FOOD ANALYTICAL SERVICE LABORATORY

FOOD AND NUTRITION RESEARCH INSTITUTE

Page 1 of 4
Revision No.: 2
Effectivity 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 Listeria
monocytogenes on varios food sa"p#es.
2. REFERENCE:
$ood and %rg &d"inistration. 2011 'hapter 10 ( %etection and
En"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
#ot0s1. 'o"posited s!.sa"p#es or ana#ytica# portions are enriched in
se#ective enrich"ent at 30
0
' for 4/ hors. The enrich"ent c#tre is
strea2ed on the prescri!ed differentia#.se#ective agar at 33
0
' for 24 h and
4/ h. 4so#ates are prified on non.se#ective agar and confir"ed !y
conventiona# and rapid tests.
4. CULTURE MEDIA AND REAENTS:
4.1 )ffered 5isteria Enrich"ent )roth p#s se#ective spp#e"ents
0'yc#ohe6i"ide, Na#idi6ic acid, &crif#avin 7'#1
4.2 *6ford Medi"
4.3 38 9heep !#ood agar
4.4 :ra" stain 2it
4.3 7ydrogen pero6ide so#tion, 38 for cata#ase test
4.; <*7, 408 so#tion
4.= 0./38 9a#ine so#tion
4./ Trypticase soy !roth ,ith 0.;8 yeast e6tract 0T9)>E1
4.? Prp#e car!ohydrate fer"entation !roth !ase, containing 0.38 0,+v1
so#tions of de6trose, esc#in, "a#tose, rha"nose, "annito#, and
6y#ose
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Reviewed by: Approved by:
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FASL
ISOLATION OF
Listeria monocytogenes
MIC-AM-007-2
Page 2 of 3
4.10 94M "edi" or "oti#ity test "edi"
4.11 Trypticase soy agar ,ith 0.;8 yeast e6tract 0T9&>E1
4.12 '7R*M Listeria &gar
4.13 &P4 Listeria <it
!. APPARATUS AND LASS"ARE:
3.1 )a#ance
3.2 )#ender+9to"acher
3.3 4nc!ator, 30@ A 33@'
3.4 9teri#e !#ender Bars or sto"acher !ags
3.3 Er#en"eyer $#as2s, 300 "#
3.; $er"entation t!es 0%rha"1
3.= 4""ersion oi#
3./ Microscopic s#ides
3.? Test T!es
3.10 'o#ony 'onter
3.11 9teri#e Petri p#ates
3.12 Pipettes
3.13 4noc#ating need#es
3.14 )acticinerator
#. PROCEDURES:
;.1 Preparation of $ood 7o"ogenate
;.1.1 Refer to &ppendi6 % for the r#es in preparation, storage and
hand#ing of the test sa"p#es.
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FASL
ISOLATION OF
Listeria monocytogenes
MIC-AM-007-2
Page 3 of 3
;.1.2 'o"posited 9a"p#es
;.1.2.1 'o##ect 10 s!.sa"p#es 0#iCid, crea" or so#id
food1 of a food #ot sa"p#e ,ith 30.g or "# portions
of each s!.sa"p#e.
;.1.2.2 Ma2e t,o co"posite sa"p#es of 230 g+"# each.
;.1.2.3 Poo# and !#end or sto"ached the first 3 s!.
sa"p#es in 230 "# )ffered Listeria enrich"ent
!roth 0containing sodi" pyrvate, ,ithot
se#ective agents1 to "a2e the first co"posite.
;.1.2.4 Ma2e the second co"posite !y co"!ining the
re"aining 3 s!.sa"p#es in the sa"e ,ay.
;.1.2.3 'o##ect 23.g ana#ytica# portions fro" each
co"posite and se for pre.enrich"ent 0;.21
;.1.2.; Retain 30 "# a#iCot of the co"posite !#end, for
possi!#e pathogen en"eration.
;.1.3 Non.co"posited 9a"p#es
;.1.3.1 Deigh a sing#e 23 g ana#ytica# portion of the food
sa"p#e
;.1.3.2 Retain 23 g sa"p#e for possi!#e pathogen
en"eration. 9tore it at 3E ' if it is not froFen or, if
froFen, in a non.defrosting freeFer.
;.2 Pre.Enrich"ent
;.2.1 &dd 23+30 "# #iCid or 23+30 g of sa"p#e to 223+430 "#
)ffered 5isteria Enrich"ent )roth ,ithot the se#ective
agents. 4nc!ate for 4 hors at 30
0
'.
;.2.2 &fter 4 hors inc!ation, add the se#ective agents and
contine inc!ating for a tota# ti"e of 4/ hors at 30
0
'.
;.3 %etection
;.3.1 &fter 24 and 4/ hors inc!ation of the enrich"ent !roth ,ith
se#ective agents, strea2 enrich"ent ho"ogenate into *6ford
&gar 0*G&1. &s an option, at 4/ hors, strea2 a#so
ho"ogenate to '7R*MListeria para##e# to *G&.
;.3.2 4nc!ate *G& p#ates at 33
0
' for 24.4/ hors. 4nc!ate
'7R*MListeria p#ates at the sa"e conditions.
;.3.3 Listeria co#onies on *G& p#ates appear !#ac2 ,ith !#ac2 ha#o
de to esc#in hydro#ysis. Listeria monocytogenes as
appear !#e.green co#onies ,ith opaCe ,hite ha#o on
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FASL
ISOLATION OF
Listeria monocytogenes
MIC-AM-007-2
Page 4 of 3
'hro" agar.
;.4 4so#ation
;.4.1 Transfer 3 or "ore typica# co#onies fro" *G& to trypticase soy
agar ,ith yeast e6tract 0T9&>E1, strea2ing for prity and
typica# iso#ated co#onies.
;.4.2 4nc!ate T9&>E p#ates at 30
0
' for 24.4/h. P#ates "ay !e
inc!ated at 33
o
' if co#onies ,i## not !e sed for ,et "ont
"oti#ity.
;.3 4dentification of Prified 4so#ates
;.3.1 Perfor" Moti#ity test, 'ata#ase test, :ra" stain and test for
7e"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 <it: Prified iso#ates "ay !e rapid#y identified sing
co""ercia# 2it 0&P4 5isteria1 and !e confir"ed as Listeria
monocytogenes.
;.;.2 'onventiona# )ioche"ica# Testing
;.;.2.1 Pic2 typica# co#ony fro" T9&>E to a t!e of T9)>E.
4nc!ate t!e at 33
0
' for 24 hors. This c#tre can
!e 2ept at 4
0
' for severa# days and sed repeated#y
as inoc#".
;.;.2.2 $ro" T9)>E c#tre, perfor" car!ohydrate
fer"entation tests. Refer to &ppendi6 '.
;.;.2.3 Dhen )#ood agar sta! test res#ts are eCivoca#,
perfor" '&MP test. Refer to &ppendi6 '.
;.;.3 Dhen the sa"p#e tests positive for Listeria monocytogenes
sing either the rapid 2it or the conventiona# !ioche"ica#
tests, en"eration is reCired. Refer to procedre M4'.&M.
00;.0 for the en"eration procedre.

7. REPORTIN OF RESULTS:
Report as Positive or Negative for L. monocytogenes in 061 g or "# of
the prodct.
$. RECORDS:
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FASL
ISOLATION OF
Listeria monocytogenes
MIC-AM-007-2
Page 3 of 3
/.1 D9.M4'.&00=.1
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