Thermo Fisher Scientific Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates User guide

Brand: Thermo Fisher Scientific

Model: Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Type: User guide

USER GUIDE

For testing of Food and Environmental samples only.

Pathatrix® 10-Pooling Salmonella spp. Kit

Linked to Selective Agar Plates

For use with the Pathatrix® Auto Instrument

Catalog Number APS500P

Publication Number MAN0006975

Revision 14 August 2012

The information in this guide is subject to change without notice.

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Limited Use Label License: Environmental testing, quality control/quality assurance testing, food and agricultural testing

The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product (a) to perform internal

research for the sole benefit of the purchaser; and (b) for environmental testing, quality control/quality assurance testing, food and agricultural testing,

including reporting results of purchaser's activities in environmental testing, quality control/quality assurance testing, food and agricultural testing for a fee

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control/ quality assurance testing, food and agricultural testing and research purposes only.

The purchase of this product does not grant the purchaser any additional rights, including (without limitation) the right to transfer or resell the product in

any form or the right to use the product as a therapeutic agent or diagnostics test component, or to use the product to perform other tests on a contract or

fee per test basis for or on behalf of third parties. For information on obtaining additional rights, please contact outlicensing@lifetech.com.

TRADEMARKS

The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners in the United States and other countries.

AOAC is a trademark and Performance Tested Methods is a service mark of AOAC International. Stomacher is a registered trademark of Seward Limited,

UK. Whirl-Pak is a registered trademark of Wheatherby/Nasco, Inc.

Contents

Pathatrix® 10-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® 10-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

Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Product Information

IMPORTANT! Beforeusingthisproduct,readandunderstandtheinformationin

AppendixD,“Safety”onpage22.

CAUTION! Salmonellaspp.isaBiosafetyLevel2(BSL‐2)organism[excludingS.typhi

andS. paratyphi,whicharebothBiosafetyLevel3(BSL‐3)].Caremustbetakenwhen

handlingsamplesthatmaycontainsalmonellae.Laboratorypersonnelmustbe

adequatelytrainedtohandlepathogensbeforebeingpermittedtoanalyzesamplesfor

Salmonellaspp.Laboratorypersonnelmustwearappropriateprotectiveequipment,

whichincludesbutisnotlimitedto:protectiveeyewear,faceshield,clothing/labcoat,

andgloves.Wasteshouldbedisposedofincompliancewithlocalandnational

legislationasappropriate.

About the kit

ThePathatrix®10‐PoolingSalmonellaspp.Kitprovidesasamplepreparationmethod

forpresence/absencetestingbasedonthedetectionofasfewas1–10cfu(colony

formingunits)/25–325goffoodsample.

UsingthePathatrix®10‐PoolingSalmonellaspp.KitLinkedtoSelectiveAgarPlates,

presumptiveresultscanbeobtained,priortoconfirmation,within38hours.A

presumptivepositiveisolateshouldbesubsequentlyconfirmedbytheuseof

subculture,aswellasappropriatebiochemicalandserologicaltestsasrequired.

Onceconfirmed,theresultsarereportedas:

•Salmonellaspp.Detectedin25–325g(samplematrices)

•Salmonellaspp.Notdetectedin25–325g(samplematrices)

SeeAppendixBonpage19foradditionalbackgroundinformation.

6Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Product Information

Kit contents

ThePathatrix®10‐PoolingSalmonellaspp.Kit(Cat.no.APS500P)containsenough

consumablecomponentsandPathatrix®beadstoprocess500 samples(50 Cartridge

runs).

IMPORTANT! NeverfreezethePathatrix®beads.Beadsthathavebeensubjectedto

freezingtemperaturesmayberenderedinactive.

Note: Partsmayshipseparatelydependingonconfigurationandstorageconditions.

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 the expiration date accordingly.

2.5 mL (50 tests) 5±3°C

Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Product Information

Materials not included in the kit

Thefollowingtableincludesmaterialsandequipmentforusing(butnotincludedin)

thePathatrix®consumableskits.Unlessotherwiseindicated,manyofthelisteditems

areavailablefrommajorlaboratorysuppliers(MLS).

Equipment, consumables, and reagents

Item Source

Equipment

Incubator, 37 ±1°C MLS

Pathatrix® Auto Instrument Life Technologies Cat. no. PATHATRIXAUTO

Consumables

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)

•Pathatrix

® Foam filters

•Pathatrix

® 10-Pool Kit—Straws (254

mm) and Syringes (10 mL)

• Nasco # B01348WA, Seward product code

BA6041/STR, or equivalent

• Life Technologies Cat. no. PFF

• Life Technologies Cat. no. POOL1010MLN

Sterile, disposable 10-µL loops MLS

Media

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.

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® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Product Information

Materials not included in the kit

Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Methods

Workflow

Procedural guidelines

•Useaseptictechniqueandgoodlaboratorypracticesatalltimes.

•Afacemaskshouldbewornwhenweighingoutpowders.

• Caremustbetakenwhenboilingagarpriortoautoclaving,andheat‐resistant

glovesshouldbewornwhenhandlinghotflasksofliquid.

• Takecarewhenhandlingplatesorvesselsthatcontainmicroorganisms.

•Avoid

generatingaerosols,aspathogenicorganismsmaybepresent.

•Usedorunusedreagents,usedmedia,sampleenrichments,aswellasother

contaminateddisposablematerialsshouldbedisposedoffollowingprocedures

forinfectiousorpotentiallyinfectiousproducts.

•Sampleenrichmentsmightbecontaminatedwithpathogenicorganisms

infectioustohumans,soallwastemustbetreatedasbiohazardousandhandled

anddisposedusingsafelaboratorypractices,inaccordanceandcompliancewith

allappropriateregulations.

Sample preparation (page 10)

Sample pooling (page 11)

Samples and consumable loading (page 12)

Sample unloading (page 14)

Detection—direct plating (page 15)

Retesting of individual samples after “Positive Pooled Sample” (page 15)

Test result interpretation and classification (page 16)

10 Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Methods

Sample preparation

Note: Certainfoodtypesandswabs/spongescanbenefitfromanalternative

enrichmentstrategy.(seeAppendixA,“A l t e r n a t i v e EnrichmentMethods”).

1. Preparea1in10dilutionofthefoodsampleintheappropriateprewarmed

(37±1°C)enrichmentmediainasterilebag.

Note: Forexample,add25goffoodsampleto225mLofprewarmedmediaor

add325goffoodsampleto2925mLofprewarmedmedia.

IMPORTANT! Itiscriticalthattheenrichmentmediaisprewarmedto37 ±1°C

priortoaddingthefoodsample.Topreventcooling,allsamplesshouldthen

immediatelybeplacedintheincubatorat37 ±1°C.

2. Homogenizethesamplebyhandmixingfor10–15seconds.Massagethesample

betweenthefingerstodisperseanylargeclumpsofmaterial(NarangandCray,

2006).

3. Incubateat37 ±1°Cforaminimumof18 hours.

Note: Werecommendthatthesesub‐samplesforpoolingareprocessed

immediatelyor,ifstorageisrequired,thatthesamplesarerefrigeratedat5±3°C.

Samplesshouldberewarmedto37 ±1°CpriortoanalysisonthePathatrix®Auto

Instrument.Theremainingenrichedsampleshouldbestoredat5±3°Cforupto

32hoursuntiltheresultsofthepooledsamplehavebeendetermined.

Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Methods

Sample pooling

1. RemovetheSampleandElutionVesselsfromtheconsumablekitpackagingand

placeintotheTubeRack(alsoknownastheSampleVesselHolder).

2. Partiallyremovethelidsfrombothvessels,makingalargeenoughopeningto

allowthesampleandwashbuffertobedispensedintothevessels.

3. PrepareasinglepooledsampleintheSampleVesselbypooling10 x5‐mL

aliquotsfromtenindividuallyenrichedsamples.

Note: Ifthesamplesarehighlyparticulateand/orcontainahighfatcontent,the

useoftheFiltaFoamsystem(Foamfilters,Cat.no.PFF)withpoolingsyringesand

straws(Cat.no.POOL1010MLN)isrecommended.Alternatively,theSeward

plainsterilebagwithinternalstrainer(SewardProductCodeBA6041/STRor

equivalent)maybeused.

4. Storetheindividualenrichedsamplesat5±3°Cforpotentialreanalysisuntilthe

testresultisconfirmed.

Note: Donotstoreformorethan32hours.

If pooled sample is negative, then

all 10 individual samples are negative.

If pooled sample is positive, then retest

all individual samples postenrichment.

10X Individual

Samples

Individual

Enrichment

of Samples

Postenriched

Samples

Pooling of

Samples

(10X 5-mL

Samples)

50-mL Pooled Sample

(1X Pathatrix®Test)

12 Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Methods

Samples and consumable loading

1. AddPBS(Cat.no.AM9624,dilutedto1X)tothefilllineoftheElutionVessel

(about35mLof1XPBS).

2. PlacethelidsontotheSampleandElutionVesselsmakingsurethatthevessels

aresealedallthewayaround.

3. Ensurethebeadsarefullyresuspendedbyagitatingthebeadvial(e.g.,vortexing

beadvialorinversionofsealedvial)andadd50μLofthebeadsuspension(beads

fromCat. no.APS500P)intothespoutonthelidoftheSampleVessel.

4. Removetheplastickitfromthebagandorientatewiththevalveplungerpointing

left.ConnectthevalvefirmlytothelidsoftheSampleandElutionVessels.

5. Holdingbothvessels,lifttheassembledvesselsandattachedkitoutoftherack

(alsoknownastheSampleVesselHolder).

Elution Vessel

Sterile 1X PBS (35 mL)

Sample Vessel Holder

(ATUBEHOLD)

Sample Vessel Spout

Sample Vessel

Capture Phase

Valve Plunger

Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Methods

Samples and consumable loading

6. PlacethevesselsintotheCartridge,pushingthemfirmlyinplacefromthebottom

upwards.

7. Next,firmlypushtheremainderofthekitintotheCartridge,ensuringthatthe

valve,thephase,andthesyringeareallheldsecurelyinthemoldedrecessofthe

Cartridge.

8. Pushthemagnetslideracrossintothelockingpositionandpressthemagnet

releasebuttononthesideoftheCartridgetoensurethekitispositionedcorrectly

andthemagnetscanfreelydisengagefromthephase.

9. Resetthemagnetsintothelockingposition.

10. InserttheCartridgeintothePathatrix®AutoInstrumentuntilitclicksintothe

lockingposition.

11. PressthenumberedbuttonabovetheappropriateCartridgetostarttherun

(approximately14minutes).TheassociatedLEDwillturngreentoindicatethe

runhasstarted.

Syringe

Magnet Release Lever

Valve Plunger

Phase

Sample Vessel

Elution Vessel

Cartridge Holder

(ACARTHOLD)

Sliding Magnet

Holder

14 Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Methods

Sample unloading

1. Attheendoftherun,theLEDwillflashredandgreenalternately.

2. PressthebuttonabovetheappropriateCartridgetoinitializethedrainingstep

(approximately1minute).

3. WhenthedrainingstepiscompleteandtheLEDisilluminatedred,removethe

Cartridgebypullingitout,awayfromtheinstrument.

4. RemovethesyringefromtheCartridgeandcarefullypullouttherestofthekit,

startingatthetopandworkingdownwards.WhenremovingtheSampleand

ElutionVesselsfromtheCartridge,holdfirmlybythevesselsthemselvesto

preventspillage.

5. PlacebothvesselsintheTubeRack(alsoknownastheSampleVesselHolderor

rack).

6. RemovethelidfromtheElutionVesseland,leavingtheElutionVesselinplacein

therack,liftawaytherestoftheconsumable,includingtheSampleVessel,and

discard.

7. PlacetheElutionVesselintherackandcapwithaflatlid(providedinthe

consumablekit).Leaveintherackfor1minutetoallowcaptureofthebeads.

8. RemovealltheliquidfromtheElutionVessel,withoutremovingthevesselfrom

therack,takingcarenottodisturbthecapturedbeads.

9. RemovetheElutionVesselfromthevesselholder,add100μLofPBSintothe

ElutionVessel,andresuspendthePathatrix®beads.

10. AppropriatealiquotsofthePathatrix®beadsuspensioncanthenbeimmediately

analyzedusingthelaboratory’schosenpathogendetectionmethod.

Note: Thebeadsuspensionmayberetainedforlatertestingifnecessary.Thebeads

shouldbestoredAWAYfrommagnets(e.g.,Pathatrix®vesselholders)at5±3°Cforup

to24hours.

Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Methods

Detection—direct plating

Note: Werecommendstreakingthesampleouttogenerateindividualcolonies,as

opposedtospreadplating.

1. Streak10μLoftheunlysedbeadsuspensionoverawell‐driedXLD(xyloselysine

desoxycholate)agarplateandanother10μLontoanappropriatesecondselective

platemedium(e.g.,BrilliantGreenAgar).

Note: Thelaboratorymaychoosewhichmediumtouse,butthesecondselective

plateshouldbeanyothersolidselectivemediumcomplimentarytoXLDand

especiallyappropriatefortheisolationoflactose‐positiveSalmonella,Salmonella

typhi,andSalmonellaparatyphistrains.

2. Allowtheplatestodryforapproximately10minutestheninvertandincubateat

attherequiredtemperaturefor18–24hoursorasrecommendedbythe

manufacturer.

Ifanegativeresultisobtainedfromthepooledsample,theindividualovernight

enrichmentscanbediscarded,asfurthertestingisnotrequired.

Apresumptivepositiveresultisdefinedastheisolationoftypical,suspicious,or

atypicalSalmonellaspp.coloniesontheagarplatesused.Apresumptivepositive

isolateshouldbesubsequentlyconfirmedbytheuseofsubculture,appropriate

biochemicalandserologicaltestsasdetailedinUSDAMicrobiologyLaboratory

Guidebook(MLG)4.04forcookedhamorFDABacteriologicalAnalyticalManual

(BAM),Chapter5,sectionsDandEfortomatoesandchocolate(see“References”on

page25).

Ifapositiveresultisobtainedfromthepooledsample,theovernightenrichmentscan

beretestedtoallowidentificationofwhichindividualsamplesinthepoolproduced

thepositiveresult(seenextsection,“Retestingofindividualsamplesafter“Positive

PooledSample””)

Retesting of individual samples after “Positive Pooled Sample”

1. Individualsamples,whichrequireretesting,shouldberewarmedto37±1°C

beforehaving10mLremovedandtransferredintoaSampleVessel.

2. Individualsamplesshouldberetestedimmediatelybyrepeatingallthestepsin

SamplesandconsumableloadingthroughDetection—directplatingdescribed

above.

16 Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Methods

Test result interpretation and classification

ThePathatrix®10‐PoolingSalmonellaspp.Kitisdesignedasasamplepreparation

methodforpresence/absencedetectionofSalmonellaspp.infoodmatrices.

UsingthePathatrix®10‐PoolingSalmonellaspp.KitLinkedtoSelectiveAgarPlates,

presumptiveresultscanbeobtained,priortoconfirmation,within38hours.

Onceconfirmed,theresultsarereportedas:

•Salmonellaspp.Detectedin25–325g(samplematrices)

•Salmonellaspp.Notdetectedin25–325g(samplematrices)

Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Alternative Enrichment Methods

Specific recommendations for high background microflora

Forcertainfoodgroups,particularlywherehighlevelsofbackgroundmicrofloraare

present,theSalmonellaassayresultscanbeenhancedbytheuseofTetrathionate(TT)

brothinsteadofBufferedPeptoneWater.Thelevelofbackgroundcontaminationon

theagarplatescanbereducedbythismethod.

ExamplesoffoodgroupsthatbenefitfromtheuseofTTbrothare:

•Rawmeat

•Rawvegetables

• Salads

•Fruits

•Readymeals

TT broth

information

Materials

• TetrathionatebrothbaseisavailablefromOxoid(ProductCodeCM0029).

•IodineSolution(tobeaddedfollowingpreparationandheatingofTTbrothbase).

Note: Preparetheiodinesolutioninadvancetoallowtheiodinetodissolve,but

useonthesamedaythatitismade.

Directions for use

1. Add77gofTTbrothbaseto1Lofdistilledwaterandbringtoaboil.

2. Coolbelow45°C.

3. Add20mLofiodinesolutionandmixwell.

Followingboiling,thepreparedbasecanbestoredforseveralweeksat5±3°C,but

oncetheiodinehasbeenadded,themediashouldbeusedthesamedayandany

excessshouldbedisposedof.

18 Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Appendix A Alternative Enrichment Methods

Specific recommendations for milk powder, chocolate, and cocoabased samples

Specific recommendations for milk powder, chocolate, and

cocoabased samples

Allmilkpowder,chocolate,orcocoa‐basedsamplescanbenefitfromanalternative

enrichment.

Useprewarmed(37°C)sterileUHTskimmilksupplementedwithBrilliantGreen

(0.002%),insteadofBufferedPeptoneWater,astheenrichmentmedia.

•Milkpowdersamplesshouldbeincubatedat37°Cfor22 ±2 hours.

• Chocolateandcocoa‐basedsamplesshouldbeincubated,asdescribedinthe

protocol,at37°Cfor19 ±1 hour.

Specific recommendations for potentially acidic/alkaline samples

SampleswhichpotentiallydeviatefromneutralpHshouldbepreparedasfollows:

1. Dilutethesampleaccordingtothesampleenrichmentprotocol.

2. Incubatefor60±5minutesatroomtemperature.

3. Mixbyhandmassaging,anddeterminethepH.

4. Ifnecessary,adjustthepHto6.8±0.2,andmixwellbeforedeterminingthe

final pH.

Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Background

Product overview

Description of

target

microorganism

Morethan2,400Salmonellaserotypeshavebeenreported,allofwhicharepotentially

pathogenic.Salmonellaisafrequentlyreportedcauseoffoodborneillness,occurringin

bothepidemicsandinisolatedcases.Salmonellabacteriaarethecausativeagentfor

Salmonellosis.Outbreakshavebeenassociatedwithrawmeatsandpoultry,eggs,milk

anddairyproducts,seafood,coconutsauces,saladdressings,cocoa,chocolate,spices,

frozenproducts,andvegetablessuchashotpeppers.

Audience ThePathatrix®10‐PoolingSalmonellaspp.Kitisforprofessionaluseonlyandis

intendedforusebyqualifiedusersinterestedindeterminingthepresence/absenceof

Salmonellaspp.infoodsamples.Usersmayinclude,butarenotlimitedto,food

producers,foodprocessors,foodmanufacturers,retailers,andmicrobiologytesting

laboratories.

Sampling protocol ThestandardfoodsamplesizeusedinthePathatrix®Autosystemis25goffood

dilutedwith225mLofenrichmentmedium.Werecommendthatsub‐samplesfor

poolingareprocessedimmediatelyor,ifstorageisrequired,thatthesamplesare

refrigeratedat5±3°C.Samplesshouldberewarmedto37 ±1°Cpriortoanalysiswith

thePathatrix®Autosystem.Theremainingenrichedsampleshouldbestoredat5±3°C

upto32 hoursuntiltheresultsofthepooledsamplehavebeendetermined.

Kit sensitivity Thesamplepreparationprocedureallowsyoutodetectasfewas1–10cfufrom

25–325gramsoffoodsample.ThelimitationofthePathatrix®10‐PoolingSalmonella

spp.Kitisintheabilityofthetargettoreproduceintheenrichmentmedium,be

capturedbythemagnet,andsubsequentlybeisolatedonselectiveagarplates.

CAUTION! ThePathatrix®kithasbeenevaluatedoncookedham,chocolate,and

choppedtomatofoodmatrices.Giventhewidevarietyofproductsandmanufacturing

procedures,werecommendthatyoucheckthatthecompositionofthematricestobe

testeddoesnotaffectthereliabilityoftheresults.

Anegativeresultdoesnotguaranteetheabsenceoftargetorganismintheoriginal

sampleandmaybeduetotheinabilityoftheorganismtoadequatelyreproduceto

requiredlevelsintheenrichmentmedium(withsubsequentoutgrowthonselective

agarplates)potentiallydueto,butnotlimitedto,competitivemicroflora,sub‐lethal

injury,ormatrixinhibition.

20 Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Appendix B Background

AOAC Performance Tested Methodssm Certification

Operating

conditions

ThePathatrix®AutoInstrumentisforindooruseonlyandforaltitudesnotexceeding

2000m(6500ft.)abovesealevel.

AOAC Performance Tested Methodssm Certification

ThePathatrix®10‐PoolingSalmonellaspp.Kithasbeenvalidatedandcertifiedby

AOACInternationalResearchInstitute(Licensenumber090203C)forthedetectionof

Salmonellaserovarsfrom10‐Pooledsamplesincookedham,chocolate,andchopped

tomatoes.ThetestoperatesinconjunctionwithapooledPathatrix®workflow.A

standard25‐gfoodsampleishandmixedbrieflywith225mLofenrichmentmedium

andincubatedforaminimumof18hoursat37°C.Afterincubation,a5‐mLsample

aliquotiscombinedwithupto9additional5‐mLsamplealiquots,andtheresulting

50‐mLpooledsampleisthensubjectedtoa15‐minutePathatrix®AutoInstrument

capturecycle.

Themethodwasvalidatedforusewiththefollowingmatrices:

• 25–325gofcookedham

•25gofchocolate

•25gofchoppedtomatoes

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® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates User guide

Brand: Thermo Fisher Scientific

Model: Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates

Type: User guide

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Thermo Fisher Scientific Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates User guide

Thermo Fisher Scientific Pathatrix® 10-Pooling Salmonella spp. Kit Linked to Selective Agar Plates User guide

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