INTERNATIONAL ISO

STANDARD 6579-1

First~dition
Z017-O~

Microbiology of the food chain -

Horizontal method for the detection,
enumeration and serotyping of
Salmonella -
Part 1:
Detection of Salmonella spp.
Microhi()ICJ9"~de /(1 chatlle uJ;,"entairl! - Mcthotit!hQri%olfto/~
ptJur la ~herche. Ie dt!llombremettt et Ie s~rol)'p(Jge des
Salrn(lflella -
PQrrJ~
1:Rt!ch~rcht!
da Sahnon~I1l'ls.DP.

R.e:feTencefHlmbu
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 ISO6S7~·1::Z017(E)

Contents

I Scope,_ ,_ j,,_ ,_ ,._

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.--1
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2 NonnatIVe references __ ..;---.

3 1
.. Prlnclple .._ ...._ ._ .._. _.. . _. __ ...._ .... ~ .._ .... _ .... _ .... 2
4.a Cen"'t'aJ_,,_._ ,,'_" ~ ~ ~~., .2
4.2 Pre·.nrlchqll:Jlt In I1lIn·~dcai"u IIqulUmedlulll ......_ .... _._ ...._ ._.. , .....2
43 Enrichment in/on $.el~ctiVetnedi~,
4.4 ·~I.tlnc \lut <Ills,'laaivl1$lllid utcdill-. _" .._,,... _, ,.._. ,_. , ,.2
4$ Cnnlirmatioll_ .....__ ,. 3
S Culture I"e<ll>l,reagents, on'd anlisern
Equlpment;md cnnllUmabltm
.., ~ 3
6
1 SalnpIIi)C_· -_.- ...__ .._ ---_ .... -"-"_'-'--- 4
8 Preparation of __ .llple.., ...... •• 1 .. " .....
9 ·I!roce.dure (s~ dlaK!'lllllS in /\onu A)-=-.. ,,~ 4
9.:1 Test RprtiQrfand inlllal suspension ....__ .... ._ ". ....4
9.2 Noo·selectivIlpre--."nchmen,- __ '__ '_1-..4
9,3. Selectiveenrichment ._ ....._ .__ .._ .. .. .• _._._._ ..._..5
<".3.1 G_eneral .." .... ,.,__ . . ,.~,,_._ ....._,. ,"_ 5
9.3.2 P...u:wuremr !UII'a,BRilnn!r"",'1."hlplc" arld'i!rivliilnment>l,ample.
ftt;J.u lh,"'rood f.'rocJul.1ion ;U't'A !'
0- 'I "'" ,t! '!'T' '" 0, ".5
9.33 Pl'oo-e'd"re.rorsamples from the primal')! prod1Jclian sta'g"'- .. _._.__ ,_ ...5
9.4 PlAtin~Qu"j' , _.. . ..... .__ ._ _.6
9.4.1 Ceneral ...__ , ... ., .._ ,,_._._._ .._ •..••• _._ ... ,," ..._ .... _." ..... _ ...... _6
IM.2 Precedur e- (or food, ."In,A1 f,ed ,",rnpl~., .ncl.e,wironmepDI •• ,nRIes
f.ornille r<Joll"rodurtibn area._. .._,,_._....,_"__ ",__ ,,, __ ,_, .... 6
1M3 p"",,,dur~ for ~nl"l!$ frnn,'he l'I'i'!I.'Y l'roduQionsbg _ __ _6
9.5 Conflr:rnatfon .. _... , ,. _ ,,_ "_0_ ~"._ . . __ . " _ ... _~, .,_,.,
9.S,1 Genel'Dl.., _.~ __ .__ ...__ ..__ ... .._,. __ i
9.5.2 Selection of colonies ro,' cenflrmatfon __ ". . ._ ..._. _, ,.._._.. .... ,,,?
9.53 81oclmlllClllle.rtlllG ~_. __ " _8
9.SA· Serologtcaltl'>'tirig_ •._ ... _ .__ .. •__ ._ '" _ •.,__ ,__ 11
'.1,5.5 Interpre.tatibttof bluc:hemk;rl lind Berolog;r.l·reactions ..__ ,,__ , ll
'1.5.6 Serolyplng , ,. _ . ""__ ,_,, , ..12
10 sw"Irn$.Unn
fir _ullll l2
11 l'er(onn:mce cllnn>cmrlSfics of tile mdhod. , _._ J2
111.1 hlterl"boratllry scudi" . ..__ _ 1?
11.2 S,l'usiliv}l)1 ,. ,,,_,,_,_,_.. 12
1'1.3 SpCt'iltclty _.._ __,, __ ._._. '12
11.4 11005<'_ ",,._." '" ..._ ..._._ .._ ...._ .. ,,,._,. ~!._.n 00 __ '" _ .. , ... _'''_" •• _. ~._." )'2
12 'feslr't!potl.. __,,' .,_" .,_,.. ,_" ..._. _. .._. ,,~. ,_ ... ,_., "._, ".-....., _, 13
AnneJtJ\ (n(orlnac;vc)Olagr.lJru ottll~ p)'OO!dureJl__ . ..__ ...__ .. _J ..

ADJleJfll ((lonr!11dvo) Culture medIa and reagents.. '_'" .•_,,_ ."__ , ..__ . ,,__ , ,.,_. ... .". , _,,' 17
Ann"", C(intonn<>livt.)M.thod validation studles'.and·perfor.llIanee cbaracb!risUc:s .. 3'l
" . I
Annex 0 (normntlve) DetectlOJl of SnltnOnl!'llnente,kd $ubspedes enteriC/I se""vars 'l'yphl
andI'Iitutypld~~ ,,_. __ .. ,,
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ISO 65":79-1:201 '1(E)

Annex' E (inmnnativej Example. ohetetth>e plating·aut"medla .__ . !lll

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 ISO 6579·1:2017(E)

It is permitted to perform the biochemical confirmation directly on a suspect, welt-isolated colony

from tho.selecnve plating medfum. Th~ purity choel<on the non-setecrlve agar medjum can tben be
performed in parallel.
Two conflrmation tests have become optional (fl-galactosidase test and indole reaction] and one
confirmation test has been deleted (Voges·Proskauer reacnon),
In this document, serological confirmation (to serogroup level] Is described. For guidance on
serotyptng (to serovar levell, reference Is made to ISO/TR 6579·3.
Tahle 1 has been Improved,
Performance testing for tbe quality assurance of the culture modi. has been added to Anne" B
Perfermance charnctertsrtcs of MSRVhave been added to Anon C.
A list ofall parts in the ISO6579 series can be found on the ISOweesue,

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 ISO6579-1:2017(£)

Introduction
ThIs document describes 3 horl20nr31 method ror the detectlon (.1' Salmonella spp, In I'ood (IncludIng
milk and mUk products, orlgin3IJy descrIbed lo ISO 6785). In animal reed, In animal faeces, and in
environmental samples ("001 the prhnary production stage (tbc tatter rwo were orlglnnlly descrlbad In
ISO 6579:Z00Z/Atnd 1:2007).
The maln changes, listed In the (oreword. introduced In thls docu ment compared to ISO6579:2002, are
considered AS minor (see ISO17468[1lI),
A procedure for the enumeration of Salmonella spp, is descrIbed In ISO/TS6579-2.[31
GuIdance for serotyplng of Salmonella spp.ls described in ISOjTR 6579-3.lZiJ

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 INTERNATIONAL STANDARD ISO6579-1:2017(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 safegum'd the health of laboratory personnet, It Is essential that tests
for detecting Salmonel/a are only undertaken In properly equipped laboratorle.< under the
centrol of a skilled microbiologist and tbnt grent CAre is taken In the dl.<posRI of nil Incubated
materials. Persons using this document should be familiar witt, nannal laboratory practice.
This document does not purport to address aU of the safety aspects, If any, associated with Its
use. It is the responsIbility of tile user to establish appropriate safety and health practices and to
ensure compliance wllh any national regulatory conditions.

1 Scope
This document spcclftes • hOl'lrontol method for the detection of Salmollollo. Ills apptlcable to the
follQwing:
products intended for human consumption and the feeding of animals:
envlronmental samples III the area of food producrlcn and food handling;
samples from the prlmnry preducnon stage such as anlmal faeces, dust, AndSWAbs.
With this horizontal method, most of the Salmonella serevars Me intended to be detected. POI' the
detection of some specific serovars, additional culture steps may be needed. For Salmonella 't'yphi and
Salmutrello Paratyphi, the procedure is described in Anne, D.
The seteetlve enrlchment medlum modUled seml·solld RapPRllort·V.sslllftdIS (MSnV) Agar Is Intended
for the detection er mente Salmollella and Is no! appropriate for the detection of non·motlieSalmollella
strains.

2 Nonnativereferences
The (allowIng documents are referred to In the texl In such a way that some or all of their content
constltutes requirements of this document, For dated references, only tbe edition cited applies. For
undated references. the latest edition of the referenced document (Including ony amendments) applies.
ISO6987 (atll,"rrs). MkrtlhlDlo.qy oJ/f1Odulld ollimal fei!tl- P,."poraliDJI afIMC romplt!.<.llIillol sasp_lIs/on
alld decimal dilutions for microblolo.gical e.yumillol/on
ISO n la, Microbiology Of Ji>od and 11IIimal/itedlng stUffs - titlJaral raquircmellts alld guie/ance {or
microbiological examinations
ISO 11133:2014, Microbiology of food, animal (eed and water - Preparation, production, storoge and
performanu C'StillQ uf culture ml!dld

3 Terms and definltions

For [he purposes of this document, the followIng terms and deflnjtlons .pply.

!'J.__ '~"" ~J.a17 - AJ1I'!shrs reSH\{cd ~f,uO ....0I.1N.tW~CI_U 1

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 ISO6579-1:2017(£)

ISOand lEe maintain tenninological databases for use in standardization at the following addresses:
IeC Elecrropedta; m",i1ableat brtp-//wwwelectrapedja prgl

ISOOnline browsing platform: available at hum/lwww Iso Ql'llit.hl'

3.1
Salmonella
microorganism which forms typical or less typica I colonies on soud selective media and wllren displays
the characteristlcs described when confirmation tests are carried outin accordance with this document

3.2
detection of Salmonella
determination of Salmonella (3..1), in a particular mass or volume of product or surface area or object
(e.g. boot socks), when tests are carried out in accordance with this document

4 Prlnetple

4.1 General
The detection of Salmonellarequires four successive stages as specified in Anne" A.
NOTE Soltnollcll0 tan be present fn ~nulil nunlhern nnd is often aecompanled hy considerably larg~J' numbers
of other linterobact~riac:eo.e or bacteria of other f1Jntilie-s.17e-enricbment i.s used to permit the deteGtion of low
nUI"bers or S(JI,nolll!/lu or Inlurl!{! So/lT1(tnttJ(a,

4.2 Pre-enrichment in non-selectiveliquid medium

Buffered peptone water at ambient temperature Is inoculated with the test portion, then ineubatcd
between l4·C and 38·Cfor lB h.
For large quantities (e.g. 11 or more), it is recommended to pre-warm the BPW to 34'C to 38·C before
mixing It with th e test portion.

4.3 Enrichment In/on selective media

Rnppaport-Vossiliadls medium witll soy. (RVS broth) or Modified Semi-solid Rappsperr-vassllladls
(MSRV) agar and Muller-Kauffmann tetrathlonate-novobiccin broth (MKTTn broth) are Inoculated
with the culture obtained In ll.
The RVS broth or the MSRVagar is incubated at 4t5'C for 24 b and the MKTTn broth at 37·C for 24 h.
For some products, it may be necessary to incubate the selective eurichment medium/medta for an
addilional24 h.
NOTE ~fSRVagar is intended for the detection of motile So/l1Jonella sarains and is not appropriate fOT the
d41U!ctton itf non-Jllorlle$ll/ll'tnnel/Q stralns,

4.4 Platingout on selectivesolid media

From the cultures obtained ill U the following two selective soltd lI1edia Arc Inoculated:
Xylose Lysine Deoxycholate agar (XLDagar);
any other solid selective medium complementary to XLDagar (for examples, See Annex lV.
The XLDagar i~ tncubated at 37 ·C and examined after 24, h. The second selective agar is tneubared
according to the manufacturer's lnstructtens.

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 ISO 6579-1:2017(£)

4.5 Confirmation
Colonies of presumptive Salmonella are subcultured arid thel r Identity Is confirmed by mea ns of
approprfate blochcrnlcal and serologlcal tcsrs.

5 Culturemedia, reagents, and antisera

For current laboratory practice. see ISO7218 and ISO 11133.
Ccmposlrlcn of culture media and reagents and their preparation are described in Annex R.

6 Equipmentand consumables
Disposable equipment Is an acceptable alternative to rCtlsnble glassware if It has suitable spectlleattcns.
Usual microblelogical laboratory equipment (see ISO 7218) and,ln particular, the following.

6,1 Appar'urusJor dry sterilization (oven) or wet steriUza!lOIl (autoclave).

As specified In ISO 7218.

6.2 Drylng cabinet or oven, capable of ollernting between 2S ·C and 50°C.

6,3 Incuoo(or(_), capable of operating in the range 34 ·C 10 3B·C and at 37·C ± 1 .c.
6.4 IncubalDr, capable of operating at 41,5 ac ± 1 ·C or water baUl capable of operating at
41,5 ·C ± 1 .c.
6.5 Water bath, capable of operating At47·C to 50 ·C,

6,6 Water batl~ capable of operating At 37·C t 1 .C,

6,7 Water bath. capable of operating at 45'C t 1 ·C

It Is recommended to use a water bath UWl: to.Ci.Z)contatnlng an antibacterial agent because of the low
Infective dose of'Salmonello.

6.8 Refrigerator, capable of operating at 5 ·C t 3 ·C.

6.9 Freezer, capable of operating .t-20·C;!; 5 ·C.

6.10 Ste.rile loops, of approximate diameter, 3 mm (10 ~IIvolume), and of 1 fll volume and Inoculation
needle or win.

6.11. pH-meter, having an accuracy of CAlibration of to,1 pH unit at 20 OCto 25 'c.

6.12 Sterile tubes, bottles, or Oasks with caps of appropriate capadty.

6.13 Sterile graduated pipettes or automatic pipettes, of nominal capacities of 2S ml, 10 ml, '1 ml,
and 0,1 rnl

6.14 Sterile PNri dishes, with a diarnct1>rof appr'oximately 90 rum and (optional) large size (diameter
allproxima!£ly 140 mill).

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ISO 6579-1:2017(8)

7 SaropUng
Salllpling',s not part ollh. Inotb01tJt1K'ei(!ed ill Ihlu(oCluncnt (see "''' specific !lIt.l·natlOnal Sl;'Indal1"i
d."lillg wi! It lh~ 1)!odU',."mP'l!rnedj. If th~t~ Is 110s"odn~'nt.m"ti"JI~1SlilIldllnl. it is n:<lUllllU~ndoId
thall,he Ilarl'leli Cllllft'I'nocl rome te nn "gl'll~l11flnl on this s~bject,
A r~ml)lmetld.d ""mplinjf method is:giv<>n in ISOll'S 17:7281.u:.J 1M fond a'nd anin"all'~d, in ISO701tl!IJ
fell' milk aniJ I(III~ l)I'.odu,CiS.If! I~() 13~o71m!to. $<ll11pl[11ll at tbe 11l'lrnQry PI'OIiu<;tiQII stalll', in
tSf) 176l14t.nl far .;an'lllmg ofcaro-..Rcs, allil in ISWI1l593tZs) '01'sumplirill M'Surf"",:.~.
It ls lmpcrtant that the I.>bor.,=y receives D sample wblcb is representattve nnd hasnot been dQlll9g~d
or cbJll)ged dllrl'ng ITolnSJlon or STorage.

8 Preparation oftest sample

Prepare th.e test sample fraln thl' laboratory sa mpll' In acecrdance ,.nth tbe specUk Jnrernarlonal
Standir.d de'3ling wuh the predur; concerned. If there is no ~Jle6fic Jnterlutiollal ~'l'.n'dlIrd. it is
reeemmended tlmt the parties concerned come to an 'IIlfrceOlcm on this subjoCt.

9 Procedure (see diagrams in Annel( A)

9.1 lJ'est portion and initial suspenslen

Pur pl'cpo""~iflh of1'hp inlrial Sw;pIl1lSil>n,In rhll gl:il,~r~1
CoIlSe"\I~D"'~ dlhillol thn.pre·fIIlll,:hIO.Dl mudlum
specified In n.:z' {buffered pollto,l" water], I'l'e'wal'lTl th ...B!'W 10 rcemreinperature l>clore use.
ss 0" volhm~)
In general. all ,,,nounl af I~ portioll (mass 0.' voluJl\e) i$ Md~d lO'~'ql1al)th:y of fIl'W [(11(\.
I\Jyield n 1'L'1.folddilmtlln. I'm; thi~, a 2S·g tO~1pm-tiull C"1Ii)(~dwith 22fi 1111
uf BPW.If"w~vl!l'.for fum"
tyl1" of saml1feo<(ell' boo!.:Sl)cks. duSt).lt'may be M=IYUlIlScanother ral'(}.
For SpetI rrcprodum, follow the procedures spedfted In IS06887 ("n IlOIrts).
Thls dnCUllIJll)luss liMO va-liJ.larrJlI1(11'U!.S' IIOl'IJrms 01 2S g. i). lllllaJJpJ' lest I'nI~Jo" O'l1Ylie used wllll0Ut
pruyldud U\31 rhe same raUc, benw*,il (lll'L1')OlIriLbtnrl1t
Ib,e o~ei1 for'(lddlUonal vatrd.1tf<ll\lllori(l<Oltf(1J)
broth 30~tc~1 pnrUul\ is nt.1lntJIill.tI. ,\ latl!el' t~S.1pnrrlcn Wan that Inil;alJy v'allilaJed ",ayll. uS<!tl
If n vaUdatlon/.ye.rlOradou slI,dy ,,~S shuwn thai there ~~e 110 nt.gIlUve e([eds (Ill tl.e lI~tect1()l1or
S(lIIl!OIJ~II(lSlIP·

NOTE L Valiilat~mtan bt: mnduc:rea oc:cnrdlng ,I> the "IJllflJJU'inl1l.

sam pl•• ("n be condllcted _"«ling.,,,
,."J'~
of ISO II5HO -VP,'iIjCnr1<,i. ror ",mUng
the I.ratpont cIe.<cri~.in ISO (lOO'r-:l,2011;1U.._'OI::ill1.
For large Qual'r!tfes (e,s. 1 I or morel, it is remtnmencilld rn pre-"""I'm thr UPW to 34 'r. til 58 ·C betOre
mhring,lt \vith the test pnrt;fali.
NOTll. When more 'ho. nne 'l.'; B resr [lnn'M from 11 <per.'''e<lln. nflmm.iar·iS w be .""min.cd nnd'1A1>.n
"'''Id.ll"e l~ aYtlIl.bt.lIt~1",,~b",j"&.l".sl lim11"It$ <toc.s ~o\ ",lfutl !he ... :<\,iI (or Ibi1l ">Ttlcular. f"."J. the lest
."'rUII'" can be PlIot.ct Mur" tnlin·lIl.llnn "n 1,,"011118(If ~'''m~JI\t as well"". pr<I<rJtl.... II>(ilS\ lbe m1lWlll.eor
p,oQltngon Ihe.<enslt1vlty"fthe me.thqd<an befnund In ISQ68S7-1P81.

9.2 Nun-selective t)l'e-enrichmenl

IUalbate the InItial suspension (!1J.J between 34·C and 38'C lti:3) for La b *'Z h,

[I t.• p~r,"iS.'ijble 10 $TV~ lire pr .. ·.~nrrch,.d ,,,",ple ultel' il.CI1l);ttioll "I fi ·C r!!.!!l r<lr~ III~JCjlllum urn h
(SlleReretell~, [3ilJ 10[~l)·
 ISO6579-1:2017(£)

9.3 Selective enrichment

9.3.1 General
Allow the selective enrichment media. RVSbroth or MSRVagar (B.l or IU), and MK'TThbroth (.B..5.) to
equlllbrate at room temperature If they were stored at a lower temperature.
Minimize the transfer of particular" material from the pre-enrichment into the selective enrichment
medi~.
After incubation, it is permissible to store the selective enrichment at 5 'C (fIJij for a maximum of 72 h
[see References llOJ to [MD.
NOTe MSRV"8"r I. Intended for the d.terllon of ruou!e Snlmoll.I/O strnln5 .nd Is not npproprlD" for ,h.
detection ofnon·r:tC)tll~SoI11KlI1el1a
stralbt.

9.3.2 Procedure for food, animal reed samples, and environmental samples from the food
production area
Transfer 0,1 011 of the culture obtained In ~ to a tube contaln!ng 10 OIlof the RVSbroth (.11.3) or to the
surface of a MSRVagar plate (M). Inoculate the MSRVagar with one to three equally spaced spots on
the surface of the medlum,
Trnnsfer 1 ml ofthe culture ebtained in ~ to R nib. conrainlng 10 ml ofMKTl'n broth (R..5).
Incubate the inoculated RVSbroth at 4.1,5·C (6.!) for 24 h ± 3 h.
Incubate the inoculated MSnVagar plates at 41.S·C rU) for 24 h ± 3 h. Do not Invertcbe plates.
Incubate the inoculated MK'l'Tn broth at 37 ·C (63) for 24 h ± 3 h.
Suspect MSRVplates will show. grey-white, turbid zone extending out from the inoculated drop.
lit dried milk products and cheese. So/molleI/o Olay be sublethally !njll,·ed. Incubate the selective
enrichment media from these products for an additlona! 24 h t 3 h (see Reference [.3!i)).
For some other products, e.g. when investigating outbreak samples, this additional incubation time may
also be beneficial.

9.3.3 Procedure for samples from tb. primary production stage

Inoculate the MSRVagar (IU) with 0.1 ml of the pre- enriched culture (.\t.Z) as one to three equally
spaced spots on the surface of the medium.
Incubate the inoculated MSRVplates at 41,5 'C (.6&] for 24 h t 3 h.
Do not Invert the plates.
Suspect MSRVplates wUlshow. grey-white, turbid zone extending out from the Inoculated drop.
Inlle plates are negauve after 24 1\, re-tncubnte for a further 24·h :t 3 h.
NOTE 5r'n.sIUvlty can be" hllproVed by u.sJng a second setecuve e:nrJchJt1e'nt procedure, e.g. M_KT'I'nbroth
Incubated at 41.5 "C for 24 hUiI

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 ISO6579-1:2017(E)

9.... Plating nul

9.4.1 General

From the selective enriched cultures (2.3), Inoculate two selective isolation agar media. The flrst
isolation medium is Xylose Lysine Deoxycholate (XLO)agar; The second isolation medium is chosen by
the testing laboratery.
Choose a second selective plating medfum which Is complementary to XLDagar and is based on different
diagnostic eharacrertsucs to those of XLOagar to Iacllltate detection of. ror Instance. lactose posltJve 01'
H2S·negative Sa/mortel/a, For examples of Isolatiol1 media. see An"," Ii,
Allow the XLO apr (R.6.) plates and the second selective plating medium to equilibrate at room
temperature if they were stored at a lower temperature, If necessary. dry the surface of the plate.
before use (see ISO 11133).

9.4.2 Procedure for (ClOd,"nl ma 1r"ed samples, and environmental samples from the food
productlon area
Prom the culture obtained in the RVS broth (2.3..Z), inoculate by means ofa10 III loop (b.J.ll) the surface
of an XLOplate (BJi) so that well-isolated colonies will be obtained. Proceed in the same way with the
second selective plating-out medium.
Ilrom the positive growth ebrained on the MSRV ngDr (2.U). determine the furthestl'oint of opaque
growth from the inoculation points and dip a 1 IIi loop ~ just inside the border of the opaque
growth. WithdrAW the loop ensuring that no large lumps of MSRVagar arc extracted. Inoculnt~ the
surface of an XI.Oplate (R.6) so that well-Isolated colonies will be obtained. Proceed in the same way
with the second selective plating-out medium.
flrom the culture obtained in the MKT1", broth (!!.3.2J, Inocurate by means of a 10 III loop (6..Ul) lhe
surface of an XLOplate eR.6.) so that well-Isolated colonies are obtained. Proceed in the same way with
the second selective plating-cut medlum,
NOTE'l To obtain well·IsolAted colonies, large Site relli dishes with plating-out .,edi. (diameter
appmxim"tely J 40 mm) OJ" tWQ normal stze plates lcliaml:!ter t'ppr()ximately 90 mm) can be used.

Incubate the X10 plates Inverted a137'C (U) for 24 b ± 3 h.

Incubate rhe secend selective pla[lng-out medium In accordance with lhe nlonuracturer's tnsrructtons,
If the selective enrichment medii! have been incubated for an additional 24 h, follow the same plating-
out procedure as described above.
'fyp!r.l colonles of Salmonelki all XLDagar have a black centre and 11lightly transparent zone of reddish
colour due t'otho colour chnngt! or the lndlcator;
NOTE2 $01100.,./10 112S·l1egatlvcv.rl.nt~ grown on XLDagar are pink wlth a dark'" pink cenrr e, IJlctosc'
pOSItive Sol",o"ollo ~1'Ownon XLD agar ore yellow willi or wltllout blackening. The occurrence of tbtse
phenorypeslssu.mmarized in I.a.hItl.

Check the second selective plating medium after the appropriate incubation time (or the presence of
cclonles which, frem their char"'terl.ttic~, are considered to be presumptive SalmQlJelln.

9.4.3 Procedure for samples from the primary production stage

From the positive growth obtained on the MSRVagar (l!.l.l), determine the furthest point of opaque
growth from the inoculation points and dip a 1 III loop (liJJlJ just inside the border of the opaque
growth. Withdraw the loop ensuring that no large lumps of MSRVagar are extracted. Inoeul.te the
surface of an XLOplate so that well-isolated colonies will be obtained. Proceed in the same way with
the second selective ptanng medium,

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 ISO 6579-1:2017(E)

tncubare the XI.D plates invel·ted at 37·C Cfi...3) for 24 h:l:3 h.

Incubate the second selective plating medium in accordance with the manufacturer's instrucnons.
Return negative MSRV plates to the41.S ·C incubator and incubate for a further 24 h t 3 b. Perform the
selective plallng procedure If. after 48 h of lncubatlon, these MSI{Vplates become poslnve,
Typical colonies of Sollll01l6110on XLD agar have B black centre and a lightly rransparcnr zone of reddish
colour due to the colour change of the indicator.
NOTE Sal,nollella H2.S-negatiV(' vertanrs grown on XL() agar are pink with a darker pink centre. Lactose-
poslnve Sol,no,lello gr-own on XLn Agar Ire yello\v with or Without blllckentng. The cccurrenee of 1hr.:ie
phen.otyp~ 15sumtUarllcd InIa.ltU::..l.

Check the second selective plating medium nfter the appropriate lncubatlon time for the presence of
colonies which, from thai r charactertstics, are considered to be presumptive Satmoneita.

9.5 Confirmation

9.S.1 General
The combination of biochemical and serological test results indicate whether an Isolate belongs to the
genus Salmonella. For charactertzatfou of Salmonella strains. full ."rotyplng Is needed, Guidance for
serotyping is described In ISO/TR 6579·31:MI.
For some of the eonrtnnarton medla as specifled III !l.5...land In IlJl to ILll. alternattve (ccmmerclal)
formulations ~lClstwhich may also be appllcabl« for blechemlcat conflrmallon of Salmollella. These
alternartve formulations are allowed. provided that the performance for the biochemical confirmation
of Salmonella Is verified before use.
For " clear dh ..tinction between positive and negative biochemical reacrtcns, it is helpful to verify the
reactions of the media of each biochenucal test with well-characterized positive and n~gative control
strains.

NOTE 1 Th~ re,mgnltloll of cnlonles of Soitllo/Jf!lIn 1$. to a large extent, 4 matter or exprrle!llce Bnd theIr
appearance can vary semcwbar. not only from serovar In serever; but also from batch to batch of the seteetlve
culture nH:diunl used.
Ir shown to be reliable. mtnlarurlzed galleries for the biochemical idenrification of Salmonella may be
used (see ISO 7218).
N01'E2 AlLernatlveprocedures CDn1M: used LOoounrm the lsolate as Sa/monel/n 'Pl'. providing tbe sulUlblllty
anhe alternative proc"dur,,;, verified ('<e. ISO7218).

9.5.2 S'e1ectlon of ~olonles for confirmation

~lark suspect colonies on each plate (.2.i). Select at lcasr one typica I or suspect colony for subculture
and cenlirmntien. If this 1$ negative. select up to four more suspect colonies ensuring that these
colonies are subcultured (rom different selective enrichment/isolation medium comhinations showing
suspect growth.
Streak the selected colonies onto the SUI' (ace of a pre-dried uon-selectlve agar medlum (ll) In a manner
which will allow well-Isolated colonies to develop. Incubate the Inoculated plates between 34 ·C and
38 ·C (6.3) for 24 h ~ 3 h.
AlternMively. if well-Isolated colonies (or" pure culture) are av~ilable on the selective plnnng media
(2d). the biochemtcal confirmation can be performed directly on a suspect. well-isolated colony
from the selective plarlng medium. The culture step on the non-selective agar rnedlum can then be
performed in parallel with the biochemical tests (or purity check ofthe colony taken From the selective
agar medium.

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 ISO6S79-1:.2017(E)

Use pure cultures for blochemlcal and serological conftrmation.

NOTE For e,p'demio1og1ralpurposes or during outbrea.k lnv~stlgatlons, confirmation of;additlonal colonies.
e.g, five typical or suspect (otonies from ellch setecnve enl"ichnlent/isolarif)n medium combination.. can be
h"".flrl.l

9.5.3 Biochemical testing

9.5.3.1 General
Inoculate the biochemical confirmation media with ench of the cultures obtained from the colonies
selected In 2A or 2.S.2, For confirmation of Salmon.llo spp. at least th e tests specified In 2..S.ll to
2.5.ll shall be performed. The tests specified 1112.5.3.5 and 2.S.J.b can also be performed when the
results of the other confirmotlon tests do not give a clear Identification.

9.5.3.2 TSI4Il"r (B.JI.)

Streak Ihe agar slant surface and stab the burt. Incubate 8137·C (A.l) for 24 h;t 3 h.
Interpret the chang es in the medium a. follows:
a) butt
yellow: glucose positive (glucose fermentation);
rod or unchanged: glucose negative (no fermentation of glucose);
black: formation of hydrogen sulphide;
bubbles or cracks. gas forn,arlon fron, glucose;
b) sian. surface
yellow: lactose and/or sucrose positive (lactose and/or sucrose fermentation);
red or unch"nge.d: lactose and sucrose negative (no fcrmclllariollofiactoso or sucrose].
The majority of the typical Salmonella cultures show alkaline (red) slants and acid (yellow) butts with
gas formation (bubbles) and (in about '10 % of the cases) formation of hydrogen sulfide (blackening of
the agar) (see Table 1).
When a lactose-posltlveSulmonella is isolated, the TSl slant is yellow, Thus, preltmlnary confirmation of
Salmollella cultures shall not be based on the results of the TSI agar test only (see ~).

NOTE KUgler·II,I". m.dlulllglvo,<$lmll.r resul,,,"sTSlosor.

9.5.3.3 Urea agar 0L2l

Streak the agar slant surface. Incubate at 37'C (!U) for up to 24 h.
I(the reaction Is positive. urea is hydrolyzed. liberating ~11111101l1a. This cha nges the colour of phenol red
to rose-plnk and tater to deep cerise. The reaction Is often apparent after 2 h to 4 h.
Typical Salmollella cultures do not hydrolyze ur-ea so tbat the colour of the urea agar wUl remain
unchanged (see Thble I).

9.5.3.4 I.-Lysine decarboxylation medium (LDC,JlJ.l.!)

I",":III.le just below the surface of the liquid medium. Incubate at 37·C (!U) for 24";t 3 h.
1Urbldlty and a purple colour after Incubatlon Indicate a positive reaction. A yellow colour indicates a
negative reaction.

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 ISO 6579-1:2017(E)

The n'ajority (If the typical Salmonelio cultures show a positive reaction ill ~bC (see Table l}.

9.S.3.S Detection of p -galactosidase (J!..ll) (optional)

The p-galactosidase test can be used to distinguish Salmonella enterico subspecies arizonae and
diorizonae and other members of the €nrorobocl"rlucIlD" (all give a positive reaction) from olhel'
subspecies of Salmone1Ja enterica (in general these give a negative reaction/see Iahle..l)..
Several procedures to perform the P.galacto.ldase test exist, An example Is given below.
Suspend. loopful of the suspected colony in a rube containing 0.25 ml of the saline solution (.I!.ll).
Add one drop of WIUL"Cand shake the rube. Place rhe rub. In • water bath set at 37 ·C (6.6) and teave
for several minutes (approxintately 5 min], Add 0/2,S ",I of the reagent for-detection of p·galactosidase
(Jl.ll) and mix.
Replace the tube IIIthe water bath set at 37·C (Ii.b) aJ,d leave for up to 24 h.
A yellow colour indlcates a positive reacnon. The reacttcn is often apparent nrter 20 min.
If prepared paper discs are used for the detection of p-galactasida se, follow the manufacturer's
instructions.

9.5.3.6 Med.lumfor lndole reaction (B.U) (optional)

The Indole test can be used when there Is. need to dllrerentlate So/moll.1l0 (generally Indole negative.
see Thb(!' 1) from Eschericblo coli and CI(rabaeter (borh Indole positive) as these organisms C3n give
typl",,1 reactions on some.of the Salmoltello Isolation media.
Innculate a rube rontlllningS ml of the tryptone/tryptophan medium (Il12.1) with rhe suspecred eol(IIIY.
Incubate at 37 ·C (!i.3) for 24 h ~ 3 h. After Incubation, ndd 1 ml of the Kovacs rcngent (B..lU).
Tho (orlllatlon of a red ring (surface I~yer) Indicates a pesmve r •• erten, A yellow-brown ring (surface
layer) Indicates a negative reactlon,

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9.5.4 Serologicaltesting

9.5.4.1 General

The pure colonies (9.5..2) showing typical biochemical reactions for Salmonella (9..5.3) are also tested
for the presence of 5{Jlmon""o 0- and H·nntlgens (and, In arras where Salmolldlu Typhl Is expected III
the food supply. also for VI-antigen) by slide agglutination using polyvalent antisera (.IL..I!). Tile pure
colonies are cultured on a non-selective ag3r medium (B..2) and tested for auto·agglutlnation. Strains
that are autc-agglutlnable cannot be tested for the presence of Salmonelk: antigens. Use the antisera
according to the manufncturer's Instructions If different from the method described belowte detect the
presence of Salmonella O·and H-antlgens (and If necessary, also for Vl-antlgen].
The following tests ~to~) arc the mlnlmullI required for serological testing ofSlIlmOllel/o spp.
Further guidance on serologlcal confirmation and on serotyping is given in ISO/TR 6579·31l!1.

9.5.4.2 Elimination of auto·.agglutfnable stralns

Place one drop of saline solution (lUJ) on a clean gl.ss slide. Using a loop, disperse part ofthe colony to
be tested In the saline 10 obtaln a homogeneous ond turbid suspension.
Rode the slide gently for 5 s to 60 s (depending on the manufacturer's instructions). Observe the
suspension. preferably against a dark background.Hrhe bacteria have formed granules IIIthe suspenslon,
this indicates auto·agglutination and serological confirmsnen will become complicated. Additional
information on the treatment of'auto-agglurtnattng stratns can be found in ISO/TR 6579·31211).

9.5.4.3 BxomlnnUon for O-anUgens

Using on. nen-auto-agglunnatlng pure colony, proceed according to~ using one drop of polyvalcm
auti-O sera (B.t4) in place of the sallne sclurion.
If agglutination occurs, this Is considered a pesltlve reactton.

9.S.4.4 Exruninatlon forVI·antigens (optional)

Using one no,,-auto·aggluth18tlng pure colony, proceed according 10 ~ using one drop of anTI-VI
scm (lUi) in place orthe saline solutlon.
If agglutin~tion occurs, this is considered n po~itivC'reaction.

9.5.4.5 Examination for O-ontlgcns

Using one non·auto-agglutinAting pure colony, proceed according to.9..S.i..Zusing one drop of polyvalent
anti-H sera (~) in place of the saline solution.
If agglutination occurs. this is considered a positive reaction.

9.5.5 Interpretanon ofblocbemlcal und serological ne"CUeDS

Iahl£ 2 gives the Interpretation of the confirmatory tests l2.!i..a and ~ carried Out on the colonies
used~.

1"' __ CrIlU_~~- All rfahl$~fd 11

....,..._.. ,
Ho._.. ' __
~~""".1.'O __ ......HJI _ ........... _MIl"s5'tOl'
~bI!UN.uII
.... ,,,
stAt~'blt;1 _"l'
ISO 6579·1:2017(E)

Table 2 -Interpretation orconfinnatory tests

Biochemical reactions Auto·aggtutination Serological reactions Interpretation
Typical No O-.nd 1I'"II.lge.. Slrllns c('Insld<rred to be
poslUv. Solt"Ohello
(ond VI jlOsitJve- lrte~ted)
Typical No O· and/or H·anttgens Pre.c;umptlveSolmnneJlu
nega_t-Ive
Typlc.1 Yes Not tested been "SO of
.,lIto·agglutination
(sec 2.S.!l.2)
No typicl.l1 reoctiQns - - Not considered co be
SlJI,nQllalIlJ

9.5.6 Serotyplng
Stralns that are confirmed as Salmoll8/1a spp. (T.lblc 2) can be further typed w serovar level, Guidance
far serotyplng Isdescribed In ISO/TR 6S79·3lH1.
If required. conflnned strams can be sent to 8 recogulzed Solmo"olla reference centre for definitive
typing (serotyping. phage typing. molecular typing). lIthe strain is sent to. reference centre. It should
be accolII",,"IQd by ~II •• Ievant l"ro'nI~lion such as confirnlMlon results. seurce from whleh the strHln
was Isolated. and whether lt concerns an isolate from an outbreak.

10 Expression of results
In accordance with the interpretation of the results. indicate Salmonella detected or not detected In a test
portion or;rg or ;rml o(prodUCI (sec ISOnl.B). or on the surface area. or In an object (e.g. boer socks).

11 Perfonnance characteristics of the method

11.1 Interlaboratory studies

The performance char~'terU-tlcs of the method were deterrnlned in interlaboratory studies- to.
determine the specificity, sensitivity, and the I.ODsoofthe method (see References [iiI and (11).The data
are summarized in ADoey C The values derived from the mtertabcrarcry studies may not be applicable
to matrix types other than those given in Annex C Furthermore, the performance characteristics as
indicated in Annex r were determined with individual test portions up to 2S g (or n11).When larger size-
test portions are used. the performance characteristics 1l1ay be different.

11.2 Sensltlvlly
The sensitivity is defined as the number of samples found positive divided by the number of samples
tested at a given level of contamination. The results are thus dependent on the level of contamination of
the sample.

11.3 Specificity
The specificity is defined as the number of samples found negative divided by the number of blank
samples tested.

11.4 LODso
The LOI)so (level of detection) Is the concentration (clu/test portion) for which the probability of
detection is SO'lb.
 ISO6S79·1:2017(E)

12 Test report
The test report shall 5jleclfy the (allowing:
th~ saml,ling method used, I( known;

the size of the test portio" and/or the ""UlI'" of the object examined;
Ihe test method used, with reference to this document, i.e.150 6579·1;
any deviation in the enrichment media or the. incubation conditions used;
all operating condlttcns nol specifled in this document. or regarded as optional, together with
details of any incidents which ",ay have influenced the results;
the results obtained.

\_,bIUNCIIIiWf g; (i:'''14)oIIIIDV.tOIGI_1"1 13
IIa"" .......
_t.'SIII .. '••
ISO 6579-1:2017(E)

AnnexA
(normative)

Diagrams of the procedures

,
 ISO6579-1:2017(E)

il 5nmple • Buffered peptone water (BPW):

~ S
~-" T~nrold dilu~on (9.1 9.2) ."d
"-;:c
.. IB h;t Z h ",34 'Cro 38'C

~ ..
C
0.1011 rulnrre-
•
1 ml culture
•
..
"C C
v ~
",
Q;'C
10 mt RVSbroth oron ~tSRVa_ga.r(9.3,2)
24 ha 3 h ot4 1.S'C! l'C
10 ml Ml<1Tnbroil! (93.2)
24h:t3 h >ta7 'C"I 'C
'" ii

.."
0
..
.§
.s
no "3.r
24 h t 3 h .1'37 'C± 1 'C (9.4.2)
~ .,.(ond lsoj"dun ag,1r
e,

r .... t least 1Iyrl",,1cr suspeei

wlony. If negative, lest ~dd.itional4
marked colonies (9.s.Z)

N.n·..,lecti,·. agar (9.5.2)

..,"
4
0 21IIt3 h al31'Cw 38'C
E
'""
s

moeh.nll""lleSting (9.5.3) Serologkal tl!$tillg(9.5.4)

Expression or''esullS
IClause 10j

Figure A.I - Diagram of procedure for detection 0"

Solmon.ella In food, animal feed, a.lId
environmental samptes lTom the rood production area

\_~f..Mll,QlldlTC1'.st...,~flON,cp**11' 15
........ ".'11>'' ••
1.
ISO6579·1:2017(E)

~ Sample + Buffered peptone warer (BPW):

"
, E
ea
T.n(oIddUulion (9.lond 9.2)
"...,
,tu 18h s 2h..134·C '038 'C
-.:
e

~
~ e 0,] ml culture in )·3 spots 01110
t E MSRVof!o'r(9.3.3)
"il (2x)24h±3haI41.5·C~1·C
~i ..

'5
'?
e
·c
.. Xwagar
24b:t 3 II. ot37 'C± 1 ·C(9.4.3)
...second isolation agar
.!!
e,

Test at leas, llypirn1 or suspect

colony. Jf negative, test additional oj.
m.rked colonl .. (9.S.2)

,
e Non-selective agar (9.5.2)
·c
"E
" 24 hi 3 h .134"C to 38'C

<::
e
.3
~ ~

Blo,homlcaJ tcstlng (9.5.3) Serological '.5Ung (9.5A]

~~
E.l<presslonof results
lebuse 10)

Figure A.2 - Diagram or procedure for detection of Salmonella In animal faeces and In
environmental samples from the primary production stage
 ISO6579-1:2017(£)

AnnexB
(normative)

Culture media and reagents

B.1 GeneraJ
The general speclflcatlons of ISO l'l133 are applicable to the preparation and performance testing of
the culture media described in this annex, If culture media or reagents are prepared from dehydrated
complete meclla!rcngcnts. or If rendy-to-use Itledia!reagenl'l: are used. follow the mnnufuctu.w·s
instructions regarding preparation, storage conditions. expiry date, and use.
The shelf life of the media indlcated In this annex has been shown in some studies. The user 5]•• 11vel'ify
this under their own storage conditions (as specified in ISO 11133).
Performance resrtng (or the quality assu ranee of the cu lture media is described in JUli,

B.2 Buffered peptone water (BPW)

B.2.1 Composition

Pepton .. 10.0g
Sodium chloride S.Og
DIsodium hydrogen phosphate dcdecahydrare 9.0g
(Na2HP04-12H20)b
Potassium dthydrogen phosphate (KtllPO.J 1.51l
WalA>r 1000lnl
:& For example, enzymatic digC$t of casein.
b Ifdlsodium hydrogen phosphate with. different water contentis used. amend
the mass orthe Ingredlcnlaccordlngly. For example. in rase of anhydrous disedlum
hydrogen phosphate (Na~HP04). use 3.57 g.

B.2.2 Preparation
Dissolve the compenents in the water by hearlng, If necessary.
Adjust the pli.lf necessary. so that arter stertltznrlcn, It Is 7.0 t 0.2 al25 ·C.
Dispense me medium inro flasks (!i.U) of suttabte capaciry to obtain the portions necessary for the test.
Sterilize for lS min In the autoclave (li.l) set at 121 ·C.
Store the medium In closed conzatners (IUl) at 5 'C (liJI) for up to.ix months.

1... _....tQpo_gf~JJ - AIII'tt)HS rest1'Ved '"-lr!UN4tllSft'lS'l',.,~tOIC'_ll' 11

,....._ _ ,.. .lCO
,., ...-... _..~pooooort... •• _HII ~_"" •• lIIJIl'!Io'IIIIU'1"
ISO 6S79·1:2017(E)

B.3 Rappaport-Vassiliadis medium with soya (RVSbroth)

B.3.1 Solution A

B.:U.l Composition

Enzymatic digest of soya S.Og

Sodfum chloride B.Og
Potasstum dlhydrogcn phcsphate (J<H1P04) 1.4 g
Dipotassium hydrogen phosphate (K2HPO.l O.Zg
Water 10001111

8.3.1.2 Prepumtion
Dissolve the components In the ware I' by heating to about 70 ·C. if necessary.
The solution sh.1I be prepared on the <1<'1)' of preparation of the complete RVSmedium.

B.3.2 Solution B

B.:i.2.1 Composition

Magnesium chloride hexahydrate (Mg02·6H20) 400g

Water 1000mi

0.3.2.2 Preparatlon
Dissolve the mrrgncslum ch Ieride In the water,
As this salt is very hygroscopic. It ls advisable to dissolve the entlre contents of MgCI~·6f1z0 [rom a
newly opened container according to the formuta, For instance, 250 g of MgC12'6U20is added to 625 ml
ofwater gfv1ng a solurton o(rotal volume 01'788 ml and a mass concenrranon of about 31.7 gper 100 rnl
of MgClz"6I1z0.
The solution nlay be kept in Adark glass boule with tight stopper at room temperature for ot least
rwo years.
B.3.3 Solution C

B.l.3.1 Composition

I
Mol""hlte green oxolole
.Water
0.4g
100ml

B.3.3.2 Preparation
Dissolve tbe malachite green oxalate In the water.
The solution Olaf be kept in .i1 dark.glass bottle at rcom temperature for at least eight months,
 ISO 6579-t:2017(E)

B.3.4 Complete medium

B.3.4.1 Composition

Solution A (8.3.1.) J 000",1

Solution 8 (Jl.l.2) 100mi
Solution C(B.J.l) 10ml

B.3.4.2 Preparation

Add to 1 000 ml of solution A.100 ml oi solutlon B. and 10 fill of solution C.

Adjust the pH. Ifnccess"ry. so that after stertllznrion itis S.2 ± 0.2 at 20·C to 25 ·C.
Dispense the medium Into lubes or n..sks (6.1Z) of suitable cap"clty to obtain the portion. necessary
for the test, e.g, 10 OJI quantities dispensed into tubes.
Sterilize for 15 min in the autoclave (6.1) set al115 ·C.
Store the complete medium In closed tubes Of flasks nt 5 ·C ~ for UI)to three months.
NOTE The tlnal medium composition i$ ."zymorlc dlges. of ""Y. 4.S &II. sodium cblorkle 7.2 &II. pntO$slum
dillydrogen phosphate (I(HzI'O•• K,HPO.) 1.+4 &I~ onhydrOU$ 111a8Jl •• Ium chloride (M8CI,) l3.+ all. or
magn,,-,Ium chloride h.""hydrate (MgCI.,.6I1Z0)28.6 glt. and maladU[C gr«!" oxalate 0.036 &lL

B.4 Modified semi-solid Rappaport-Vassiliadis (MSRV)agar

NOTE See Reference lUI.
B.4.1 Solution A

B.4.1.1 Composition

Enzym~ticdigest of animal and plant tissue 4,6g

Add hydrolysate of casein 4,6 g
Sodium chloride 7.3 g
Potassium dihydrogen phosphate (KH2PO..) l.Sg
W~rer 8901111

0.1.1.2 Preparation
Dissolve the compon~nr:s in the water by heating-to about 70 oC,If necessary.
The solution shall.be prepared on the day of preparation cf the complete MSRVagar.

8.4.2 Solution 8

B.4.2.1 ComposlUon

Magnesium chloride hexahydrate (MgCI2·6H20) 400g

Water 1000011

0.4.2.2 Preparntlon
Dissolve the magnesiumchloride in the water.

.......~~IJ"'_
~"'".\I'_"'_."'~
~_
..

.. C!t_'""_
(P'J~J.\UJ- All "Shn;rC!$t:r"cd
•• ~_ttlI
... .....;.-,,,-
......~ .,.......,,,
\_;"~!IiIlO'~",c.o~fOI~.W'IM'''' 19
ISO6579-1:2017(E}

As this salt is very hygroscopic. it is advisable to dissolve the entire contents of MgC12·6Hl0from a
newly opened conralner flecordl))g to the forrnulu, For lnstance, 250 gor MgC12·6H20Is added to 625 ml
of water giving a solution of tetal velume of 788 ",I and a mass cencenrraticn of about 31.7 g per lOO ml
ofMgCI2'6H20,
The solution may be kept in a dark glass bortle with tight stopper at room temperature for at least
two years.

8.4.3 Solution C

8.4.3.1 Comllasltlon

Mal3Cbtle green oxallltc 0.4g

Water 100ml

8.4.3.2 Preparation
Dissolve the malachite green oxalate ill 'he water.
The solutIon may be kept illa dark glass bottle at room temperature for at least eight months.

B.4.4 Base medIum

0.4.4.1 Composition

Solution A (lUJ) 890ml

Solution B (lL1.ZJ 100 ml
Solution C(1U.l) to 011
Ag.'" 2,7g
• It may be necessary to determtneexpertmentatty the concentration of'agar
needed fur lho oprlrnal.swarmlng of Satmonell« (o.g.when using a botch ofagar wIth
unknowngel strength).

B.4.4.2 Preparation
Add to 890 ml of solution A. 100 ml of solution B. and 10 ml of solution Cand 111i" by agit.tiou.
Add and suspend the "liar.
Adluslthe pll, Ifnecessary, so that after stertlizatlcn, ttls 5.2 (5.1 105.4) at 20'C to 25 'C.
Heat to boiling with agitation. Do not autoclave,
00 not hold the medium at hIgh temperatures longer than necessary.
Cool the medlum to 47·C III 50 'C (.6.5].

8.4.5 Novobiocin solution

8.4.5.1 Composition

Novobiocin sodium salt 0.05g

Water LO lUI
 ISO 6579-1:2017(E)

B.4.5.2 Preparation
Dissolve tho novoblodn sodIum salt In the water,
Sterlllzc by filtratIon through ~ triter with 0 pore size 0(0.22,,,n.
The solution may be stored for up to fOUT weeks at 5 ·C (t>..8) OT in small portions (e.g. of 2 ml) at -20 ·C
(ti9) for up to ODeyear.

8.4.6 Complete medIum

B.4.6.1 Collll1051tion

ease medium I.llM) 1000mi

Novobiacin solution (~ 2ml

B.4.6.2 Preparation
Aseptically. add 2 ml of the novobiocin solution (.!!d.5) to 1 000 ml of bas. medium (8.4.4) at 47'C to
50 ·C. Mix carefully. The final concentration of novohlocin In the ccmpleee medium is 10 mgtl.
The final pH shall be 5.•2 (5.1 to 5.4) at20·C to 25 ·C.
Pour the tnedtuut Into sterlle Petri dishes (6J.1) up to a volume of 15 ",I to 20 tnt in dishes with a
dlameterof90 mm.
Allow the medium to solidify before moving and handle with care,
Store the plates, with surface upwards. and protected from drying for up to two weeks at S'C (6Jl]ln
the dark.
Do nor mverr the plates as Ihe semi-solid agar is 100 Hquld to do so.
Any plates in wbich the semi-solid agar has liquefied or fragmented shall not be used.
tmmedlately, before usc a.nd only If visible moisture Is apparent. dry the surface of the agar plates
cnrefully. for example. by placing them with the lids off and the agar surface upwards in a laminar air
nolV cabinet, Thke care not 10 overdry the medium.
NOTS 1 The <011111051001\ of MSRV"gar .• s described in Reference UlJ. contains 20 IU8/1of novobiocin.
However, fro III a M(cntJDc pclnt of vjew. 10 ITlg/lnuvobloc:ln ls preferred, Studtrs have $hown larger trllgr-aUot1
zones on ~'SRVagar with a lower concentranon of novobiodnWl and the (negative) Innuence of novobiocin on
bacterl.1 motllityJUI
NOTE2 The tlnol medium composition Is .n~otk dinesr of onlm.lond ill.nt nssue ••6311.oeld hydrolysate
of casein ••6 gil. sodium chloride (NoCI)7.3gil. potassium dlhydmgcn phOsl,h.telKllzl'O.J 1.5gil. ilIIhydrou.
magnesium chloride (Hgeh) 10.9 gil. or mngne1)llIIn rhlorid. hexahy.drot. (IoIgCI2·6I1,O)28.6 g/l. mal.chlt.
green ox.la", 0.04 gil. novubloclnsodlutn S1llt0.01g/l. ~nd agar 2.7a/I.

___ D,._ eMSiQ i.Q1l - All rlllllIs~rve<l ... -,'_- . _. 21

l_btIUiilljl41"tao"'~'DICI_I"
~"""IIfI"'''_''_''''_eo
w......-......._..,"'_"_.....,_ ..... -..,,, ~"'."',"":"0If,""1.:a1~
ISO6579-1:2017(E}

8.5 Muller-Kauffmanntetrathionate-novoblocln (MKlTn) broth

NOTti See Reference IUJ.

B.S.l Base medium

B.5.1.1 Comllosltlon

Meat extract 4.,3g

Enzymatic digest of casein 8.6g
Sodium chloride (NnO) 2,6g
Calcium carbonate (f.aC03J 38.7g
Sodhim thiosulfatepentahydrate (Na2~03·5H20) 47.8 g
Oxbile for bacteriological use 4·,78 s
Brilliant green 9.6mg
W~ter 1000 ml

B.5.1.2 Preparation

Dissolvo the components or tho dehydrntod comptere medturn ill the water by heating. with frequent
agitation. until the medium starts to boll. Avoid overh •• ting,
Adjust the pH, Ir necessary, so that It Is 9,0 ± 0,2 at 2S ·C.
Th(lroughly mix tho medium,
The MSCmedium may be stored in closed fI.sks (fW.Z) ot S'C (6Jl) for up to three months.

8.5 ..2 Iodine-iodide solution

B.S.2·.1 Composition

Iodine 20.0g
Potassfum Iodlde (KI) 2S,Og
WRter lOO nIl

B.5.2.2 Preparation
Completely dissolve the potassium Iodide In 10 ml of water. then add the Iodin. and dilute to 100 1111
with sterile war"r. 01)nnt heat,
Star" the prep. red solution In a (tlghtly) closed container (!U2) In the dark for up to one year.

0.5.3 NovobiocIn solution

B.S.l.1 Composition

Novobiodn sodium salt 0,04g

Woter 5 nil

t:# .5tAl.l'...aP.J.fi)
t-tt;!JN.at~1 ~7 - All tlgt1l$I't$H'\'ed
.......... _f· ....
" ... '"
 ISO6S79-1:2017(E)

B.5.3.2 Preparation

Dissolve the novobiocin sodlu III salt In the water,

Sterifize by (11Im!i"" through a filter with a pore size of 0,22 I'm,

The solution may be stored for up to four weeks at 5 ·C (f>.ll) or in smaU portions (e.g. of 5 ml) at -20 ·C
(63) for up to one ye~T.

B,S.4 Complete medium

B.S.4.1 Composltlon

B~semedium 0L.'iJ) 1 0001111

Iodine-Iodide solution (ll.S.2) 20nJ!
Novobiocin solution (B.S.J) 5 ml

B.5.4.2 Preparatlon
Aseptic"IIy, ndd 5 1111of the novobiocin solution [R.!i.3) to 1000 rnl of base medium fB.5.1)- Mix, then
add 20 ml of the ledlne- Iodide sclutlen (ll.S.2). Mix well. The final concenlratlon or novobiocin In the
complete medium is W mgjl.
Dispense the medium aseptically into containers (2.Il) of suitable capacity to obtain tho porrions
necessary for the test, e.g, 10 1111
quantities dispensed into tubes. After preparation, the pH of complete
MKTl'n broth will be approximately B,O.Ifthe complete medium is not used immediately, store it in the
dark at 5 'C (0.8). The pH may drop during storage due to chemical reacrtons, Do not use the complete
nwdiumlrthe pll drop~ below 7,0.

8-6 XyloseLysine Deoxycholate agar (XLD agar)

NOTE See Wel'eru:ellll-

B.6_1 Compo$ltlon

Yeas! exrrac: 3,0 g

Sodium chloride (NaCJ) 5,Og
Xylose 3,75 g
Lactose 7,S s
Sucrose 7,5 g
L.-l_yslnehydrochloride 5,0 e
Sodium thiosulFate 6,8g
Iron(ll1) ammontum citrate O,8g
Phenol red O,08g
Sodium deoxycholate 1.,0 g
Agar 9gto]8g'
Water 10001111
" Oel,elldlng on the gel strength olthe agnr.
B.6.Z Preparation
Dissolve lhe components or the dehydrated complete medium In the water by heating, with frequent
agitation, untU the medium sta rts to boO. Avoid overheating.

1"'.... _GP'*,_~..J.S:QlUj,7 - All rtghl~reserved 23

""_,,,U'IIII_"'_I01"~
,..._ ..... _ .... .. R_...
\_leHt..JN4L'*'CII'st.v~fl(ltl(l'l~.1'
fI;o,,"~.".~'lIm"1'
ISO 6579-1:2017(£1

Adjust the pH, Ifnecessary, so tl,ol tile final pH shall be 7,4 :t 0,2 at 25 ·C,
Pour tbe base medium into tubes or flasks (P.l2) of appropriate capacity,

B.6.3 Preparation of the agar plates

Cooltbe medium to 47 ·C to 50 ·C In a water bath (6..5.). mlx, and pour Into sterile Petri dishes (2.H).
A lIow to solidify.

Immediately before USA>, dry the "gar plates carefully (preferably with the lids offand the agar surface
downwards) In the oven lQ.D set between 2S'C nnd 50'C unttl thc surface oftJle agar Is dry.
Store the poured plates protected from drying, at 5·C (tiJI) forup to four weeks.

8.7 Nutrient agar (example or non-selective medium)

0.7.1 Composition
Ment extract 3,Og
Peptone 5,Og
Sodium chlorldo (Nael) (optional) 5,Og
Agar 9gto 18g'
Water l 000 ",I
• Depending on the gel Strength of the agar,

B.7.2 Preparadon
Dissolve the ccmponents or the dehydrated complete medium in the water by heating, witb frequent
~8ltatlon,
Adjust the pH. Ifnecessary, so that .ftcrst.rfll:rotlon,It *
Is 7,0 0,2 at 25 ·C.
Transfe r the culture medium into tubes or flasks (6.12) of apprepriate capaelry,

Sterilize for 15 min in the autoclave (6.l.) .etol121 ·C.

0.7.3 Preparalion of nutrient agar plates

CooJ Ih. medium 1'0 47 ·C to SO'C III a wnter bnth (6.!i), mtx, ~nd pour Illto steril. Petri dishes (ti.,U).
Allo\~ to solidify.
hnl\ledi~tcly before lise, dry tho agar plate s carefully (preferably with the lids off alld the agar surface
downwards] in rne oven (5.;!)set between 25·r. and SO·r. until the surface of the agar is dry.
Store the poured plates protected from drying, at 5·C (fiJI) for up to four weeks.
 ISO6579·1:2017(£)

B.8 Triple sugar/Iron agar (TSIagar)

8.8.1 Composition

Meat extract 3,Og

Yeast extract 3,Og
Peptone 20,Og
Sodium chloride (NaCI) S,Og
Lactose lO,Og
Sucrose 10,Og
Glucose 1,Og
Iron(lll) cltrate 0,3 g
Sodium thiosulfate 0,3 g
Phenol red 0,024 g
Agar 9g.IOJBg·
Waler 1 000 1111
• Depending an the gel strength of the agar.

8.8.2 Preparation
Dissolve tbe components or the dehydrated complete medium in the water by beating with frequent
agitation,
Adjust the pH, if necessary, so tbatarrer stertlizancn, it is 7.4 ± 0,2 at25 ·C.
Dispense the medium Into tubes or bottles (J>,ll) In quantities of 10 mi.
Sterilize for 15 min in the autoclave m.n sot at 12l ·C.
AllolYto set In a slOping posltto» to gfve a butt of depth 2,5 em to about 5 cm.
Store the poured rubes protected (Tom drying. at 5·C (jIJl) for up to four weeks.
NOTE As an alternative. a double ~ugar/lron agar- can be used (Kligier-Halna).

._,.,....C>~......,J.S,Q.hOJJ
fttw.__ "l~,,"_ __ .I6_'O
- All d8l1tlJ reM:I'Vcd "_0ltI""",Q11.1Q0" .1~nCUt:1Ib'111I'f 2S
............
,_w ..... ,•
...~ ....
'""_ ..,,~_~_ .._ttI
ISO 6579-1:Z0'17(E)

8,9 IJrea agar (Christensen)

1J.9.1 Base medlum

B,9.1.1 Composition

Pep,on" 1,0 g
Glum$~ l,Og
Sodium chloride (N.Ct) 5,1) g
Potassium dih_yQ.rogenphosphate ('KH:tPO...) 2,Og
Phcnoll'ed 0,010&
A~ar <;I !fro
18 gb
W,ater 1000 ml
• ('o......ample,enzyntat'j~digest of gelatine.
II Dependingon the gel strength cf the agar.

8,9.1.2 Pnp9.l"dtioD

Ol.solve the tOllll"lhtmts or the d.hydrat"d cOUlI'II!,tcb.~e In the water by h~.tlf1gwith fr1!'I"""t
agitation.
Adjust the pH, Ifnecessatly, so that after sterilization, It Is 6.B'± U,Zot'25 .c.

POllrthe basetuedlum Into tubes or n,nsk.s (CLll) of appropriate OlpaOty,

Storlli7.l!for 1~
min In tbQ nuwdmlC ~ ser ar 12~ ·C,
Tho base Uledlum may be stered In closed cubes or nnsi<.sgt S·C for up to three months.

8.9.2 U.n'JlsoluUc)o

8.9.2·.1 Compo.<itlon

IJr~a 4-0C) i
Water, to a nllol volume or 1000 nil

8.1),2.2 Pl'l!parnUon

DI$501vefhe IIt'''-110 the water, SUlrfll~. by filrrarron tJlt'OUllhA fIIr~I' wltl) n Jl<Ir. sliu of 0.22 ,10'.
See ISO lU33,

8.9.3 Complete medium

8.9.3.1 Composition

llaS1! (illhl.) 950011

Urea solutton lflli.2J SO ml
 ISO6579·1:2017(E)

B.9.3.2 Preparation
Add, under aseptic condttlons, the urea solution to the base prevIously melted and tben cooled to 47 ·C
to 50·C.
Dispense the complete medium Into sterlle tubes (fi.ll) In quantities o( 101111.
Allow to set III a sloping position.
Store the poured tulles protected (rom drying, a.t 5·C (A.B) for up to four weeks.

B.I0 L·Lyslne decarboxylation medium (LDC)

B.I0.1 Composition

L·Lysillcmonohydrcchlortde 5,0 g
Yeast extract 3,Og
Glucose 1,0g
Bromocresol purple O.015g
Water 1000,01

8.10.2 Preparation
Dissolve tho cemponents In the water by heating, If necessary.
Adjust the pli, if necessary, so thatafter sterilization. it is 6,8:t 0,2 at 25 ·C.
Transfer the medlum ln quantities of 2101 to 5 ml to narrow lubes (tUZ) with snow caps.
Sterilize for 15 min in the autoclave (2JJ set at 121 ·C.
Store the poured tubes at 5 ·C (ti.lI) for up to three months.

B.11 p·GaJactosidase reagent (optional)

Additional to the reagent descrtbed below, toluene Is needed for thep·galactosldase test.

8.11.1 Buffer solution

B.Ul.t Composition

Socllurn dlhydrcgen phosphate r,..,.H~P04) 6,98

Sodium hyd"oxlde, 10 11101/1
sclurlon "pprox.301I
Water, to a final volume of SOml

B.11.1.2 Preparation
Dissolve the scdlum IIlhydrogen phosphate In approxhnately 4S ml of water In a volumetric flask.
Adlust the pH to 7.0 ± 0,2 At 25·C with the sodium hydroxide solution.
Add water to a finn I volume of 50 ml.
The buffer solution may be stored In dosed flasks at 5·C (ti.lI) for up to six months.

~JmQI.7-
t_Hf"'()ooM.,. .. All rf8hrs ~red 7.7
.. _ .. ,,.,,_,,,"_
..14.0
HoI_ .. W_l,_,_t'"'_ ......,.....,,,
ISO6S79-1:2017(E)

B.11.2 ONPGsolud.on

0.1,1.2.1 Composition

o·Nltrophenyl p·D·galactopyrnnosldc (ONPG) O.OOg

Watu 15011

0.1l.2.2 Preparation
Dissolve the ONPGin the water at appro~imately 50 ·C.
Cool the solution.

B.l1.3 Complete reagent

0.1l.3.1 Composition

Buffu solution (R.1LQ Sml

ONPG5011l1l0n (1!.ll.lJ 15011

0.11.3.2 Preparation
Add the buffer solution to the ONPGsolution.
Store the complete reagent In closed flasks ~1 at 5 ·C (MJ for up to til ree months. Discard the
complete reagent as soon as the colour changes to yellow.

B.12Medium and reagent for indole reaction (optional)

B.12.1 Tryptone/tryptophan medium

0.12.1.1 Collll)OsltiOD

Tryptone 109
Sodium chloride (N.CI) Sg
D~-Tryptoph.n 19
Water 1000 ml

D.12.L2 Prepararlon
Dissolve the components in the bOithlg water,

Adjust the pH, Ifneccssary. sc that after sterilization, it I. 7.5 i 0.2 at 25 ·C.
Dtspense the medium Into tubes (§J.l) In qunntltl"" of 5 ",I,
Sterilize for 15 nun In the autoclave (6.1) ser ar 121 oe.
Store the poured lubes at 5 'C (ll.l!J for up to three months.

-..-tnOM4tIiSl'CII'''U' rn'~~~7-Al1rfgbts~d
ll .. _ .......... f,IYII.... U
 ISO6579·l:2017{E)

B.12.2 KovlICS reagent

B.12.2.1 Composition

4·Dimethylamirl(lhenzoldehyde 5g
Hydrochloric.dd.p = l,18g/ml to 1.19 g/ml 25ml
2·Methyl·2·but.nol 75ml

B.U.2.2 Preparation

Mix the components.

Store the complete reagent in closed flasks (6.ll) ill the dar'l, at 5'C f.6JO for up to six months,

B.13 Saline solution

8.13.1 Composition

Sodium ehlorlde (NaCt) 8.5g

Water 1 QOOml

8.13.2 Preparation
Dissolve the sodium chloride In the water,
Adlust tb~ pli.lfllccessary. $0 that arter stertllzatlon, Ills 7.0:l 0.2 at 25 ·C.
Dispense the solution Into flasks or tubes f6.lll of suitable capaclty to obtain the portions necessary
for the test,
Sterlllze far 15 mill 10 tire autoclave ~) set at 121 ·C.
Store the solution IIIclosed flasks/tubes alS DCf.6JO for up to sl~ months.

B.14Antisera
Several typos or agglllrinOllng sera containing antibodies for OM or several O.antrgeus are available
commercially. i.e. antisera containing one or more '0" groups (called monovalent or polyvalent anti-O
sera),8ntl~VI sere.and anttsera containing antibodies ror one or several II-factors (called monovelent or
polyvalent alltl·H sera).

B.15 Performance testing for the quality assurance of the culture media
The definition of selectlvlty and productivity Is specifled In ISOlUl3.ln general. rollow the procedures
for performance testing described in ISO 11133. For performance testing of selective liquid media and
MSRV "gM.
use the same lncculurn volume as specified in 2.3.2. For MSRV agar the Inoculum should
contain 1()3cfu to 104cfu for deterrnlning productivity and 104cfu to 106 cfu for determlnl ngselectivlty
(see ISO11133). For the other media. the inoculum levels for the target and the non-target organlams
ore specified In ISOll133,2014. 5.4.

i...__ Opo_~~!7 - All righTS: rest:fVcd "-blUNGtllsttlSUI~tole'_ll' 29

"~""._'1~~
.........
__ """"''''_'''''
_.''''' ..,__ .,.. :&0 ~_""""'.~I'GJJl1"
ISO 6S79-1:20l7(E)

Tablc B.l - Pcrformancc tcstlllgfor the quality assurance of the culture media
MediuIli Function Incubation Control srratns WDCM Criteriab
nu_mb~rsa
BPW Productivity 10h ~ 2 hI Snitnanella l'Yllhlmul'lutnC.d 00031 Thrhldlty (1-2)
34'C [0 38'C SO/,,,,olle/lu Enleritfdt5c,d 00030
MKTTn Productivity Sulmonell" Typhlmurhllnc.d 00031 >JO ctrar;u::tt:rlstic
broth Sa/ulOIlella Enteritfdisc-,d 00030 coIoutes on XLD ag~r or
other medium of choice
..-escherlc:hla t!olld 000"12Clr
00013
24h~3h/ ,. Pscud(1",onns (f~rvD("t)$Q OOOZS
37'C:l 'C
SciectMty Esc:herkhlu col,d 000'12 or Partlallnhlbldon
DODD
CIllOr.nlnnlunn TSA
Enceroc,ocCllS (ol!cn/;sd 00009 or <10 colonies OD TSA
00087
RVS broth Pl'oductlvlty Salmonella Typhhnur lumc.d 00031 >10characterlsdc
$ol,",m,II" EnJj)I'ltldl~<,d tolonl<son XLDag.ror
00030
other medium of choice
.y- Escherfchlo cnltd 000'12 or
00013
24h± 3 hI .. Pseudomonas Ot!Tl{9inoso tlOOZS
~1,5"* i-c
s"lectlvl,y Escltu/c/,fo allie' 00012 or Pardo) Inhlbltk>n
00013 S10() <01001..<on TSA
Enterococcus /a",al/S< 00009 or <JO colonies on ·rSA
OOtl87
MSRV ProductlvllY Salmonella Typhlmurlwnc.d 00031 CreY·IA"hlte.turbld eone
agar exccndlng out (rom
tnoculated drop(s),
seuuonene Enterltldisc,tI 00030 After 2~ h to -w h, the
turhld ..,"0($) will b.
(almost) fully tlligrated
over the ~late, I)ns5ibl.
II!xtl'tI: characu!rl$tic
2x(24 h ± 3 h)/ rolonlcs atter
4.1,5·C t 1 'C Sllbclliruring on XLD
agar
S.Ic:ctlVlcy /isel .. r/chln "",id 00012 or P~~lhl. growth m tbe
00013 place ottbe inoculated
drort<) without n nrrhid
zone
ElltllTOCOCClI<
rncr:tJII'" D0009 nr 110growth
(lOOS7
•c.,nt.,C1dcti1Usj\VDOf: straJn cal3lolllt" at w",w.w(rr jnr" fur Infflrm:;JUIlIlnn culture
IUfcf' to the ref<-:,.enCf:.
WMtd Dlr.a Cunlr'e for boficroor'llni~ms •
cuHtCl_lnuQuln nunlh~ and

• Gn'J\vth iscatrgod"led AS0: no gro\vlh, I: \lo'eilkgrowth (pilrtlilllnhlbition), and 2: good growth (see ISO Il133-1.
e Scme o.atiollal restrktiOI1,'i .and dlrrction£ Inay require th£! use of.a dlffereo1 $emv;.r:, Ma1.¢ereference tn n..wonal
requ'rcl1'lcnts relatJng to lhf: fhoke- of So,,"oltftllu serovars,
d $lr:lII" fre.e 01' chol(lt'; one 0' lileslr;lln!l h....lC to be used QIC:Ii
mJulmum.
 ISO6579-1:2017(£)

Table s.t (continued)

Medium Function Incubation Controlstrains WDCIII Crlterlail
nu.mber$)
XLD agar Producllvicy Sol",<>nell. '!Yphlmurlumc,d 00031 Good growth (2) of
So/mOl1oll. El1t~rl.ldl~t.d 00030 (010ni.5 wnh blatk
centre and a lightly
trans-patent zone of
reddish colour due
24 h ~ :~hl to rhe colour change
(1f the luedlum
37'Ct'I'C
SeI,ellviCY ""cherie;';" colid 000120r Growth or partial
00013 inhibition (H) of
yellow eolontes
EutwocoCCI/$ J{rfX(JII.~ 0000901' TOlallnhlbltlon (0)
00087
Nutrtcnt Prnduct1Vfcy 24 h l' 3 hi Salmonolla '!Yphhl1urlulllc,d 00031 Good stO\oJth
ag.r 34'C 10 38'(; Sal1nooeJla E11teritidfs4:.4
00030
l Refer 11)l11t:rerereoee SI",*I" C,I:.taJosltcat )YWl!.w(C'~Jrt(Qror tnrtu',nalifl" on culture (oUecllon srraln nlHllhcl'11 tlnd
contact tlC:l.l15: WOCM: \Vol'ld n.tit cemre for "Herool'ganistus,

• Gn)wth is catr8oril~ -as0: no Growth, tz weak ,ro\vlh (parti;al inhibition). and 2! &podgrewth [see ISO lI13)).
e Some nation.J restrtcttens olneJcUrrctlons may requfre.' the WI! of ~ different scrm'ir, Make: reference: In natjonal
rtqulf'f.:nlenrS !'el.ttfns to the (holoe or SbluUJlfeiStl Stl'J)Yrtl'S,
511'(1111'ree ot chQlee: nne (I(llu: $'tr;ilnll hJl$IQ be u..;ed.u,
" IIlhunHIIU..

I_ ....... ~_~~JJ -AIII"ghls·n!SeI'Ved t._blUNGIIiS10#S'!"'~fOle1_", 31

,.,..,._.. e. .11li"._IOo
~ ...... _.. .ltOO
,_..,_~_ •• __ H!I &O'.............. '~'II ..... 1'1
ISO 6579·1:1017(,£)

Annex C
(informative)

Method validation studies and performance characteristics

C.l Performance characteristics RVSbroth and MKTTnbroth

InwrnAt[OMllnierlaboratory .tociics were organlz~d 1n :woo In lilt>'ralliO O( the Europeal1l"'0jecl SM'l'
CT '11.1 Z098 (see RcJereilces lli] and 1:J1ill. l'n,,-.~srudies involved 11 taborarortes in nine countries in
Europe and 10 lnbnrarorles In USA and were carrted OUl an fresh cheese curd, dried egg·PQwder, raw
poultry meat, aod a ref.renu lI1ate,lal. ThL' food samples were ~a.ct1r.slMl al low And high levelS of
contamination. plus n ncgati.V(1.conrrol.
T~e method submitted to-the interlaboratory studies was that of ISO'6579:2002 (see Reference (11),
Including selective enrlcbment in RVS broth ;rnd MK'Mn broth. Tbo procedure for detection of
Salmollella in food sarnpl es as-described in ISO6519:2002.15 comparable to the prpcedureas described
in thisdocumeut,
The 93.b.lelIoPlle performance charaereristlcs derived from thiS collaboliU:Jve test are $hown per
type of samplu In Tabhw C,l to ~ Data oblalrW<!by some ~dbol'lItorles hllYebeen enrludcd from the
calcutartons 00).1all the basts 01 deal'ly id.ntiliecJ rechnical reasons (deviations to die protocol).

TableC.l- ResuJUofdatil analysis obtained with Cnsh cheese curd samples

Parameter Freshcbu'" Fresh clte.e-st'! Fresh cheese.
curd curd turd
(hl,".k) (low Ie..,. (hIgh level
t;:ullt'aruLua.dntl r' centa ntlnatJcm}'f
Number ufpar-tictpating eeltaboratoes 2·3 23 ~3
t;ulOber or ""'pies pcr toUtihota[Or 5 S 5
Nulllh<ll'
ofcollooorororsratained .iter evaluDtlonMlhe 21 21 2J
data
t;d.nbof nfump.I"" I·.tamed o(ter ellllluadllllll(lru. data lOS IO!> lOS
Test por.tjon 1;i~e. ill g 25 25 2S
Spc<-tlIclly.In % 100 - -
Sen.<ltivity; In % - 14,3 83,8
LOOS<!(95 % eOIlIld"'I," 1II'''''''''II.ln
•
d"/'_ ,)l0rUoll -
.. t:ed with ,'tlllrnn",,1111 ~"'ltc"idctJ
CkeClj)C:~':"I1P,-" weee artIl!d,lllUy C;OCif-.1111hll llxtnll~
S.7 (4.0 to 8,1)
pOr.ltI~"u'3h:l).

J.1r.KtprvbJtbk nlJll\Mr (t.JPN) results of the.artifici"Uy ('fIutilmjmtttd s'amp}.e$were the fnrtowirt&=

MPN/Z~I
Low level O,7lU.Z to 2.'1
l1igtHrvtl ~7.Zt7,5 to 95.0)
 ISO6579-1:2017(E)

Table C.2 - Results of data analysis obtained wltl. dried ""powder samples
Parameter Trial I dried Trial I dried Trial! dried Trial II dried
e&&JlOwder ell&powder eupowder eupowder
{blank) (low level (hIgh Jtovel (low level
contam ination}' CODGllnina_tionft cont-aminarion)a
Nunlber of parttdpatlng CQUaborlllO-rS 26 26 26 9
Nnmiler of .~mple. per collobor.tor 5 5 5 5
NlUIlbcr- of collaberaters retained 21 21 21 8
after ~W1Jua[Jonof the data
Numher of SiI!nlpipf) retained aftf.r 105 lOS 104 4-0
evalulltion of the datil
'rest portion s.lze..In g 2S 25 ZS 2S
Sp.dOclty.ln % 100 - -99 nd
Sensitivity. b. % - 98,1 nd
I.ODS<1 (95 % cnnfldence Inrerv II). In
c(u/'." portion
- 6.0 (+.1 In 1.7)

nd. not dct-ermin«i.

• EgGpowder samples were artifkl;dty ccoramlnared \vitb SalmotJt!lIa Panama,
MPH I'e~uh'lnfthe~II'tlfk:'JUy(GnNlnlluted Ample-II- werethe (1'lIIM\lln~
MPN/25g
,'rtalllC)w W:vC!1 9.6(2.2 t. 2(.)
lOria-II high level ItS l22,S to 495J
"rbl II 10\" lete' 0.7(0.2 In 2.3)

of dnta analysis obtained with row poultry men~sampl es

Table C.3 - Re&U)t.<
1>"rnltlclC!,' T,'I"II Trial I raw Trl"III'i1w Trillill rdW TrlolU ... w
raw poultrym ea t poultry moat poultry meat pGultry mear
J)uuhry
(low level (high level (low I<ovol (11lg" 1"".1
meat c.antamlnatlan}tf ton[alntnatton~contall1lnatlonJs contalnlnad()n)a
(blAnk)
Number of partkipiJtlng 25 2S 2S 13 13
coUQ.bornwrs
Number of samples per S 5 5 6 6
collabornfnr
Number of ccllaberarcrs 20 20 ZO 13 13
rerelned Grrer evalunrton
of th. data
Number of samples 100 99 100 78 78
retelned after evalll.rlon
of the data
'rest portion . ;Ile.ln g 2S 2S 25 2S 2S
Sl'edficlty. in % 100 - - nd nd

l._,"UN.oIIifQ''''''~fOlC1.tO'114I'f 33
..... IIIo... __ MllII •• jA
 ISO6579·1:2017(E)

Table C.3 (C(Jlldfluedj

Parameter Trial I Trial I row Trl:.11 ra\\' Trill I IIrow TrI.11I raw
raw poultry meat poultry meat poultry moat poultry meat
poultry
(Inw""'ol (high 10v.1 (",wl.vrl (high 10v.1
meat contamlnalfon). c:onta,ulnaUon}i contaminatlon)- c:untaluJnarlon)ll
(bI.•nk)
Sensitivity. in'WI - 98 100 nd nd
LODso(95 'II> confidence
Interval}, In du/test
- nd lid 2,2 (1,5 ro3,2)

pol'tinn
nd;::. not determtned.
• I/nultry meat samples wftr., artlOclally c:ontnn,lnJlI,odwith Sol,nnf'ldlu 'typhllurlunlln TrlnlI I1l1d\VI!J'e
naturally centamlnated with Soimrlhella spp.In Trbili.
MPHresults or the contamInated samples weroi
~IPNIZ5g
TrI.1 I low 1eve1 3,7 (1 to 9.5)
Trloll high level 5,8 (1 ttl 25)
Trl.II1IQw level 0,2 (0,04", 0,9)
TrI.11I high 1eve1 1,0 (2.2 to 4.5)

Table C.4 - Results or data analysis oblalned with rererence materials

Parameter Reference materla1
(capsules conraining approx, S cfu ofs. l'yphimtlnum)
Numbot .n.bol'1l",rIQ$ hAving returned re.sull$ 26
Numbe of samptes per laboratory 5
NtHT1hc of excluded I:.boratorles 1
Nunlbel' offaboralOries retained after exclusion 25
N,"nh4'.lJ'-llfaccupred ~n"IJlte5 125
SllcdHdcy.ln %
Sensitivity. in %
-
94.4

C.2 Performance characteristics of MSRVagar for detection of Salmonella spp, in

food and animal feed
In 2003, • validation study confonning to ISO 16140:2003JSJ was performed (see Reference [i)) to
compare recovery using MSRVagar alone with the method described in ISO 6579:20021.11(using the
selective enrichment media, RVSbroth and MKTThbroth). The r esufrs of the method comparison part
or this st.udy are summarized Iii Tables r S and £.Ii, As part of the study, an Interlaboratory comparison
study was also performed of which results are surnmartzed belew Tables C 5 and.!:...ti.

.,.~1-_if,o~
'.......
N..._"'1'1 ., ..
"'-'blc.u.lCJIIiIOfGl'",,,,~~7
.....
_,.. ... _,...."IU_I~ - Allrfghrs ~'fld
.....,._..,~._'U.,__ _ ........._".
.....__
 ISO6579-1:2017(E)

Table C,S- Number oftested samples In the method comparison study on validation or
MSRVagar
Food categertes Positive Negative Total
n.e, a.c, 101.1
Pofearand meat products 26 13 39 40 79
Dairy products 13 18 31 36 61
Fish. sen food, veget~bfes 4 28 32 32 6+
Egg products. pastry 3 29 32 3'1 66
f.nvlronmentaI5llInj"lles 0 30 ao :il 61
To.ar 46 UO 164 173 337
11.c:.. naturallyCnntiunlnillcd; ac, ;a: ltr,Ulclalty cnlltll"th"lttd.

TobIe C,6- Re..wlts .,rllletbod cOllll",rlson study oflhe validation study orMSRVogor
food catrl0riH" AC(%) SP(%) SE(%)
Meat nnd OlenI produ",. 96 98 95
Dairy I'rnduOl-< HID 100 IDO
Fish. seafood, vegetables 95 94 97
Po" prl)dUCI~,pastl'y 98 100 97
Envtronmt"ncal samples 98 100 97
All,Qmpl"" 98 98 97
AC= reliUiw .:.r.c:ur.cy; SP. relarwe .cpeciliclty: SE.: Mlilu_.senskivJry.

Other results of the method comparison study are as follows:

eight deviaring results (five negative deviations and three positive deviatlons], not significant;
relAtive detecrton level ror the five matrlees W'$ 0,49 to 0,78 So/mom,}I" cells per 2S 8 or OIl:
IncJuslvicylCSled on 5S Sal/non.IID food lsolates (10 cJu/l1ll to 90 cfll/miln BPW):
~xclusivitytested on 48 non-Salmonella strains interfering ....nth Salmonella detection methods
(10' ro 1.06du/ml in BPW);
results Inclusivicy/exch,sivity study showed method specificity, Three Salmonella strains were
not detected (2x S, P'''''typhl A. Ix S, Enteritidis) and two strains of Enterobocter cloacae goy.
presumptive positive results.
ResultS of die Inlerlaboratory comparlson study are as follows:
29 participating laboratnries rrom 10 different countries;
each lab tested 24 artificially contaminated skim milk powder samples At three diffel'em
contaruination levels (0 du/2S g.10 ciu/2S g. 30 <fullS g), Eightre.pltcalesamplcs percontamination
level were tested:
duptleate restlng with MSRVagar and ISO6579;2002111;
240 resultsper method;

no outliers;
for.1I contamination levels: AC,56. 51): 100 %. and confidence InterVilI: 98 %;
IWO false·po~itive results, most likely due to crcss-ccntaminancn.

•__ ~ I!)JSfUOJ.7 - All rights ftiel'\'ed ~_lftlU6tnII(QOI""""~'lOUeI _". 3S

"....
............
!..-.... 't'GII._ ..........'-""O
_~jIIIMO!I.l.. w.._ .._HtI
"".._ ..• "I~""cw."1•
 ISO 6S79·1:2017(E)

C.3 Performance characteristics of MSRVagar for detectlon of Salmonella spp. In

animal faeces and in environmental samples from the primary productlon stage
The precision data ot MSRVagar for detection of Salmonella spp, in animal faeces and in environmental
samples from the primary production stage were calculated from three different Interlaboratory
studies organized by the fURL·Salmonel/a, RlVM.The Netherlands. This concerned studies organized
in 2008Wil,20'l2UlI and 20 13.lUI The samples tested in the three studies were respectively.chicken
faeces, pigfaeces, and boot socks. Thesampleswere each tested at two different levelsof contamination,
plus a negative controL All studies were funded by the European Commissionand the latter study was
also performed as part of the CEN mandate M381.
The method submitted to the Interlaboratory studies WDS that or ISO6S79;2002/Al1Id 1;2007121forth.
detection of Salmonello ln samples from the primary production stage Including selective enrichment
011 MSRVngar.This method has bee n lnrorpcrated In this document.
The values of the performance characteristics derived trcm the interlaboratory studies are shown per
type 01 sample In Tables C.7 to J:.2. Data obtaln~d by sorue collaborators have been cxchldcd from the
calculations olily all the basis of clearlyidentilfed technical reasons (deviationsto the protocol].

Table C.7- Results of data analysis obtaIned wltb chkken faeces samples
l'OlranleLU' Chlck.n (•• <6 +
Bi;mk STMS' STM44' 51>7" 5E91'
Numbe ro (pa rtlclpatlng ""llobo ... tors 32 32 32 32 32
N\,m"",' of •• mplesper ,,,II.h,,r",or 5 5 5 5 5
Nurnbercf collaborators rctaln~daftu cvaluallub 19 19 19 19 19
oft he data
Number of samples retatned Rf~r evaluation of tbe 95 95 95 95 9S
dare
Test portion size, in g 10 10 10 10 10
Spcctlklty.ln % 100 - - - -
Sensitivity per serevar and level, in %
LOOseper serovnr (95 'l(, con(!dMcelnterv.I). In
-- 96.8
1.0(0.7to L4)
100 67.4 100
~.3 (3.3 105.6)
du/test portion
1.001;4<w~r.1I{CIS'!(, cnnRd,·nc. Int.tvol). In
du/teSt portion
- 2.~(2.1 til 3.0)

• Chkl(tfl'l 'lIec:cs s.:lIflll,Tdwere art Intfl.lIy c;onl;',,,h'tJlr.d \vllh "t(~..t'IiCCmat,,"III~ \""lb Ihi: .rl)l'luwI"~pUr"I"S~ndlevels;
Sn'm(l11~1MTyphlmurlum (STMJ i1tR~el nf!> Cfll/lf!!1Ot pqrtton and a level r)r44 dU/lt:Sl fl"I'c-inn:
.,.,I'I!OI1I!f1nf.ntf!rflidi,~ (SE) ~·l.. lf!velnf? trll/,cst poretnn.aIUJ It level (I'" ((u/t't$! pon-IQn,

1... ~jj.'
......
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__ ... y_..._'11~"'O
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 ISO6579-1:2017(E)

Table C.B- Results of data a 118 lysis obtained with 1,Ig faeces samples
Parameter Pig faeces +
Blank 51)6' SD37> ~"~1I0' STMSH'
Number of ,,"rtklp.tlng collabor"tors 33 33 33 33 33
Number of 5aIJlp1e~per-collaborator 5 5 5 5 5
Number of conaboraters ret>Jncd amr 26 26 26 26 26
evoluAtlon of the-data
Number or 5Aluples retalned arter cvaluildon 130 '130 130 130 130
of the data
're<t portion size. In g 25 25 25 25 2S
Spedfldty.ln '*' 99.2 - - - -
Scnsitivlrypcr serovar and level. in 'Ito
L0Ds. per serovar' (95 % conftdenee Interval).
-- 88.S 97.7
2.8 (2.2 [0 3.5)
91.5 98.5
3.8 (3.0 to 4.7)
In <(u/t .. [ portlnn
!.ODs. overall (95 % ccufldence In[6\'ol).ln - 3.2 (2.8 to 3.8)
cfu/lost portlnn
, l'ig farces samples \VUe arti(JclallycontaminatC!'d with rercnnce matertals with the foUO'\.tngstralns acd levels:
SdI'''(I,lC'Ilu Derby (5D) »1 ~ leval o( 6 clU/lesl pon,"n and alcvdcf3? c(u/tc.t p"l'tlon:
Sob"ond!a Typhhnurium (mf) at .. ~\'clof todu/test portion and iii t~el or58 r:(u/test portion.

Table C.9- Results of data analysis obtained willi boot sock samples
Parurt1~t.er Boot ~GGks
..10 glaying hen "nvlronment.1
material.
B"Il1I, STM9> STM81'
Number of participating collaborators 36 36 36
Number or $:UUpICN per t:ullalu,r::.tor 8 9 9
Nunlher oflnboTotoJies: rereined after 3J 33 33
eVl'llulltlon (If the daca
Number of samples retained .after 264 264 264
evaluation nfthedata
Sample,-Ize 800, Boor ~odc:s
sn<ks
Specificity. In % 99.6 - -
SenlCJtlvlty per level. In % - 9~',7 99.1
1.00.0 (95 % contidenre Interval], - 3.8 [3,Z to +.4)
Indu/sample
, The boot .UIrdc: s:.unple.s were lIrtl(icU..Uy cenramtneted with il diluted culture or
Sobnollf!llo TyphlnulI'lum (STW) at a: level 0(9 cfu/nnlrle and it level of 81 cfu/$amptr.

, Il>.®J.IlJ.7 - AllrI"IIS ..... ,"'.d l_~411c1ff0!'..r..v~tJeuC1 !ilMtll' 37

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".1IIt1~
 ISO 6S79-1:2017(E)

Annex D
(normative)

Detection of Salmonella enterica subspecies enterica serovars

Typhi and Paratypbi

0.1 General
Certain serovars of Salmonella are not consistently detected by the method described In this document.
This annex spedOes addltlollal steps to be taken when the detection or Solmon.flo enterico subspecies
enterica serovars Typhl and Paratyphi Is of spedflc concern. The full method described in this document
shall also be performed.
NOTE Srrnlns of serevnr G3111narum (b1nvitY'Sgnllfnllrum and Ilullnrum) are of nn s'Bnlficance to humau
health and so thelr Isolation Is not Included In this document (see Refel'enceltlll.l1ow.ver.lrtheS<! biovars are
specifienlly sought. Selenite Cystine medium (SC) cnn be used in .ddition tn RVShroth and MKTl'n hroth a.
de~('rlbedIn this annt!x.

D.2 Detection of Salmonella Typbj and Salmonello Paratyphl

0.2.1 Principle

WARNING - Salmonella lYllhl and Salmonella Pllratyphl are lJamrd Group 3 organisms.
Approl,rlate containment facilities shau.ld be used when baudlhlg these strains.

0.2.1.1 General

Seell

0.2.1.2 Pre-enrfchment In non-selective liquid medium

See 1.2.

0.2.1.3 Enrichment In selective liquid lIIedIn

See;!,;!. Selenite cystine medlum (SC) is inoculated with the culture obtained in il in addition '0
illoculatioll ofRVS broth and MKTTnbroth.
The SCmedium is Incubated at 37'C (6.3) for 24 hand 48 h.

0.2.1.4 Plating out and Identification

See ti. Bismuth sulphite agur (BS) is Inoculated with the cultures obt~lned In U and D2.L1ln
addltlon to XlO agar.
The 5S Agllr IRincubated nr 37'C (6.;l.) and examined after 24 h••md again, if necessary, after 411h.

0.2.1.S Cnnnrmlltion orlde.ntlty

See~.

-...~-,..-""-
..~-~-.,...--......
~"""\I_"'''' •. I*.-o
-.,~
 ISO6579-1:2017(£)

D.3 Culturemedia
D.3.1 Selenite cystine medium (SC)
WARNING- SodIum bydrogen selenite Is potentially teratogenlcand may barm the unborn child.
Appropriate protective precauUonsshould be (liken when prepnrlng8nd handling this medium.

0.3.1.1 Buse medium

0.3.1.1.1 Compos ilIon

Peptone- 5,Og
Lactose 4.0g
Disodiurn hydrogen phosphate dudccahydl'Olle 10,0 g
(Na2HPO •. 12H,O)
Sodium hydrogen scl""lt. 4.0 g
Wat,er i 0001111
• For example, enzymatic digest of caseln.

0.3.1.1.2 Preparation

Dissolve the sodium hydrogen selenite In the water, then add lh. romalnln.g Ingredients. Ueat to boiling
to dissolve.

0.3.1.2 L-Cystlne solution

0.3.1.2.1 Composition

L-Cystine O,lg
Sodium hydroxide solution,c(NaOH) = 1. 11101/1 15 ml
Sterllo water Allprox.85 011

0.3.1.2.2 Preparation
Add the components to a sterile 100 ml one-mark volumetric flask (0.4.2). Dilute to the mark with
sterile warer, 00 not stertltze,

O.l. 1.3 Co",pl~te medium

0.3.1.:1.1 Composltlon

Base (l13J..l) I 0001111

L-Cystltle soludon (D.Jll) 100m]

D.3.1.3.2 Preparation
Add the L-cysthlc sclurtcn aseptically to the bas c, Adjust the plf. if necessary, so that the rinal ptl will be
7,0 ~ 0,2 at 25 ·C. Disp"nse the medium aseptically into sterile tubes or flASks(§J.l) IDAchievea depth
of at lcast 5 em. Sterilize by steaming for 15 min. Do not autoclave.
Store the poured tubes at 5·C (fUI). The medlum may be used until "red precipitate occurs.

Ifi ......... ~_I:J~1) - AU "''''116 reM:I"Vcd 39

1'I_e.It*u_b ....,
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ISO6579·1:2017(E)

D.3.2 Blsmuth sulfite agar (BS)

D.3.2. t Composition

Enzymatic digest of anin ta 1 tissues 10,Og

Meat extract 5,Og
Dextrose S,Og
Dlsodlum hydrogen phosphate [anhydrous) (Nazll PO.) ",Og
Ferrous sulfate (anhydrous) 0,3 g
Bismuth sulfite (Indicator) B,Og
Brillrantgreon 0,025 g
Agar ZO,Og
Water l 000 OIl

D.3.2.2 PreparntJon

Add the components to the wafer and heat with frequentagitanollllntil boiling. Continll. to boll gently
for 30 s to 60 s to dissolve the agar and obtain a uniform suspension (precipltate wlll not dissolve}. Cool
to 47'C to SO'C, then gently "glial. to suspend the precipitate.
Adjust the pH, Ifnecessary, so that itis 7,7 ± 0,2 at 25 ·C.
Pour 20 ml to 25 1111 Into Petri dishes (6..11) and allow to set. Correctly prepared plates will be a pale
straw colour with a smooth crenm-Ilke opacity.
Prepare the plates the day before use and store In the dark at ambient temperature.
Dry the plates, Ifnecessary. before use. Do not overdry.

D.3.3 Performance testing

The deOnltlon or selectivity and productivity Is spedfled In ISO 11133. The lnocuhrm volume should be
tbe same as that used in the method for that medium and should contaln the number of target or non-
target organtsms specified In ISO11133:2014,5.4.
 ISO6579-1:2017(E)

Table 0.1- Perlormance testing for Il,e qualtty assurance or the culture media
Mrdlum Func:UOD l'It:u.blatiun (Qnl"-ul ..l-n,ln.J WDCM Critrr.,,·
hu._tnbe~
Selt'ohtr Productivity Sctbrrotlft11o '~fphlnlul'lun,(.11 (10031 >1n chllrxrel'ltlk:
cystl"" So/motlena Ente:ritidisc.d
colOtllc$ I)JJ. XtD Igal' or
00030
"th~ r medaim of
choke
+ £SC'hcrid/(f (olP 000120r
24h±3h/ (10013
37"C"'C ...~eudrNQo"as aeru,qn'lDSO ooezs
5<l«lIvl., t..dttt,'It:"/u co,''' OOOI2.or JI'.1rUallnllll)lllon
00013
'Sloo (:o-Ionlc$ on TSA
£nter'ocor-l1l$ fi,t.(ulfsd 0(1009.,· <-IOcolnnles nit "tSA
00087
Bismuth I)rl')d:uctlyhy S«ImQllrllo Typl.tlnlurfu_mf~d n0031 Good gl'ow,h (Zl or
sulphite brown~grey. or blac:1(
fig'aT
S"lm"ftdla Eltlerhtd";U 110030
eolonk-:t:1uruaUy with
" met.1l1ic sheen arlcl'
24 h bocomlng
uniformly black "(Ie'I'
46b
b(24bk3h)/
S~JMtivitYI 37~C±r-c £SCherid!lo colid OOOl2nr Growth er-parna
!\'j)Cclllcltl 00013 '""'0"I0Il (0 to I) or
duUgreenorbrown
cnlonlell wh_htu.ll
metallle sheen
S('lenlvlty E", tJ'OC«hlSfi,«ull,(d (10009 or Totallnhlb'tlQu (O)
00067
•WncM:
Refcr to d,c r,"korellclt.tr:!I'l (2U1"tI\lot~l'YWWwkr ....'o(orinfcII'ltI2tlan 0" culture co.lIl!(tirnllltl'lll."",tmtn a,ndD)nUld-detllU-c;
\vortd n~f.aCnltre (Pf Mk:TOOri!ilntUl.l'i,

• StTain to b~u!CedlUI:.minimum.
C Sofl\t ~1.I(I"1I1lutrkC'tllI' II", dl!'tCUOlu '''.r NI(IIIh'tIII~ use of • dl((crtIU"lrO'/II', M..u 1'~(t"'*'(C'0 nal.Jo~1 ''C''(ltUreIftC,u.
reblihg to d~c:hoiclL" (If Sot.-Jlldlu ~;lIrs.

•• Sr,'.,'" "'Of!
ofchotco: OM oftllot'U';1lllCbl,lON:uJU:d o:c.1t I1Illt.. \,.,..
Growth I. e~t~rilttd aJOt noJ;r-owtll.•I: wf!:IIk,,'owth(partb_lluhlbiHol!),a.nd2:jloods:,~vthbC'e: ISOtll3»),

D.4 Equipment and consumables

0.4.1 General
In addtticn to the equipment And ecnsumables described In Clause 6 add the followtng.

0.4.2 Volumetric nasks. sterile. 100 ml nomInal capacity.

D.S Procedure
0.5.1 General
In .ddltlon to the procedure described In Clause 9 add the following steps.

0.5.2 Enrichment in selective liquid media

Transfer 1 Il1lllft,hcculture obrntned in ~ (011 tube containingl() ml of Sl: medium (.I1.3.J).
Incubate the inoculated SCmedium at 37 ·C (1;.3) fDr 24 h ± 3 hand 48 h ± 3 h.

'_~"'Q ~.~J.%IlO~7
........__~.a -1011rfgl".resel'Vod
\I__ ...
41
'.'''-_'. _ .. \1...... __ •• _ ...
 ISO 6579-1:2017(E)

0.5.3 PlatIngout

0.5.3.1 After incobation (or 24 h ± 3 hand 48 h ± 3 h usiog the culture obtained in the SC broth (0.5.2),
use a loop (UQ) to inoculate from the upper one-third of the broth onto the surface of an Xl.Dagar plate
UM.l so that well-Isolated colonl..s are obtnlned.
0.5.3.2 Proceed in the sallie way with BSngar (J2.J.l) using a sterlle loop as above.

0.5.3.3 Incubate the XI.Dand OS agnr piatcsat 37'C (n,;u for 24 h ±3 h.

0.5.3.4 After Incubation (or 24 h ± 3 hat 37 ·C (6.3), exa mlne the OSagar plates for the presence of
typical colonies of Solmollel/a which are black. grey, or brown with or without a metallic sheen. The
surrounding medium Is usually brown at first becoming black with tncreased Incubation. MArk their
position on the bottom of the dish. If typical colonies are present. select five colonies for confirmation.
Relncubate the OSagar plates for a further 24 h (48 h .. 3 h In total) and examine again for typical or
atypical colonies which are green with little or no darkening of the surrounding medium.

0.5.3.5 After incubation for 24 h ± 3 h at 37 ·C. ex.. mine the XI.Dagar plates as described in 9.!1:.2.
Typical colonies have a black centre nnd a lightly transparent zone of reddish colour due to the colour
change of the indicator: Salmonella H2$.-.negative variants such as Salmonella Paratyphi A are pink with a
darker pink centre, Marie their pCI$ldollon the bottom of tho dish.

If no typical or atypical colonies arc detected. re-tncubare the plates for a further 24 h at 37 ·C (Ii.3,)
(48 b ± 3 h in tol<ll)and examine again.
NOTE Not .U chroloocenlc egor medla mlly t>:lststIII tho recovery of 501111011.1/0 1ypbl olld 5a//II/)/w}/a
Paratyph).

D.6 Confirmation
0.6.1 Geneml

See2.S.

0.6.2 Interpretation of the biochemical tests

Sa/moll./Io l'Yphi and Salmo/HIlla Pannyphl generally show the reaeuens given in Tahle J.

1.... ~ __ ""~ .....,...

".......""'I!'fl"._ .... _ ...,.. lIlO
NoI'..-....._I\I....,._ ... ~ ..... t..o. ...
 ISO6S79-1:Z017(E)

AnnexE
(informative)

Examples of selective plating-out media

NOTti1 Derivedfl'omIl.f.r~ncc llD.l. Rel""duced by permission oflhelloy.1 So<;iety"fCh=ist''Y, Co,nbridge.

United Kingdom.
NOT6l Thlslist .(sel.clive )l1.tlng-ou,m.d~ I. not e'''llUSrive.

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43
ISO 6579-112017(&)

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47
ISO6579·1:2017(E)

BibUograpby

[1) ISO 6579:2002, Microbiology of food and animal feeding stuffs - Horironto! method for the
dewel/oll o/Salmotlel/n spp,
[2] ISO 5579:2002/Amd 1:2007, Microbiology of food and animal fcedillg scuffs - Horizomo! method
for the detecdoll of Salmonella spp. / Amendment 1: Ann~x D: Detection of So/mc1Ilel/a spp. In
animal faeces and In environmental samples from the prtmary production stage
(3) ISOITS 6579-2, MicrobiOlogy of food and clllimol fud - /lorIZ<l"t.ol merhod for th~ del«l}o.,.
enumeration anel serotyping oJ'Salmonella - Parr 2: EnumeraTion by a miniaturized most
probnblc number technique
[4] ISO6785:2001, Milk and milk products - Decution ofS"'mo"e/la·spp.
[5] ISO 16140:2003. Microbiology of food O1Idunlmal feedlllg stuffs - Protocol for the validation of
alternotive methods
[6) ISO 16140·1. Mrcroblolo[/Y of the rood chailt - Method vulldatlon - Part I: Vocabulary

l7) ISO '16140·2, Microblolugy uJ' the fiwd elwin - M.tJwd vutidutio« - Port Z: Protoc»! for lhe
validoc/on%lccmar/ve (proprietary) methuds against a reference method

lB) AfSSA, ZOO1.Evaluation of mfcroblologkol methods for ".teer/oll gild [01 enumeration of
microbiologIcal contaminants In food. Final report: Contrad SMT4/CT96 2098. Coartiiuation by
Agellcl! F''''JIl~ise de S~,urllii Sanltolre des Aliments. AFSSA,France, February 2001
(9) COLOMBO S.. CROCIANI l., HORRY ]i. fS016140 validation a/the MSRV method [or Salmon.llaspp.
Detectlolllllfood alld envtron-mento! samples. Poster at lAPP, ROlne. 2007
[10] COOl(£ V.M.• MILf.5 R.,., PRICE R.G.• RICllAROSOrl A.C. A novel chromogenic mer ngnr medium
for detection of sallnonel/a~.Appl. ~·"vlron. Microbial. 1999, 65 pp. 807-,2
(U) CUl.'rURE MEDtA FORFOODMICROBIOLOGY.
In: Progress In Industrial Microbiology. (CORIIV ,.S.L.,
CUNTIS G.O.W., BAIRD R.M. CdS.). Elsevier, Amsterdam. Vol. 34, 1995
[12] SMEDTl)r, ,.M .. BOLDEROtlKR.F.• RAPPOLDII .. LAUTSNSCHI.iIEGER O. RopldS.,Imonelia deteaton
infoods by motility enrichment on aroodified seml'$o/XJ Rappaport.Vo'sfliadls medium.]. Food Prot.
1986. 49 (7) pp. 510-514
[13) EWING W.H. Edwards and F,wing's Identification of F.nterobacteriaceae. Elsevier Science
publishing Co., Inc, New Yorl', Fourth Edition. t986

l14J EWING, W.H. AND BALL, M.M. The biochemical reacUanso{thenenusSalmonelia. National Center
(or Disease Conlroland Prevention, Atl3nm. 1')%
[15] FELosmE P. RecoveJ)'orSalmonelia ill Selected Foods by the ISO 6S79 Salmonella Culture Procedure
(llIll rhO' AOIIC '''tltrnucloIJelI O/pciQIMuthod of Al1oiy$i:r. Col/abol'Otivc Study.I.IIQAC 1111.2001
(l6J KmlI'E.RS A.F.A..•YEENMAN C.• MOOIIMAN K.A. £U tnteriaboratory comparison stu~· Veterinary·
XI, 2008. 8oLcedoiogieDI detection o{Salmonella i" chicken faeces. National Institute for Public
Health and the Environment, Bilthoven, The Netherlands. RIVM Report no.: 3306(l4 011/ 2008.
A".,liable at: bn:p'ltwwwdym ollbjbUQlhCcklrafllwrten/U0604011 pdf (visited 2016·12·14)

[171 KmlPERS A,F.A•• & MOOI}MAN K.A. 2013. £U Interlaboratary

comparison study Veterinary·
XV (2012) • Oetectloll a/Salmonell. In plOfa"'.'" Natlon.,llnstitute
for Public Henlth .nd the
Environment. Bllthoven, The Netherlands. RIYM Report no.: 330604 028/ 2013. Available at:
bttp'/6yww,dym nJlhlbHatlieeklrallportenf330604028 pdf (visited 2016·12·14)
 ISO6S79-1~2017(E)

[18] MAI.l.INSONE.T., MI~~elt R.G.,dE Il~Z£ND~ C.E., FlrRRIS K.Il.,dtGR.Mlt·HM.SON '.,JoSEPH S.W.
Improved plari'l9 media for the detection afSalmonella species with typical alld crrypico/ hydrogell
sulfide/>roduccioll.J. Vet Dio911./nvest. 2000, 12 pp.83-87
('19] MANUALO, DIAGNOSTIC TESTS ANI) VACCINES FORTERRESTRIAL ANIMALS, 2009 ((liE Tcrl·estri.1
Manual) Chapter 2.3:11 Fowl 1Yphoid and Pullorum Disease, pp, 538-48, Available at:
bttp'//\ywwoje int/n)antlal.Q[.diagnosrjc~tests·and-yaccines·fQr.terrestrial-aniDlaJs/(visited
2016·[2·14}
(20] MOOIIMANK.A. Culture media for the Isolalfon of SalmolU1ila.In: Handbook or Culture media fer
Food and Wawr Mlcroblology,{CORRY"E.L., CURTISG.D.W.BAIRDR.M.eds.). Third Edition, 2012
(21] KUIIPHRS A.F.A., & MOOIIMANK.A. EU IIlterlaborota,,· c()I)'pariGO"study llrimary pl'Oductto,,·
XVI ( 2013) - Dctecaol) ojSalmonnll. (1) cMckell fa""" .. adherillfJ to boo« socks. National lnstirutc
for Public Health and the Environment. Bilrboven. The Nerhcl·lands. RIVM Report no.: 330604
03lj2014. Available ~t: hnmllwww,dymnllblbliorhcckiI'DPQ<lrtQn/33!l6Q4Q31.pdt (vlsited
2016·12-14)
l22J SOUTOUIUNAO.A.• SSM[NOVAE.A., PAR,ENOY" V.V.. DANCHlI'IA.. BERTlN P. Control of bactertal
motility by environmental factnrs in polarly flagellated and pedtlchous bacteria Isolated from
l.ak. 8.,iI(.I. Appl.llnvlron. Mlcroblol. 2001, 67 (9) pp. 3852-3859
(231 V6&NMANC.. KORYER1-1.. MOOIjMANK.A. Improvements I" the method fortf.lectioll of Salmonella
spp. III (1IIlmolluullS. Notlonalillstlrule for Public lIealth and the Envlrenrnent, SUthoven, The
Netherlands. RIVM report 330300 010, 2007. Available at: hll!>://www dym nl/blbU,,!b..,k/
,"QPorn'll/3a03aaO] Q pdr (visited 2016·12·14)
[24] ISO/TR 6579-3, Microhiology afthe JO()dchan, - Ht>rizolltal method for the detection eJlumerotiQII
ana SJII'otypi',g of Sa 1111Oil gila - Pao-t3: Guld.lin"" for scrolyping or 5011110110/10 spp,
(251 ISO 18593, Microbiology ol food and onlmalleedl"l/ stllffs - Horitontal methods for somplilIjJ
techniques Jrom surfoces USingCOIIUlelplotesand swabs

[261 ISO/TS 17728, Microbiology of the rood chain - Sampllll9 {lUhniques for microbiological ollolysis
(J{/'oud alllt fl!<!d samples
(27] ISO 707, Milk alld mill< products-« GuidO/Iceall sampling
(281 ISO 13307, Microbiology of food and animal reed - Primary production stage - Sampling
I.cchnlqu.s

(291 ISO 17604, Microbiology of tile food cnatn - carcass samplilllJ [or microbiolo.l}ico/ analysis
(30J D'AOUSTJ.Y.MAISHM£NT C., BRUGENERD.M.,CONLEYD.R., LOIT A.• MILLINGM.AND PURVIS
U. Detection of Salmonella in refrigerated preenrichment and enrlchment breth cultures. J. Food
Prot. 1980. 43 (5) pp. 343-345
[311 D'AOUST'.Y.SECKERS fl.l.. BOOTHROYDM.•MATESA., MCKEEC.R.. MORANA,B., SADOP.•SPAIN
G.Il.. SPEIIBER W.H.. VASSILIADISP.. WAGNERD.Il.AND WI8ERG C. ICMSFmethods studies. XIV.
Comparative study on recovery of Salmonella from refrigerated preenrichment BJWenrictunent
broth euttures.j, Food Prot. 1983. 46 (5) pp. 391-399
(32] BECKERS 11.1.• van LEUIDEN F.M. AND PETERS It Her effect "an koelen van bebrcede
vool·ophoplngs. en selectteve ophoplngsbaulIIon op de Isolatlc van S<lImullc/lo. (In Dutch), De
Ware(n). Chemlcus. 1984, 14 pp. 75-BO
[33J DAVIESR.H. BIlDI'ORDS. AND SHANKl>'TERS. Enhanced culture techniques far the detection or
Salmonella. Vet.Roc. 2001, pp. 539-540
[34J COMMUNITY REFllRENCE LABORATORYFOR SIILMONIiLM. Newsletter 200B. Vol. 14, no. 2,
page 8. Available at: http;llww\y eurlsalolonrlla ru/dsresollrce1.type:pdf&dh;·posU·jon-:lnJjnc&
a)Jj<'ctfd=rly:tDp'181
223&yp[sjgnld=&sIIbgbird DPDlC(visited 2016·[2-14)

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[35] BECKhl<H" EOERHARDT S.. Mi\HTLBAII6R E. ComparatilTl! studies on rhe detecticn of Sa/monel/ae
In milk 9nd milk products using 9 horlwnllli (ISO 6579:2002) and a vertical (ISO 6785/101'
93:2001)lnternntional Srnnda,·d. Arch. LebmrsmiClolhyg. 2003. S4 PI" 118-121
[36] European Union lI.ference Laboratory i'or Salmollslla.
Newsletter 201S, Vol. 21, no. 4·, pase 4. Available at:
http"/vJ'vW Plrdsa I[Donella PJl/dsreSQllrce1t)rpe=pd fBI dj sposi tinlJ =jnljne&objprHd:rivlup'
299BSO&vrrsionjd~&$lIbob.jtctl1jln,e!:! [visited 2016-12·14):

[37] ISO17468.MiCl'obhllugyof tile food challl - Tee/mical requirements audgult/allce all eswblishmellL
or revision Of Q sWlfdardlled reference method
[38] ISO6887-1:20) 7, Microbiology of th~ food cllOin - Preparation of usc samples, initial suspeuston
and decimal dilutiolls for mi~robiologi<:1)1examtnatton - Pan 1: Gelft!rolrules for the prtlparatioll
of the initial suspension and decimal dilutions
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