Procedure for Isolation of Listeria monocytogenes

8/12/2019 Procedure for Isolation of Listeria monocytogenes

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FOOD ANALYTICAL SERVICE LABORATORY

FOOD AND NUTRITION RESEARCH INSTITUTE

Page 1 of 4Revision No.: 2Effectivity date:Mar 1, 2013

ANALYTICAL METHODS MIC-AM 007-2

ISOLATION OF Listeria monocytogenes

1. PURPOSE/SCOPE:

The procedre descri!es the "ethod for detection of Listeriamonocytogeneson varios food sa"p#es.

2. REFERENCE:

$ood and %rg &d"inistration. 2011 'hapter 10 ( %etection andEn"eration of Listeria monocytogenes in $oods. )acterio#ogica#&na#ytica# Mana# *n#ine. http:++.cfscan.fda.gov+-e!a"10.ht"#.&ccessed 2+1/+2013.

3. PRINCIPLE:

The "ethod is !ased on the ana#ysis of the sspected conta"inated food#ots. 'o"posited s!sa"p#es or ana#ytica# portions are enriched inse#ective enrich"ent at 300' for 4/ hors. The enrich"ent c#tre isstreaed on the prescri!ed differentia#se#ective agar at 30' for 24 h and4/ h. so#ates are prified on nonse#ective agar and confir"ed !yconventiona# and rapid tests.

4. CULTURE MEDIA AND REAENTS:

4.1 )ffered 5isteria Enrich"ent )roth p#s se#ective spp#e"ents'yc#ohe6i"ide, Na#idi6ic acid, &crif#avin 7'#

4.2 *6ford Medi"

4.3 8 9heep !#ood agar

4.4 ra" stain it

4. 7ydrogen pero6ide so#tion, 38 for cata#ase test

4.; E

4.? Prp#e car!ohydrate fer"entation !roth !ase, containing 0.8 +vso#tions of de6trose, esc#in, "a#tose, rha"nose, "annito#, and6y#ose

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Reviewed by: Approved by:

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FASL

ISOLATION OF

Listeria monocytogenes

MIC-AM-007-2

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4.10 9M "edi" or "oti#ity test "edi"

4.11 Trypticase soy agar ith 0.;8 yeast e6tract T9&>E

4.12 '7R*M Listeria&gar

4.13 &P Listeria


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;.1.2 'o"posited 9a"p#es

;.1.2.1 'o##ect 10 s!sa"p#es #iCid, crea" or so#idfood of a food #ot sa"p#e ith 0g or "# portionsof each s!sa"p#e.

;.1.2.2 Mae to co"posite sa"p#es of 20 g+"# each.;.1.2.3 Poo# and !#end or sto"ached the first s!

sa"p#es in 20 "# )ffered Listeria enrich"ent!roth containing sodi" pyrvate, ithotse#ective agents to "ae the first co"posite.

;.1.2.4 Mae the second co"posite !y co"!ining there"aining s!sa"p#es in the sa"e ay.

;.1.2. 'o##ect 2g ana#ytica# portions fro" each

co"posite and se for preenrich"ent ;.2;.1.2.; Retain 0 "# a#iCot of the co"posite !#end, for

possi!#e pathogen en"eration.

;.1.3 Nonco"posited 9a"p#es;.1.3.1 Deigh a sing#e 2 g ana#ytica# portion of the food

sa"p#e;.1.3.2 Retain 2 g sa"p#e for possi!#e pathogen

en"eration. 9tore itat ' if it is not froFen or, iffroFen, in a nondefrosting freeFer.

;.2 PreEnrich"ent

;.2.1 &dd 2+0 "# #iCid or 2+0 g of sa"p#e to 22+40 "#)ffered 5isteria Enrich"ent )roth ithot the se#ectiveagents. nc!ate for 4 hors at 300'.

;.2.2 &fter 4 hors inc!ation, add the se#ective agents andcontine inc!ating for a tota# ti"e of 4/ hors at 300'.

;.3 %etection

;.3.1 &fter 24 and 4/ hors inc!ation of the enrich"ent !roth ithse#ective agents, strea enrich"ent ho"ogenate into *6ford&gar *G&. &s an option, at 4/ hors, strea a#soho"ogenate to '7R*MListeriapara##e# to *G&.

;.3.2 nc!ate *G& p#ates at 30' for 244/ hors. nc!ate'7R*MListeriap#ates at the sa"e conditions.

;.3.3 Listeria co#onies on *G& p#ates appear !#ac ith !#ac ha#ode to esc#in hydro#ysis. Listeria monocytogenes as

appear !#egreen co#onies ith opaCe hite ha#o on______________________________________________________________________________________

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'hro" agar.

;.4 so#ation

;.4.1 Transfer or "ore typica# co#onies fro" *G& to trypticase soyagar ith yeast e6tract T9&>E, streaing for prity andtypica# iso#ated co#onies.

;.4.2 nc!ate T9&>E p#ates at 300' for 244/h. P#ates "ay !einc!ated at 3o' if co#onies i## not !e sed for et "ont"oti#ity.

;. dentification of Prified so#ates

;..1 Perfor" Moti#ity test, 'ata#ase test, ra" stain and test for7e"o#ysis on typica# co#onies fro" T9&>E p#ate. Refer to&ppendi6 ' for the "entioned tests.

;.; 'onfir"ation of Listeria monocytogenes

;.;.1 Rapid


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Procedure for Isolation of Listeria monocytogenes