se the folowing URL: www iso org / iso/ for word html

This document was pr p r d b the Euro ean C mmitt ee for Stan ardization (CEN), Te h ical

Committ ee CEN /TC 2 5, Fo d a alys i s — Hori zontal meth ds , in cola oration with ISO Te h ical

Committ ee TC 34, Fo d produc ts , Subcommitt ee SC 9, Mic ro iolo y, in ac ordanc with the a r ement

on t ech ical co pe ation betwe n ISO an CEN(V ien a Agr ement)

This f irs edition of ISO 6 7 -1 canc ls an r plac s ISO 6 7 :2 0 an ISO 6 8 :2 0 , w hich ha e be n

t ech icaly r vised It also incorp rat es ISO 6 7 :2 0 /Amd 1:2 0 an ISO 6 7 :2 0 /Cor 1:2 04

The main chang es, compar d t o ISO 65 9:2 0 , ar the folowing

— ISO 6 8 has be n incorp rat ed in this document

— Samples from the primary prod ction s ag e ha e be n ad ed t o the sco e

— Det ection of S lmo ela Ty hi an S lmo ela Paraty hi is desc ibed in An ex D

— Desc iptions of pr p rations of initial suspensions ha e be n r mo ed an r fe enc s made t o

r lev nt p rt of ISO 6 8 , w heneve pos ible

— The t empe atur rang e for incub tion of non-sele tive media has be n ext en ed from 3 °C ± 1 °C

t o 34 °C t o 3 °Cwithout urthe t ole anc

— F or sele tive enrichment, the e is a choic betwe n using the broth or the semi-sold a ar of

Ra p port Vas i adis medium (RVS or MSRV) for fo d, animal fe d samples, an for environmental

samples from the fo d prod ction ar a

— The inoculation of the isolation medium has be ome les pr sc iptive; the o je tive is t o o tain

wel-isolat ed colonies aft er incub tion

— F or conf irmation, it is ac epta le t o pe form the t es s on only one suspe t colony ( inst ead of one

suspe t colony of each medium combination) If thisisolat e t es s neg tive for S lmo ela, four mor

suspe t isolat es from dife ent media combinations shal be t est ed

— It is pe mitt ed t o pe form the biochemical conf irmation dir ctly on a suspe t, wel-isolat ed colony

from the sele tive plating medium The purity che k on the non-sele tive a ar medium can then be

pe formed in p ralel

— Two conf irmation t es s ha e be ome o tional (ß -g lact osidase t es an in ole r action) and one

confirmation t es has be n delet ed (Vog es-Proska ue r action)

— In this document, se olo ical conf irmation (t o se o roup level) is desc ibed F or guidanc on

se oty ing (t o se o ar level), r fe enc is made t o ISO/TR 6 7 -3

— Ta le1 has be n impro ed

— Pe formanc t es ing for the q alty as uranc of the cultur media has be n ad ed t o Annex B

— Pe formanc charact eris ics of MSRV ha e be n ad ed t o An ex C

A l s of al p rt in theISO 6 7 se ies can be fou d on the ISO websit e

This document desc ibes a horizontal method for the det ection of S lmo ela sp in fo d ( inclu ing

mi k an mik prod ct , originaly desc ibed in ISO 6 85), in animal fe d, in animal fae es, an in

environmental samples from the primary prod ction s ag e ( he latt er two we e original y desc ibed in

Micr obiolog y ofthe food chain — Horizontal method

Part 1:

WARNING — In order to safe uard the he lth of laboratory per o nel, it is es ential that tests

for detecting S lmonela are o ly undertaken in pro erly eq ipped lab ratories unde the

co trol of a ski led mic o iologist and that gre t care is taken in the disposal of al incubated

mat erials Per o s using this document sh uld be fami iar with normal lab ratory practice

This document does not purport to addres al of the safety aspects, if any, as ociated with its

use It is the respo sibi ity of the user to establ sh appropriate safety and he lth practices and to

ensure complance with any natio al re ulatory co ditio s

This document spe if ies a horizontal method for the det ection of S lmo ela It is a plca le t o the

folowing:

— prod ct int en ed for h man consumption an thefe ding ofanimals;

— environmental samples in the ar a of fo d prod ction an fo d han l ng;

— samples from the primary prod ction s ag e such as animal fae es, d s , an swa s

W ith this horizontal method, mos of the S lmo ela se o ars ar int ended t o be det ect ed F or the

det ection of some spe ific se o ars, ad itional cultur st eps ma be ne ded F or S lmo ela Ty hi an

S lmo ela Paraty hi, the proc d r is desc ibed in An ex D

The sele tive enrichment medium modif ied semi-sold Ra p p rt- Vas i iadis (MSRV) a ar is int en ed

for the det ection of motie S lmo ela an is not a pro riat e for the det ection of non-motie S lmo ela

s rains

2 Normati ve referenc es

The folowing document ar r fe r d t o in the t ext in such a wa that some or al of their cont ent

cons itut es r q ir ment of this document F or dat ed r fe enc s, only the edition cit ed a ples F or

un ated r fe enc s, the lat es edition of the r fe enc d document ( inclu ing any amendment ) a ples

ISO 6 8 (al part ), Mic ro iolo y o fod a d a imal fe d — Prep ratio o tes t s amples , initial s us pens io

a d dec imal diutio s fr mic ro iolo ic al ex min tio

ISO 7 1 , Mic ro iolo y o fod a d a imal fe din s tu fs — G ene al r q ir ment a d g ida c e fr

mic ro iolo ic al ex min tio s

ISO 1 1 3:2 14, Mic ro iolo y o fod, a imal fe d a d wate — Prep ratio , produc tio , s tora e a d

pe frma c e tes tin o c ultur media

3 Terms and definitions

F or the purposes of this document, the fol owing t erms and def initionsa ply

ISO an IEC maintain t erminolo ical data ases for use in s an ardization at the folowing ad r s es:

— IECEle tro edia: a aia le at ht p:/ www ele tro edia org

— ISO Onlnebrow sing plat orm:a ai a le at ht p:/ www iso org o p

3.1

S lmone lla

mic o rg nism w hich forms ty ical or les ty ical colonies on sold sele tive media an w hich display s

the charact eris ics desc ibed w hen conf irmation t es s ar car ied out in ac ordanc with this document

3.2

detectio of S lmonela

det ermination of S lmo ela (3.1), in a p rticular mas or volume of prod ct or surfac ar a or o je t

(e.g b ot socks), w hen t es s ar car ied out in ac ordanc with thisdocument

The det ection of S lmo ela r q ir s four suc es ive s ag esas spe ified in An ex A

NOTE Salmo ela can b present in smal n mb r an is oft en ac ompanied by considera ly larg r n mb r

of other Ente o a te iac ea or b cteria of other families Pre-enrichment is used t o permit the detection of low

n mb r of Salmo ela or injured Salmo ela

4.2 Pre-enr ichment in non-selecti ve l quid medium

Bufe ed pept one wat er at ambient t empe atur is inoculat ed with the t es p rtion, then incub t ed

betwe n 34 °C an 3 °C for 1 h

F or larg e q antities (e.g 1 l or mor ), it is r commen ed t o pr -warm the BPW t o 34 °C t o 3 °C befor

mixing it with the t es portion

4.3 Enrichment in/ n selecti ve media

Ra p p rt- Vas i iadis medium with so a (RV S broth) or Modif ied S mi-sold Ra p p rt- Vas i iadis

(MSRV) a ar an Muler-Ka ufmann t etrathionat e-no o iocin broth (MKTT broth) ar inoculat ed

with the cultur o tained in 4.2

The RVS broth or the MSRV a ar is incub t ed at 41,5 °C for 24 h and the MKTT broth at 3 °Cfor 24 h

F or some prod ct , it ma be ne es ary t o incub t e the sele tive enrichment medium/ media for an

ad itional 24 h

NOTE MSRV a ar is int en ed for the detection of motile Salmo ela strains an is not ap ropriate for the

detection of non-motile Salmo ela strains

4.4 Plating out on selecti ve sol d media

From the cultur s o tained in 4.3, the folowing two sele tive sold media ar inoculat ed:

— Xy lose L ysine Deo ycholat e a ar (XLD a ar);

— any othe sold sele tive medium complementary t o XLD a ar (for ex mples, se Annex E)

The XLD a ar is incub t ed at 3 °C an ex mined aft er 24 h The se on sele tive a ar is incub t ed

ac ording t o the man factur r’s ins ructions

4.5 Confir mation

Colonies of pr sumptive S lmo ela ar subcultur d an their identity is conf irmed b means of

a pro riat e biochemical an se olo ical t es s

5 Culture media, reag ents, and antisera

F or cur ent la orat ory practic , se ISO 7 1 an ISO 1 1 3

Comp sition of cultur media an r ag ent and their pr p ration ar desc ibed in An ex B

Disposa le eq ipment is an ac epta le alt ernative t o r usa le glas war if it has suita le spe if ications

Usual mic o iolo ical la orat ory eq ipment ( e ISO 7 1 ) and, in p rticular, the fol owing

6.1 A pparatus for dry steriization (o en)or wet ster ilzatio (autoclave)

As spe if ied in ISO 7 1

6.2 Dr ying cabinet or o en, ca a le of o erating betwe n 25 °Cand 50 °C

6.3 Incubator(s), ca a le of o er ating in ther ang e 34 °C to 3 °C an at 3 °C ± 1 °C

6.4 Incubator, ca a le of o er ating at 41,5 °C ± 1 °C or wate bath ca a le of o erating at

41,5 °C ± 1 °C

6.5 Wate bath, ca a le of o erating at 47 °C to 5 °C

6.6 Wate bath, ca a le of o erating at 3 °C ± 1 °C

6.7 Wate bath, ca a le of o erating at 45 °C ± 1 °C

It is r commen ed t o use a wat er b th (6.4 t o 6.7) containing an antibact erial ag ent be ause of the low

infe tive dose of S lmo ela

6.8 Refr ig erator, ca a le of o erating at 5 °C ± 3°C

6.9 F reezer, ca a le of o er ating at -2 °C± 5 °C

6.10 Ster ile loo s, of a prox imate diamete , 3 mm (1 μl v lume), an of 1 µl v lume an inoculation

ne dle or wir e

6.11 pH-meter, ha ving an ac uracy of calbration of ±0,1 pH u it at 20 °Cto 2 °C

6.12 Ster ile tubes, bot les, or flasks with ca s of a pr opriate ca acity

6.13 Ster ile g raduated pipet es or automatic pipet es, of nominal ca acities of 2 ml, 1 ml, 1 ml,

and 0,1 ml

6.14 Ster ile Petr i dishes, with a diamete of a pr ox imately 9 mm an (o tional) lar g e siz (diamete

a pr oximately 140 mm)

7 Sampl ng

Samplng is not p rt of the method spe if ied in this document ( e the spe if ic Int ernational Stan ard

dealng with the prod ct conc rned) If the e is no spe if ic Int ernational Stan ard, it is r commen ed

that he p rties conc rned come t o an a r ement on this subje t

A r commen ed samplng method is given in ISO/TS 1 7 8

for sampl ng of surfac s

It isimportant that the la orat ory r c ivesa sample w hich is r pr sentative and has not be n damag ed

or chang ed d ring transp rt or st orag e

8 Preparation oftest sample

Pr p r the t es sample from the la orat ory sample in ac ordanc with the spe if ic Int ernational

Stan ard dealng with the prod ct conc rned If the e is no spe ific Int ernational Stan ard, it is

r commen ed that the p rties conc rned come t o an a r ement on this subje t

9 Proc edure ( ee diag rams in A nne x A)

9.1 Test por tion and initial suspension

F or pr p ration of the initial suspension, in the g ene al case, use as diuent the pr -enrichment medium

spe ified in B.2 (bufe ed pept one wat er) Pr -warm the BPW t o ro m t empe atur befor use

In g ene al, an amou t of t es portion (mas or v lume) is ad ed t o a q antity of BPW (mas or volume)

t o yield a t enfold diution F or this, a 2 g t es portion is mix ed with 2 5 ml of BPW Howeve , for some

ty e of samples(e.g b ot socks, d s ), it ma be ne es ary t o use anothe ratio

F or spe if ic prod ct , folow the proc d r s spe if ied in ISO 68 7 (al p rt )

This document has be n v l dated for t es p rtions of 2 g A smal e t es p rtion ma be used without

the ne d for ad itional v ldation/ e if ication pro ided that the same ratio betwe n (pr - enrichment

broth an t es p rtion is maintained A larg er t es p rtion than that initial y v l dated ma be used

if a v ldation/ e if ication s u y has shown that the e ar no neg tive efe t on the dete tion of

S lmo ela sp

NOTE 1 Validation can b con ucted ac ording t o the ap ropriate parts of ISO 16 140 Verif ication for po ling

samples can b con ucted ac ording t o the prot ocoldes rib d in ISO 68 7-1:2 17, An e D

[3 ]

F or larg e q antities (e.g 1 l or mor ), it is r commen ed t o pr -warm the BPW t o 34 °C t o 3 °C befor

mixing it with the t es portion

NOTE 2 When more than one 2 g test portion from a specif ied lot of prod ct is to b e amined an when

evidence is availa le that combining t est portions do s not afect the result for that particular fo d, the test

portions can b po led More information on po ling of samples as wel as a proced re t o test the influence of

po ling on the sensitivity of the method can b fou d in ISO 68 7-1

[3 ]

9.2 Non-selecti ve pre-enrichment

Incubat e the initial suspension (9.1) betwe n 34 °Cand 38 °C (6.3) for 1 h ± 2 h

It is pe mis ible t o st or the pr -enriched sample aft er incub tion at 5 °C (6.8) for a ma imum of 7 h

( e R efe enc s [3 ]t o [34])

9.3 S lecti ve enr ichment

9.3.1 General

Alow the sele tive enrichment media, RVS broth or MSRV a ar (B.3 or B.4), an MKTT broth (B.5) t o

eq i brat e at ro m t empe atur if they we e st or d at a lowe t empe atur

Minimiz the trans e of p rticulat e mat erial from the pr -enrichment int o the sele tive enrichment

media

Aft er incub tion, it is pe mis ible t o st or the sele tive enrichment at 5 °C (6.8) for a ma imum of 7 h

( e R efe enc s [3 ]t o [34])

NOTE MSR a ar is inten ed for the det ection of motile Salmo ela strains an is not ap ropriate for the

detection of non-motile Salmo ela strains

9.3.2 Pr oc edure for fo d, animal fe d samples, and environmental samples fr om the fo d

production ar ea

Trans e 0,1 ml of the cultur o tained in 9.2t o a tube containing 1 ml of the RV S broth (B.3) or t o the

surfac of a MSRV a ar plat e (B.4) Inoculat e the MSRV a ar with one t o thr e eq aly sp c d spot on

the surfac of the medium

Trans e 1 ml of the cultur o tained in 9.2 t o a tube containing 1 ml of MKTT broth (B.5)

Incubat e the inoculat ed RVS broth at 41,5 °C (6.4) for 24 h ± 3 h

Incubat e the inoculat ed MSRV a ar plat es at 41,5 °C (6.4) for 24 h ± 3 h Do not invert the plates

Incubat e the inoculat ed MKTT broth at 3 °C(6.3) for 24 h ± 3 h

Suspe t MSRV plat es wi show a gr y-w hit e, turbid zone ext en ing out from the inoculat ed dro

In dried mik prod ct an che se, S lmo ela ma be sublethaly injur d Incub t e the sele tive

enrichment media from these prod ct for an ad itional 24 h ±3 h ( e R efe enc [3 ])

F or some othe prod ct , e.g w hen inves ig ting out br ak samples, this ad itional incub tion time ma

also be beneficial

9.3.3 Pr oc edure for samples from the pr imar y pr oductio stag e

Inoculat e the MSRV a ar (B.4) with 0,1 ml of the pr -enriched cultur (9.2) as one t o thr e eq aly

sp c d spot on the surfac of the medium

Incubat e the inoculat ed MSRV plat es at 41,5°C(6.4) for 24 h ± 3 h

Do not invert the plates

Suspe t MSRV plat es wi show a gr y-w hit e, turbid zone ext en ing out from the inoculat ed dro

If the plat es ar neg tive aft er 24 h, r -incub t e for a furthe 24 h ± 3 h

NOTE S nsitivity can b improved by using a secon selective enrichment proced re, e.g MKT n broth

incub ted at 41,5 °C for 24 h

[3 ]

9.4 Plating out

9.4.1 General

From the sele tive enriched cultur s (9.3), inoculat e two sele tive isolation a ar media The f irs

isolation medium is Xy lose L y sine Deo ycholat e (XLD ) a ar The se ond isolation medium is chosen b

the t es ing la orat ory

Cho sea se on sele tive plating medium w hich is complementary t o XLD a ar an is based on dife ent

dia nos ic charact eris ics t o those of XLD a ar t o faci itat e det ection of, for ins anc , lact ose p sitiveor

H2S-neg tive S lmo ela F or ex mples of isolation media, se An ex E

Alow the XLD a ar (B.6) plat es an the se on sele tive plating medium t o eq i ibrat e at ro m

t empe atur if they wer st or d at a lower t empe atur If ne es ary, dry the surfac of the plat es

befor use ( e ISO 1 1 3)

9.4.2 Proc edure for fo d, animal fe d samples, and environmental samples fr om the fo d

production area

From the cultur o tained in the RVS broth (9.3.2), inoculat e b means of a 1 µl lo p (6.1 )the surfac

of an XLD plat e (B.6) so that wel-isolat ed colonies wi be o tained Proc ed in the same wa with the

se on sele tive plating -out medium

From the positive growth o tained on the MSRV a ar (9.3.2), det ermine the furthes p int of o aq e

growth from the inoculation p int an dip a 1 µl lo p (6.1 ) jus inside the b rde of the o aq e

growth W ith raw the lo p ensuring that no larg e lumps of MSRV a ar ar extract ed Inoculat e the

surfac of an XLD plat e (B.6) so that wel-isolat ed colonies wi l be o tained Proc ed in the same wa

with the se on sele tive plating -out medium

From the cultur o tained in the MKTT broth (9.3.2), inoculat e b means of a 1 µl lo p (6.1 ) the

surfac of an XLD plat e (B.6) so that wel-isolat ed colonies ar o tained Proc ed in the same wa with

the se on sele tive plating -out medium

NOTE 1 To o tain wel -solat ed colonies, larg size Petri dishes with plating-out media (diamet er

ap ro imately 140mm) or two normal size plates (diamet er ap ro imat ely 9 mm)can b used

Incubat e the XLD plat es inve t ed at 3 °C(6.3) for 24 h ± 3 h

Incubat e the se on sele tiveplating -out medium in ac ordanc with the man factur r’s ins ructions

If the sele tive enrichment media ha e be n incub t ed for an ad itional 24 h, folow the same plating

-out proc d r as desc ibed a o e

Ty ical colonies of S lmo ela on XLD a ar ha e a black c ntr an a l ghtly transp r nt zone of r d ish

colour d e t o the colour chang e of the in icat or

NOTE 2 Salmo ela H2 -ne ative variants grown on X D a ar are pin with a darker pin centre

Lactose-positive Salmo ela grown on X D a ar are yelow with or without blackening T e oc ur ence of these

p enoty es is summarized in Ta le 1

Che k the se on sele tive plating medium aft er the a pro riat e incub tion time for the pr senc of

colonies w hich, from their charact eris ics, ar conside ed t o be pr sumptive S lmo ela

9.4.3 Proc edure for samples fr om the pr imar y pr oductio stag e

From the positive growth o tained on the MSRV a ar (9.3.3), det ermine the furthes p int of o aq e

growth from the inoculation p int an dip a 1 μl lo p (6.1 ) jus inside the b rde of the o aq e

growth W ith raw the lo p ensuring that no larg e lumps of MSRV a ar ar extract ed Inoculat e the

surfac of an XLD plat e so that wel-isolat ed colonies wi l be o tained Proc ed in the same wa with

the se on sele tive plating medium

Incubat e the XLD plat es inve t ed at 3 °C(6.3) for 24 h ± 3 h.

Incubat e the se on sele tive plating medium in ac ordanc with the man factur r’s ins ructions

R eturn neg tive MSRV plat es t o the 41,5 °C incub t or an incub t e for a furthe 24 h ± 3h Pe form the

sele tive plating proc d r if, aft er 48 h of incubation, these MSRV plat es be ome p sitive

Ty ical coloniesof S lmo ela on XLD a ar ha e a black c ntr an a l ghtly transp r nt zone of r d ish

colour d e t o the colour chang e of the in icat or

NOTE Salmo ela H2 -ne ative variants grown on X D a ar are pin with a darker pin centre

Lactose-positive Salmo ela grown on X D a ar are yelow with or without blackening T e oc ur ence of these

p enoty es is summarized in Ta le 1

Che k the se on sele tive plating medium aft er the a pro riat e incub tion time for the pr senc of

colonies w hich, from their charact eris ics, ar conside ed t o be pr sumptive S lmo ela

9.5 Confir mation

9.5.1 General

The combination of biochemical an se olo ical t es r sult in icat e w hethe an isolat e belongs t o the

g en s S lmo ela F or charact erization of S lmo ela s rains, ful se oty ing is ne ded Guidanc for

se oty ing is desc ibed in ISO/TR 6 7 -3

[24]

F or some of the confirmation media as spe if ied in 9.5.3 an in B.8 t o B.1 , alt ernative (comme cial)

formulations exis w hich ma also be a plca le for biochemical conf irmation of S lmo ela These

alt ernative formulations ar al owed, pro ided that the pe formanc for the biochemical conf irmation

of S lmo ela is ve ified befor use

F or a clear dis inction betwe n p sitive an neg tive biochemical r actions, it is helpful t o ve ify the

r actions of the media of each biochemical t es with wel-charact eriz d p sitive an neg tive control

s rains

NOTE 1 T e reco nition of colonies of Salmo ela is, to a larg e tent, a mat er of e perience an their

ap e rance can vary somewhat, not only from serovar t o serovar, but also from b tch to b t ch of the selective

culture medium used

If shown t o be r l a le, miniaturiz d g le ies for the biochemical identif ication of S lmo ela ma be

used ( e ISO 7 1 )

NOTE 2 Alternative proced res can b used to conf irm the isolate as Salmo ela sp providing the suita ility

of the alternative proced re is verif ied (se ISO 72 8)

9.5.2 S lectio of c olonies for c onfirmatio

Mark suspe t colonies on each plat e (9.4) S le t at leas one ty ical or suspe t colony for subcultur

an conf irmation If this is neg tive, sele t up t o four mor suspe t colonies ensuring that these

coloniesar subcultur d from dife ent sele tiveenrichment/isolation medium combinationsshowing

suspe t growth

Str ak the sele t ed colonies ont o the surfac of a pr -dried non-sele tive a ar medium (B.7) in a manne

w hich wi l alow wel-isolat ed colonies t o develo Incub t e the inoculat ed plat es betwe n 34 °C an

3 °C(6.3) for 24 h ± 3 h

Alt ernatively, if wel-isolat ed colonies (of a pur cultur ) ar a ai a le on the sele tive plating media

(9.4), the biochemical confirmation can be pe formed dir ctly on a suspe t, wel-isolat ed colony

from the sele tive plating medium The cultur st ep on the non-sele tive a ar medium can then be

pe formed in p ralel with the biochemical t es s for purity che k of the colony taken from the sele tive

a ar medium

Use pur cultur s for biochemical an se olo ical conf irmation.

NOTE For epidemiolo ical p rposes or d ring outbre k investig tions, conf irmation of ad itional colonies,

e.g f ive ty ical or suspect colonies from e ch selective enrichment/isolation medium combination, can b

b nef icial

9.5.3 Biochemical testing

9.5.3.1 General

Inoculat e the biochemical con rmation media with each of the cultur s o tained from the colonies

sele t ed in 9.4 or 9.5.2 F or conf irmation of S lmo ela sp at leas the t es s spe if ied in 9.5.3.2 t o

r sult of the othe conf irmation t es sdo not give a clear identification

9.5.3.2 TSI agar (B.8)

Str ak thea ar slant surfac an s a the but Incub t e at 3 °C (6.3)for 24 h ± 3h

Int erpr t thechang es in the medium as folow s:

a) but

— yelow: glucose p sitive (glucose fe mentation);

— r d or unchang ed:glucose neg tive (no fe mentation of glucose);

— black: formation of hydrog en sulphide;

— bub les or c acks: g s formation from glucose;

b) slant surfac

— yelow: lact ose an / r suc ose p sitive (lact ose and/ r suc ose fe mentation);

— r d or unchang ed:lact ose an suc ose neg tive (no fe mentation of lact ose or suc ose)

The majority of the ty ical S lmo ela cultur s show alkal ne (r d) slant an acid (yelow) but s with

g s formation (bub les) an ( in a out 9 % of the cases) formation of hydrog en sulf ide (blackening of

the a ar) ( e Ta le 1)

When a lact ose-p sitive S lmo ela is isolat ed, the TSI slant is yelow Th s, pr lminary conf irmation of

S lmo ela cultur sshal not be b sed on the r sult of the TSI a ar t es only ( e 9.5.3.1)

NOTE Kligler -Hajna medium gives similar results as TSI a ar

9.5.3.3 Urea agar (B.9)

Str ak thea ar slant surfac Incub t e at 3 °C (6.3) for up t o 24 h

If the r action is p sitive, ur a is hydrolyz d, lbe ating ammonia This chang es the colour of phenol r d

t o rose-pin an lat er t o de p c rise The r action is oft en a p r nt aft er 2h t o 4 h

Ty ical S lmo ela cultur s do not hydrolyz ur a so that the colour of the ur a a ar wi r main

unchang ed ( e Ta le 1)

9.5.3.4 L-L sine decar box ylatio medium (LDC, B.10)

Inoculat e jus below the surfac of the lq id medium Incub t e at 3 °C(6.3) for 24 h ± 3 h

T rbidity an a purple colour aft er incub tion in icat e a positive r action A yelow colour in icat es a

neg tive r action

The majority of the ty ical S lmo ela cultur s show a p sitiver action in LDC ( e Ta le 1).

9.5.3.5 Detection of β -galactosidase (B.11)(o tional

The β -g lact osidase t es can be used t o dis inguish S lmo ela ente ic a subspe ies ari zo ae an

diari zon e an othe membe s of the Ente o ac te iac eae (al give a p sitive r action) from othe

subspe ies of S lmo ela ente ic a ( in g ene al these give a neg tive r action, se Ta le 1)

S ve al proc d r st o pe form the β -g lact osidase t es exis An ex mple is given below

Suspen a lo pful of the suspe t ed colony in a tube containing 0,2 ml of the sal ne solution (B.1 )

A dd one dro of t oluene an shake the tube Plac the tube in a wat er b th set at 3 °C (6.6) an lea e

for seve al min t es (a pro imat ely 5 min) Ad 0,2 ml of the r ag ent for det ection of β -g lact osidase

(B.1 ) and mix

R eplac the tube in the wat er b th set at 3 °C(6.6) an lea e for upt o 24 h

A yelow colour in icat es a p sitive r action Ther action is oft en a p r nt aft er 2 min

If pr p r d p pe discs ar used for the det ection of β -g lact osidase, fol ow the man factur r’s

ins ructions

9.5.3.6 Medium for in ole r eaction (B.12)(o tio al

The in ole t es can be used w hen the e is a ne d t o dife entiat e S lmo ela (g ene aly in ole neg tive,

se Ta le 1) from Es che ichia c ol an Citro ac te (b th in ole p sitive) as these org nisms can give

ty ical r actions on some of the S lmo ela isolation media

Inoculat e a tube containing 5 ml of the try t one /try t ophan medium (B.1 1) with the suspe t ed colony

Incubat e at 3 °C(6.3) for 24 h ± 3 h Aft er incub tion, ad 1 ml of the K ov cs r ag ent (B.1 2)

The formation of a r d ring ( urfac la e ) in icat es a p sitive r action A yelow-brown ring ( urfac

la e ) in icat esa neg tive r action

TSI g s from glucos −

e

h

TSI hy dro en sulfide

pro uc d

f

Vf

9.5.4 S rolog ical testing

9.5.4.1 General

The pur colonies (9.5.2) showing ty ical biochemical r actions for S lmo ela (9.5.3) ar also t est ed

for the pr senc of S lmo ela O- an H-antig ens (an , in ar as w he e S lmo ela Ty hi is ex e t ed in

the fo d sup ly, also for V i-antig en) b slde a glutination using p ly alent antise a (B.14) The pur

colonies ar cultur d on a non-sele tive a ar medium (B.7) an t est ed for aut o-a glutination Strains

that ar aut o-a glutina le can ot be t est ed for the pr senc of S lmo ela antig ens Use the antise a

ac ording t o theman factur r’ s ins ructions if dife ent from the method desc ibed below t o det ect the

pr senc of S lmo ela O- and H-antig ens (an if ne es ary, also for V i-antig en)

The folowing tes s (9.5.4.2 to 9.5.4.5) ar the minim um r q ir d for se olo ical tes ing ofS lmo e lla sp

Furthe guidanc on se olo ical confirmation an on se oty ing is given in ISO/TR 6 7 -3

[24]

9.5.4.2 Elminatio of auto-ag glutinable str ains

Plac one dro of salne solution (B.1 )on a clean glas slde Using a lo p, dispe se p rt of the colony t o

bet est ed in the sal ne t o o tain a homog eneous an turbid suspension

Rock the slde g ently for 5 s to 6 s (depen ing on the man factur r’s ins ructions) Obse ve the

suspension, pr fe a ly a ains a dar k b ckgrou d If the b cteria ha e formed gran les in the suspension,

this in icat es a ut o-a glutination and se olo ical confirmation wi be ome complcat ed Additional

information on the tr atment of a ut o-a glutinating s rains can be fou d in ISO/TR 6 7 -3

[24]

)

9.5.4.3 Examination for O-antig ens

Using one non-aut o-a glutinating pur colony, proc ed ac ording t o 9.5.4.2 using one dro of p lyv lent

anti-O se a (B.14)in plac of the sal ne solution

If a glutination oc urs, this is conside ed a positive r action

9.5.4.4 Examination for Viantig ens (optio al

Using one non-aut o-a glutinating pur colony, proc ed ac ording t o 9.5.4.2 using one dro of anti-V i

se a (B.14) in plac of the salne solution

If a glutination oc urs, this is conside ed a positive r action

9.5.4.5 Examination for H-antig ens

Using one non-aut o-a glutinating pur colony, proc ed ac ording t o 9.5.4.2 using one dro of p lyv lent

anti-H se a (B.14)in plac of the sal ne solution

If a glutination oc urs, this is conside ed a positive r action

9.5.5 Inter pretatio of biochemical and ser olog ical reactio s

Ta le 2 gives the int erpr tation of the conf irmat ory t es s (9.5.3 an 9.5.4) car ied out on the colonies

used (9.5.2)

Biochemical r actions A ut o-a glutination S rolo ical r actions Int erpr tation

aut o-a glutination

(se 9.5.4.2)

Salmo ela

9.5.6 S rotyping

Strains that ar conf irmed as S lmo ela sp (Ta le 2) can be furthe ty ed t o se o ar level Guidanc

for se oty ing is desc ibed in ISO/TR 6 7 -3

[24]

If r q ir d, conf irmed s rains can be sent t o a r co niz d S lmo ela r fe enc c ntr for def initive

ty ing ( e oty ing, phag e ty ing, mole ular ty ing) If the s rain is sent t o a r fe enc c ntr , it hould

be ac ompanied b al r lev nt information such as confirmation r sult , sour e from w hich the s rain

was isolat ed, an w hethe it conc rns an isolat e from an out br ak

10 Ex pres ion of results

In ac ordanc with the int erpr tation of the r sult , in icat e S lmo e lla detected or not detect ed in a t es

p rtion of x g or x ml of prod ct ( e ISO 7 1 ), or on the surfac ar a, or in an o je t (e.g b ot socks)

11 P erformanc e characteristics ofthe method

11.1 Inter laborator y studies

The pe formanc charact eris ics of the method wer det ermined in int erla orat ory s u ies t o

det ermine the spe if icity, sensitivity, an the LOD

5

of the method ( e R efe enc s [6]an [7]) The data

ar summariz d in Annex C The v lues de ived from theint erla orat ory s u ies ma not be a pl ca le

t o matrix ty es othe than those given in An ex C Furthe mor , the pe formanc charact eris ics as

in icat ed in An ex C we e det ermined with in ivid al t es p rtionsup t o 2 g (or ml) When larg er siz

t es p rtions ar used, the pe formanc charact eris ics ma be dife ent

11.2 S nsiti vity

The sensitivity is defined as the n mbe ofsamples fou d p sitive divided b the n mbe of samples

t est ed at a given level of contamination Ther sult ar th s depen ent on the level of contamination of

the sample

11.3 Specificity

The spe if icity is def ined as the n mbe of samples fou d neg tive divided b the n mbe of blank

samples t est ed

12 Test r eport

The t es r p rt hal spe ify the folowing:

— thesampl ng method used, if k own;

— thesiz of the t es p rtion an / r the natur of the o je t ex mined;

— thet es method used, with r fe enc t o this document, i.e ISO 6 7 -1;

— any deviation in the enrichment media or the incub tion con itions used;

— al o e ating con itions not spe if ied in this document, or r g rded as o tional, t og ethe with

detai s of any incident w hich ma ha e influenc d the r sult ;

— ther sult o tained

A nnex A

(normative)

Figure A.1 — Dia ram of procedure for detectio of S lmone lla in foo , animal fe d, an

enviro mental samples from the fo d pro uction are

Figure A.2— Dia ram of proced re for detectio of S lmonela in animal fa ces and in

enviro mental samples from the primary productio stag e

A nne x B

(normative)

The g ene al spe ifications of ISO 1 1 3 ar a plca le t o the pr paration an pe formanc t es ing of

the cultur media desc ibed in this an ex If cultur media or r ag ent ar pr par d from dehydrat ed

complet e media/ reag ent , or if r ady-t o-use media r ag ent ar used, folow the man factur r’s

ins ructions r g rding pr p ration, st orag e con itions, ex iry dat e, an use

The shelf l fe of the media in icat ed in this annex has be n shown in some s u ies The use shal ve ify

this un e their own st orag e con itions (as spe ified in ISO 1 1 3)

Pe formanc t es ing for the q alty as uranc of thecultur media is desc ibed in B.1

9,0 g

Potas ium dihydrog en phosphat e (KH

2PO

If disodium h drogen p osp ate with a dife ent w ater ontent is used, amen

the mas of the ingr dient ac ordingly F or ex mple, in case ofanhydrous disodium

hydrog en phosphat e (Na

2

HPO

4), use 3,5 g

B.2.2 Preparation

Dis olve thecomp nent in the wat er b heating, if ne es ary

A djus the pH, if ne es ary, so that aft er st eri ization, it is 7,0 ± 0,2 at 2 °C

Dispense the medium int o flasks (6.1 ) of suita le ca acity t o o tain the p rtions ne es ary for the t es

St eri iz for 1 min in the aut ocla e (6.1)set at 1 1 °C

St or the medium in closed containe s (6.1 ) at 5 °C (6.8)for upt o six months

B.3 Rappaport- Vas i iadis medium with soya (RVS broth)

4

Dipotas ium hydrog en phosphat e (K

2HPO

4

B.3.1.2 Preparatio

Dis olve the comp nent in the wat er b heating t o a out 7 °C, if ne es ary

Thesolution shal be pr p r d on the da of pr p ration of the complet e RV S medium

Dis olve the ma nesium chloride in the wat er

As this salt is ve y hygrosco ic, it is advisa le t o dis olve the entir cont ent of Mg l

Dis olve themalachit e gr en o alat e in the wat er

The solution ma be kept in a dark glas b t le at ro m t empe atur for at leas eight months

A dd t o 1 00 ml of solution A, 1 0 ml of solution B, an 1 ml of solution C.

A djus the pH, if ne es ary, so that aft er st eri ization it is 5,2± 0,2 at 2 °C t o 2 °C

Dispense the medium int o tubes or flasks (6.1 ) of suita le ca acity t o o tain the portions ne es ary

for thet es , e.g 1 ml q antities dispensed int o tubes

St eri iz for 1 min in the aut ocla e (6.1)set at 1 5 °C

St or the complet e medium in closed tubes or flasks at 5 °C (6.8) for up t o thr e months

NOTE T e f inal medium composition is enzymatic dig st of soya 4,5 g l, sodium chloride 7,2 g l, potas ium

dihydro en p osp ate (KH

2PO

4+ K

2HPO

4) 1,4 g l, an ydrous ma nesium chloride (MgCl

2) 1 ,4 g l, or

ma nesium chloride he ahydrat e (MgCl

2

⋅6H

2O)2 ,6 g l, an malachite gre n o alate 0,0 6 g l

B.4 Modified semi-sol d R appaport- Vas i iadis (MSRV) agar

NOTE S e R eference [1 ]

B.4.1 S lution A

B.4.1.1 Compositio

E zymatic dig es of animal an plant tis ue 4,6 g

Potas ium dihydrog en phosphat e (KH

2PO

4

B.4.1.2 Preparatio

Dis olve the comp nent in thewat er b heating t o a out 7 °C, if ne es ary

The solution shal be pr p r d on the da ofpr p ration ofthe complet e MSRV a ar

As this salt is ve y hygrosco ic, it is advisa le t o dis olve the entir cont ent of Mg l

Dis olve themalachit e gr en o alat e in the wat er

The solution ma be kept in a dark glas b t le at ro m t empe atur for at leas eight months

It ma be ne es ary t o det ermine ex e imental y the conc ntration of a ar

ne ded for the o timal sw arming ofS lmo ela (e.g w hen using a b t ch of a ar with

u k own g el s r ngth)

B.4.4.2 Preparatio

A dd t o 8 0 ml ofsolution A, 1 0 ml of solution B, an 1 ml of solution C an mix b a itation

A dd an suspen the a ar

A djus the pH, if ne es ary, so that aft er st eri ization, it is 5,2 (5,1 t o 5,4 ) at 2 °Ct o 2 °C

Heat t o b i ng with a itation Do not autoclave

Do not hold the medium at high t empe atur s long er than ne es ary

B.4.5.2 Preparatio

Dis olve theno o iocin sodium salt in the wat er

St eri iz b filtration through a f ilt er with a p r siz of 0,2 μm

The solution ma be st or d for up t o four we ks at 5 °C (6.8) or in smal portions (e.g of 2 ml) at - 20 °C

(6 9)for upt o one year

Asepticaly, ad 2 ml of the no o iocin solution (B.4.5) t o 1 0 0 ml ofb se medium (B.4.4) at 47 °C t o

5 °C Mix car fuly The final conc ntration of no o iocin in the complet e medium is 1 mg l

The f inal pHshal be 5,2 (5,1 t o 5,4 ) at 2 °C t o 2 °C

Pour the medium int o st erie Petri dishes (6.14) up t o a v lume of 1 ml t o 2 ml in dishes with a

diamet er of 9 mm

Alow the medium t o soldify befor mo ing an han le with car

St or the plat es, with surface upwards, an prot ect ed from drying for up t o two we ks at 5 °C(6.8) in

the dark

Do not invert the plat es as thesemi-sold a ar is t oo lq id t o do so

Any plat es in w hich the semi-sold a ar has lq ef ied or fra ment ed shal not be used

Immediat ely, befor use an only if visible mois ur is a p r nt, dry the surfac of the a ar plat es

car ful y, for ex mple, b placing them with the lds of an the a ar surfac upwards in a laminar air

flow ca inet Take car not t o o e dry the medium

NOTE 1 T e composition of MSRV a ar, as des rib d in R eference [1 ], contains 2 mg l of novobiocin

However, from a s ientif ic point of view, 1 mg l novobiocin is prefer ed Stu ies have shown larg r migration

zones on MSR a ar with a lower concentration of novobiocin

[2 ]

an the (ne ative) influence of novobiocin on

b cterial motility

[2 ]

NOTE 2 T e f inal medium composition is enzymatic dig st of animal an plant is ue 4,6 g l, acid hydroly sate

of casein 4,6 g l, sodium chloride (NaCl) 7,3 g l, potas ium dihydro en p osp ate (K

2PO

4) 1,5 g l, an ydrous

ma nesium chloride (MgCl

2) 1 , 9 g l, or ma nesium chloride he ahydrate (MgCl

2

⋅6H

2O) 2 ,6 g l, malachit e

gre n o alat e 0,04 g l,novobiocin sodium salt 0,0 g l, an a ar 2,7 g l

B.5 Mul er-Kaufmann tetrathionate-novobiocin (MKTTn) br oth

NOTE S e R eference [14]

B.5.1 Base medium

B.5.1.1 Comp sitio

C lcium carb nat e (C CO

3

Sodium thiosulfat e pentahydrat e (Na

2S

2O

Dis olve the component or the dehydrat ed complet e medium in the wat er b heating, with fr q ent

a itation, u ti the medium s art t o b i Avoid o e heating

A djus the pH, if ne es ary, so that it is 8,0± 0,2 at 2 °C

Thoroughly mix the medium

Theb se medium ma be st or d in closed flasks (6.1 ) at 5 °C(6.8) for up t o thr emonths

B.5.2 Iodine- odide solution

Complet ely dis olve the p tas ium iodide in 1 ml of wat er, then ad the iodine an diut e t o 1 0 ml

with st eri e wat er Do not heat

St or the pr par d solution in a ( ightly) closed containe (6.1 ) in the dark for up t o one year

B.5.3 Novobiocin solution

B.5.3.1 Comp sitio