Thermo Fisher Scientific Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates User guide

Type
User guide
USER GUIDE
For testing of Food and Environmental samples only.
Pathatrix® 5-Pooling Salmonella spp. Kit Linked
to Selective Agar Plates
For use with the Pathatrix® Auto Instrument
Catalog Number APS250P
Publication Number MAN0006973
Revision 14 August 2012
The information in this guide is subject to change without notice.
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UK. Whirl-Pak is a registered trademark of Wheatherby/Nasco, Inc.
© 2012 Life Technologies Corporation. All rights reserved.
Contents
3
Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Product Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
About the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Kit contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Materials not included in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Sample pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Samples and consumable loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
Sample unloading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
Detection—direct plating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Retesting of individual samples after “Positive Pooled Sample” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Test result interpretation and classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
APPENDIX A Alternative Enrichment Methods . . . . . . . . . . . . . . . . . . 17
Specific recommendations for high background microflora . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
TT broth information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Specific recommendations for milk powder, chocolate, and cocoabased samples . . . . . . . . . . . . . 18
Specific recommendations for potentially acidic/alkaline samples . . . . . . . . . . . . . . . . . . . . . . . . . . 18
APPENDIX B Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Description of target microorganism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Audience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Sampling protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Kit sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Operating conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
AOAC Performance Tested Methodssm Certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
APPENDIX C Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
Related materials from Life Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Contents
APPENDIX D Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Specific chemical handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Obtaining Certificates of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
5
Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Product Information
IMPORTANT! Beforeusingthisproduct,readandunderstandtheinformationin
AppendixD,“Safety”onpage22.
CAUTION! Salmonellaspp.isaBiosafetyLevel2(BSL2)organism[excludingS.typhi
andS. paratyphi,whicharebothBiosafetyLevel3(BSL3)].Caremustbetakenwhen
handlingsamplesthatmaycontainsalmonellae.Laboratorypersonnelmustbe
adequatelytrainedtohandlepathogensbeforebeingpermittedtoanalyzesamplesfor
Salmonellaspp.Laboratorypersonnelmustwearappropriateprotectiveequipment,
whichincludesbutisnotlimitedto:protectiveeyewear,faceshield,clothing/labcoat,
andgloves.Wasteshouldbedisposedofincompliancewithlocalandnational
legislationasappropriate.
About the kit
ThePathatrix®5PoolingSalmonellaspp.Kitisasamplepreparationmethodfor
presence/absencetestingbasedonthedetectionofasfewas1–10cfu(colonyforming
units)/25–325goffoodsample.
UsingthePathatrix®5PoolingSalmonellaspp.KitLinkedtoSelectiveAgarPlates,
presumptiveresultscanbeobtained,priortoconfirmation,within36hours.
Apresumptivepositiveisolateshouldbesubsequentlyconfirmedbytheuseof
subculture,aswellasappropriatebiochemicalandserologicaltestsasrequired.
Onceconfirmed,theresultsarereportedas:
Salmonellaspp.Detectedin25–325g(samplematrices)
Salmonellaspp.Notdetectedin25–325g(samplematrices)
SeeAppendixBonpage19foradditionalbackgroundinformation.
6Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Product Information
Kit contents
Kit contents
ThePathatrix®5PoolingSalmonellaspp.Kit(Cat.no.APS250P)containsenough
consumablecomponentsandPathatrix®beadstoprocess250 samples(50Cartridge
runs).
IMPORTANT! NeverfreezethePathatrix®beads.Beadsthathavebeensubjectedto
freezingtemperaturesmayberenderedinactive.
Note: Partsmayshipseparatelydependingonconfigurationandstorageconditions.
Item Quantity or
volume Storage
Pre-sterilized Sample and Elution
Vessel Packs
50 each Room temperature
Pre-sterilized Capture Phase Packs 50 each Room temperature
Pre-sterilized Flat Cap Lids 50 each Room temperature
Anti-Salmonella spp. Antibody-
Coated Paramagnetic Beads
The beads have a shelf life of 12 months and are labeled with an expiration date accordingly.
2.5 mL (50 tests) 5±3°C
7
Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Product Information
Materials not included in the kit
Materials not included in the kit
Thefollowingtableincludesmaterialsandequipmentforusing(butnotincludedin)
thePathatriconsumableskits.Unlessotherwiseindicated,manyofthelisteditems
areavailablefrommajorlaboratorysuppliers(MLS).
Equipment, consumables, and reagents
Item Source
Equipment
Incubator, 37 ±1°C MLS
Pathatrix® Auto Instrument Life Technologies Cat. no. PATHATRIXAUTO
Consumables
See page 10 and Appendix A on page 17 for recommendations about enrichment media
choice. The media is supplied by several manufacturers [e.g., Oxoid (product codes shown),
Difco, and Merck] in a dehydrated form and should be prepared according to the
manufacturer's instructions.
Sterile bags for enrichment (Whirl-Pak® or
Stomacher® bag, or equivalent)
Nasco # B01196WA, Seward product code
BA6041, or equivalent
Optional for high-particulate or high-fat-
content samples:
Sterile filter bags for enrichment (Whirl-
Pak® or Stomacher® bag, or equivalent)
or
•Pathatrix
® Foam filters
•Pathatrix
® 5-Pool Kit—Straws (254 mm)
and Syringes (10 mL)
Nasco # B01348WA, Seward product code
BA6041/STR, or equivalent
or
Life Technologies Cat. no. PFF
Life Technologies Cat. no. POOL510MLN
Sterile, disposable 10-µL loops MLS
Media
Buffered peptone water Oxoid product code CM0509
For samples with high background
microflora:
Tetrathionate (TT) broth base Oxoid product code CM0029
For milk powder, chocolate, or cocoa-based
samples:
•UHT skim milk
Brilliant green (CAS 633-03-4)
Food retail store
•MLS
Selective agar
XLD agar Oxoid product code CM0469
Brilliant green agar (modified) Oxoid product code CM0329
Reagents
PBS, 10X, pH 7.4 Life Technologies Cat. no. AM9624 or AM9625
8Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Product Information
Materials not included in the kit
9
Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Methods
Workflow
Procedural guidelines
•Useaseptictechniqueandgoodlaboratorypracticesatalltimes.
•Afacemaskshouldbewornwhenweighingoutpowders.
• Caremustbetakenwhenboilingagarpriortoautoclaving,andheatresistant
glovesshouldbewornwhenhandlinghotflasksofliquid.
• Takecarewhenhandlingplatesorvesselsthatcontainmicroorganisms.
•Avoid
generatingaerosols,aspathogenicorganismsmaybepresent.
•Usedorunusedreagents,usedmedia,sampleenrichments,aswellasother
contaminateddisposablematerialsshouldbedisposedoffollowingprocedures
forinfectiousorpotentiallyinfectiousproducts.
•Sampleenrichmentsmightbecontaminatedwithpathogenicorganisms
infectioustohumans,soallwastemustbetreatedasbiohazardousandhandled
anddisposedusingsafelaboratorypractices,inaccordanceandcompliancewith
allappropriateregulations.
Sample preparation (page 10)
T
Sample pooling (page 11)
T
Samples and consumable loading (page 12)
T
Sample unloading (page 14)
T
Detection—direct plating (page 15)
T
Retesting of individual samples after “Positive Pooled Sample” (page 15)
T
Test result interpretation and classification (page 16)
10 Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Methods
Sample preparation
Sample preparation
Note: Certainfoodtypesandswabs/spongescanbenefitfromanalternative
enrichmentstrategy.(seeAppendixA,“A l t e r n a t i v e EnrichmentMethods”).
1. Preparea1in10dilutionofthefoodsampleintheappropriateprewarmed
(37±1°C)enrichmentmediainasterilebag.
Note: Forexample,add25goffoodsampleto225mLofprewarmedmediaor
add325goffoodsampleto2925mLofprewarmedmedia.
IMPORTANT! Itiscriticalthattheenrichmentmediaisprewarmedto37 ±1°C
priortoaddingthefoodsample.Topreventcooling,allsamplesshouldthen
immediatelybeplacedintheincubatorat37 ±1°C.
2. Homogenizethesamplebyhandmixingfor10–15seconds.Massagethesample
betweenthefingerstodisperseanylargeclumpsofmaterial(NarangandCray,
2006).
3. Incubateat37 ±1°Cforaminimumof16 hours.
Note: Werecommendthatthesesubsamplesforpoolingareprocessed
immediatelyor,ifstorageisrequired,thatthesamplesarerefrigeratedat5±3°C.
Samplesshouldberewarmedto37 ±1°CpriortoanalysisonthePathatrix®Auto
Instrument.Theremainingenrichedsampleshouldbestoredat5±3°Cforupto
32 hoursuntiltheresultsofthepooledsamplehavebeendetermined.
11
Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Methods
Sample pooling
Sample pooling
1. RemovetheSampleandElutionVesselsfromtheconsumablekitpackagingand
placeintotheTubeRack(alsoknownastheSampleVesselHolder).
2. Partiallyremovethelidsfrombothvessels,makingalargeenoughopeningto
allowthesampleandwashbuffertobedispensedintothevessels.
3. PrepareasinglepooledsampleintheSampleVesselbypooling5x10mL
aliquotsfromfiveindividuallyenrichedsamples.
Note: Ifthesamplesarehighlyparticulateand/orcontainahighfatcontent,the
useoftheFiltaFoamsystem(Foamfilters,Cat.no.PFF)withpoolingsyringesand
straws(Cat.no.POOL510MLN)isrecommended.Alternatively,theSewardplain
sterilebagwithinternalstrainer(SewardProductCodeBA6041/STRor
equivalent)maybeused.
4. Storetheindividualenrichedsamplesat5±3°Cforpotentialreanalysisuntilthe
testresultisconfirmed.
Note: Donotstoreformorethan32hours.
If pooled sample is negative, then
all 5 individual samples are negative. If pooled sample is positive, then
retest all 5 individual samples postenrichment.
5X Individual
Samples
Individual
Enrichment of
Samples
Postenriched
Samples
Pooling of Samples
(5X 10-mL Samples)
50-mL Pooled Sample
(1X Pathatrix® Test)
12 Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Methods
Samples and consumable loading
Samples and consumable loading
1. AddPBS(Cat.no.AM9624,dilutedto1X)tothefilllineoftheElutionVessel
(about35mLof1XPBS).
2. PlacethelidsontotheSampleandElutionVesselsmakingsurethatthevessels
aresealedallthewayaround.
3. Ensurethebeadsarefullyresuspendedbyagitatingthebeadvial(e.g.,vortexing
beadvialorinversionofsealedvial)andadd50μLofthebeadsuspension(beads
fromCat. no.APS250P)intothespoutonthelidoftheSampleVessel.
4. Removetheplastickitfromthebagandorientatewiththevalveplungerpointing
left.ConnectthevalvefirmlytothelidsoftheSampleandElutionVessels.
5. Holdingbothvessels,lifttheassembledvesselsandattachedkitoutoftherack
(alsoknownastheSampleVesselHolder).
Elution Vessel
Sterile 1X PBS (35 mL)
Sample Vessel Holder
(ATUBEHOLD)
Sample Vessel Spout
Sample Vessel
Capture Phase
Valve Plunger
13
Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Methods
Samples and consumable loading
6. PlacethevesselsintotheCartridge,pushingthemfirmlyinplacefromthebottom
upwards.
7. Next,firmlypushtheremainderofthekitintotheCartridge,ensuringthatthe
valve,thephase,andthesyringeareallheldsecurelyinthemoldedrecessofthe
Cartridge.
8. Pushthemagnetslideracrossintothelockingpositionandpressthemagnet
releasebuttononthesideoftheCartridgetoensurethekitispositionedcorrectly
andthemagnetscanfreelydisengagefromthephase.
9. Resetthemagnetsintothelockingposition.
10. InserttheCartridgeintothePathatrix®AutoInstrumentuntilitclicksintothe
lockingposition.
11. PressthenumberedbuttonabovetheappropriateCartridgetostarttherun.The
associatedLEDwillturngreentoindicatetherunhasstarted.
Syringe
Magnet Release Button
Valve Plunger
Phase
Sample Vessel
Elution Vessel
Cartridge Holder
(ACARTHOLD)
Sliding Magnet
Holder
14 Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Methods
Sample unloading
Sample unloading
1. Attheendoftherun,theLEDwillflashredandgreenalternately.
2. PressthebuttonabovetheappropriateCartridgetoinitializethedrainingstep
(approximately1minute).
3. WhenthedrainingstepiscompleteandtheLEDisilluminatedred,removethe
Cartridgebypullingitout,awayfromtheinstrument.
4. RemovethesyringefromtheCartridgeandcarefullypullouttherestofthekit,
startingatthetopandworkingdownwards.WhenremovingtheSampleand
ElutionVesselsfromtheCartridge,holdfirmlybythevesselsthemselvesto
preventspillage.
5. PlacebothvesselsintheTubeRack(alsoknownastheSampleVesselHolderor
rack).
6. RemovethelidfromtheElutionVesseland,leavingtheElutionVesselinplacein
therack,liftawaytherestoftheconsumable,includingtheSampleVessel,and
discard.
7. Placeaspareflatlid(providedintheconsumablekit)ontopoftheElutionVessel
intherackfor1minutetoallowcaptureofthebeads.
8. RemovealltheliquidfromtheElutionVessel,withoutremovingthevesselfrom
therack,takingcarenottodisturbthecapturedbeads.
9. Add100μLofPBSintotheElutionVesselandresuspendthePathatrix®beads.
10. AppropriatealiquotsofthePathatrix®beadsuspensioncanthenbeimmediately
analyzedusingthelaboratory’schosenpathogendetectionmethod.
Note: Thebeadsuspensionmayberetainedforlatertestingifnecessary.Thebeads
shouldbestoredAWAYfrommagnets(e.g.,Pathatrix®vesselholders)at5±3°Cforup
to24hours.
15
Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Methods
Detection—direct plating
Detection—direct plating
Note: Werecommendstreakingthesampleouttogenerateindividualcolonies,as
opposedtospreadplating.
1. Streak10μLoftheunlysedbeadsuspensionoverawelldriedXLD(xyloselysine
desoxycholate)agarplateandanother10μLontoanappropriatesecondselective
platemedium(e.g.,BrilliantGreenAgar).
Note: Thelaboratorymaychoosewhichmediumtouse,butthesecondselective
plateshouldbeanyothersolidselectivemediumcomplimentarytoXLDand
especiallyappropriatefortheisolationoflactosepositiveSalmonella,Salmonella
typhi,andSalmonellaparatyphistrains.
2. Allowtheplatestodryforapproximately10minutestheninvertandincubateat
therequiredtemperaturefor18–24hoursorasrecommendedbythe
manufacturer.
Ifanegativeresultisobtainedfromthepooledsample,theindividualovernight
enrichmentscanbediscarded,asfurthertestingisnotrequired.
Apresumptivepositiveresultisdefinedastheisolationoftypical,suspicious,or
atypicalSalmonellaspp.coloniesontheagarplatesused.Apresumptivepositive
isolateshouldbesubsequentlyconfirmedbytheuseofsubculture,appropriate
biochemicalandserologicaltestsasdetailedintheUSDAMicrobiologyLaboratory
Guidebook(MLG)4.04forcookedhamorFDABacteriologicalAnalyticalManual
(BAM),Chapter5,sectionsDandEfortomatoesandchocolate(see“References”on
page25).
Ifapositiveresultisobtainedfromthepooledsample,theovernightenrichmentscan
beretestedtoallowidentificationofwhichindividualsamplesinthepoolproduced
thepositiveresult(seenextsection,“Retestingofindividualsamplesafter“Positive
PooledSample””)
Retesting of individual samples after “Positive Pooled Sample”
1. Individualsamples,whichrequireretesting,shouldberewarmedto37±1°C
beforehaving10mLremovedandtransferredintoaSampleVessel.
2. Individualsamplesshouldberetestedimmediatelybyrepeatingallthestepsin
SamplesandconsumableloadingthroughDetection—directplatingdescribed
above.
16 Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Methods
Test result interpretation and classification
Test result interpretation and classification
ThePathatrix®5PoolingSalmonellaspp.Kitisdesignedasasamplepreparation
methodforpresence/absencedetectionofSalmonellaspp.infoodmatrices.
UsingthePathatrix®5PoolingSalmonellaspp.KitLinkedtoSelectiveAgarPlates,
presumptiveresultscanbeobtained,priortoconfirmation,within36hours.
Onceconfirmed,theresultsarereportedas:
Salmonellaspp.Detectedin25–325g(samplematrices)
Salmonellaspp.Notdetectedin25–325g(samplematrices)
A
17
Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Alternative Enrichment Methods
Specific recommendations for high background microflora
Forcertainfoodgroups,particularlywherehighlevelsofbackgroundmicrofloraare
present,theSalmonellaassayresultscanbeenhancedbytheuseofTetrathionate(TT)
brothinsteadofBufferedPeptoneWater.Thelevelofbackgroundcontaminationon
theagarplatescanbereducedbythismethod.
ExamplesoffoodgroupsthatbenefitfromtheuseofTTbrothare:
•Rawmeat
•Rawvegetables
• Salads
•Fruits
•Readymeals
TT broth
information
Materials
• TetrathionatebrothbaseisavailablefromOxoid(ProductCodeCM0029).
•IodineSolution(tobeaddedfollowingpreparationandheatingofTTbrothbase).
Note: Preparetheiodinesolutioninadvancetoallowtheiodinetodissolve,but
useonthesamedaythatitismade.
Directions for use
1. Add77gofTTbrothbaseto1Lofdistilledwaterandbringtoaboil.
2. Coolbelow45°C.
3. Add20mLofiodinesolutionandmixwell.
Followingboiling,thepreparedbasecanbestoredforseveralweeksat5±3°C,but
oncetheiodinehasbeenadded,themediashouldbeusedthesamedayandany
excessshouldbedisposedof.
18 Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Appendix A Alternative Enrichment Methods
Specific recommendations for milk powder, chocolate, and cocoabased samples
A
Specific recommendations for milk powder, chocolate, and
cocoabased samples
Allmilkpowder,chocolate,orcocoabasedsamplescanbenefitfromanalternative
enrichment.
Useprewarmed(37±1°C)sterileUHTskimmilksupplementedwithBrilliantGreen
(0.002%),insteadofBufferedPeptoneWater,astheenrichmentmedia.
•Milkpowdersamplesshouldbeincubatedat37±1°Cfor22 ±2 hours.
• Chocolateandcocoabasedsamplesshouldbeincubated,asdescribedinthe
protocol,at37±1°Cfor19 ±1 hour.
Specific recommendations for potentially acidic/alkaline samples
SampleswhichpotentiallydeviatefromneutralpHshouldbepreparedasfollows:
1. Dilutethesampleaccordingtothesampleenrichmentprotocol.
2. Incubatefor60±5minutesatroomtemperature.
3. Mixbyhandmassaging,anddeterminethepH.
4. Ifnecessary,adjustthepHto6.8±0.2,andmixwellbeforedeterminingthe
final pH.
B
19
Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Background
Product overview
Description of
target
microorganism
Morethan2,400Salmonellaserotypeshavebeenreported,allofwhicharepotentially
pathogenic.Salmonellaisafrequentlyreportedcauseoffoodborneillness,occurringin
bothepidemicsandinisolatedcases.Salmonellabacteriaarethecausativeagentfor
Salmonellosis.Outbreakshavebeenassociatedwithrawmeatsandpoultry,eggs,milk
anddairyproducts,seafood,coconutsauces,saladdressings,cocoa,chocolate,spices,
frozenproducts,andvegetablessuchashotpeppers.
Audience ThePathatrix®5PoolingSalmonellaspp.Kitisforprofessionaluseonlyandis
intendedforusebyqualifiedusersinterestedindeterminingthepresence/absenceof
Salmonellaspp.infoodsamples.Usersmayinclude,butarenotlimitedto,food
producers,foodprocessors,foodmanufacturers,retailers,andmicrobiologytesting
laboratories.
Sampling protocol ThestandardfoodsamplesizeusedinthePathatrix®Autosystemis25goffood
dilutedwith225mLofenrichmentmedium.Werecommendthatsubsamplesfor
poolingareprocessedimmediatelyor,ifstorageisrequired,thatthesamplesare
refrigeratedat5±3°C.Samplesshouldberewarmedto37±1°Cpriortoanalysiswith
thePathatrix®Autosystem.Theremainingenrichedsampleshouldbestoredat5±3°C
forupto32hoursuntiltheresultsofthepooledsamplehavebeendetermined.
Kit sensitivity Thesamplepreparationprocedureallowsyoutodetectasfewas1–10cfufrom
25–325gramsoffoodsample.ThelimitationofthePathatrix®5PoolingSalmonella
spp.Kitisintheabilityofthetargettoreproduceintheenrichmentmedium,be
capturedbythemagnet,andsubsequentlybeisolatedonselectiveagarplates.
CAUTION! ThePathatrix®kithasbeenevaluatedoncookedham,chocolate,and
choppedtomatofoodmatrices.Giventhewidevarietyofproductsandmanufacturing
procedures,werecommendthatyoucheckthatthecompositionofthematricestobe
testeddoesnotaffectthereliabilityoftheresults.
Anegativeresultdoesnotguaranteetheabsenceoftargetorganismintheoriginal
sampleandmaybeduetotheinabilityoftheorganismtoadequatelyreproduceto
requiredlevelsintheenrichmentmedium(withsubsequentoutgrowthonselective
agarplates)potentiallydueto,butnotlimitedto,competitivemicroflora,sublethal
injury,ormatrixinhibition.
20 Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates
Appendix B Background
AOAC Performance Tested Methodssm Certification
B
Operating
conditions
ThePathatrix®AutoInstrumentisforindooruseonlyandforaltitudesnotexceeding
2000m(6500ft.)abovesealevel.
AOAC Performance Tested Methodssm Certification
ThePathatrix®5PoolingSalmonellaspp.KithasbeenvalidatedandcertifiedbyAOAC
InternationalResearchInstitute(Licensenumber090203C)forthedetectionof
Salmonellaserovarsfrom5Pooledsamplesincookedham,chocolate,andchopped
tomatoes.ThetestoperatesinconjunctionwithapooledPathatrix®workflow.A
standard25gfoodsampleishandmixedbrieflywith225mLofenrichmentmedium
andincubatedforaminimumof18–24hoursat37°C.Afterincubation,a10mL
samplealiquotiscombinedwithupto4additional10mLsamplealiquots,andthe
resulting50mLpooledsampleisthensubjectedtoa15minutePathatrix®Auto
Instrumentcapturecycle.
Themethodwasvalidatedforusewiththefollowingmatrices:
• 25–325gofcookedham
•25gofchocolate
•25gofchoppedtomatoes
Temperature and humidity requirements
Condition Acceptable range
Temperature 5 to 40°C
Humidity Maximum relative humidity 80% for
temperatures up to 31°C, decreasing to 50%
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Thermo Fisher Scientific Pathatrix® 5-Pooling Salmonella spp. Kit Linked to Selective Agar Plates User guide

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