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    Procedure For Isolation of Listeria Monocytogenes

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    clairealbertini
    Step by step procedure for isolation of listeria monocytogenes in foods according to US FDA standard method

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    Procedure For Isolation of Listeria Monocytogenes

    Uploaded by

    clairealbertini
    105 views5 pages
    Step by step procedure for isolation of listeria monocytogenes in foods according to US FDA standard method

    Original Title

    Procedure for Isolation of Listeria monocytogenes

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    © © All Rights Reserved

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    Step by step procedure for isolation of listeria monocytogenes in foods according to US FDA standard method

    Copyright:

    © All Rights Reserved
    Download as DOC, PDF, TXT or read online from Scribd
    Download as doc, pdf, or txt
    105 views5 pages

    Procedure For Isolation of Listeria Monocytogenes

    Uploaded by

    clairealbertini
    Step by step procedure for isolation of listeria monocytogenes in foods according to US FDA standard method

    Copyright:

    © All Rights Reserved
    Download as DOC, PDF, TXT or read online from Scribd
    Download as doc, pdf, or txt
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    The key takeaways are that this procedure describes the method for detecting Listeria monocytogenes in food samples through enrichment, isolation on selective and differential media, and confirmation through biochemical and rapid tests.

    The purpose of this procedure is to describe the method for detection of Listeria monocytogenes on various food samples.

    The main steps involved in the procedure are sample preparation, pre-enrichment, detection through plating on selective and differential media, isolation, and identification and confirmation of purified isolates.

    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
    ______________________________________________________________________________________
    Reviewed by: Approved by:
    This document shall not be copied in whole or in part; if not stamped in red, this document is uncontrolled.
    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.
    ______________________________________________________________________________________
    This document shall not be copied in whole or in part; if not stamped in red, this document is uncontrolled
    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
    ______________________________________________________________________________________
    This document shall not be copied in whole or in part; if not stamped in red, this document is uncontrolled
    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:
    ______________________________________________________________________________________
    This document shall not be copied in whole or in part; if not stamped in red, this document is uncontrolled
    FASL
    ISOLATION OF
    Listeria monocytogenes
    MIC-AM-007-2
    Page 3 of 3
    /.1 D9.M4'.&00=.1
    ______________________________________________________________________________________
    This document shall not be copied in whole or in part; if not stamped in red, this document is uncontrolled

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