Thermo Fisher Scientific Pathatrix E. coli O157:H7 Kit User guide

USER GUIDE

For testing of Food and Environmental samples only.

Pathatrix® E. coli O157:H7 Kit (Individual

Samples) Linked to Selective Agar Plates

For use with the Pathatrix® Auto Instrument

Catalog Number APE50SD

Publication Number MAN0007092

Revision 12 October 2012

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

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INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT

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UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN

CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF.

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

or other commercial consideration. No other right is hereby granted expressly, by implication, or by estoppel. This product is for environmental testing, quality

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 outlicensi[email protected]om.

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® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Product Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

About the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

Kit contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

Materials not included in the kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

■CHAPTER 1 Pathatrix® E. coli O157:H7 Kit –

Same-Day–Enrichment Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

Sample enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

Samples and consumable loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

Sample unloading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

Detection – direct plating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

Test result interpretation and classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

■CHAPTER 2 Pathatrix® E. coli O157:H7 Kit –

Overnight-Enrichment Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

Sample enrichment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

Samples and consumable loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Sample unloading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Detection – direct plating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Test result interpretation and classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

4Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Contents

■APPENDIX A Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Description of target microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Audience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Sampling protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Kit sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

Operating conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

AOAC Performance Tested Methodssm Certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

■APPENDIX B Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Related materials from Life Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

■APPENDIX C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Specific chemical handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Obtaining Certificates of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Product Information

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

AppendixC,“Safety”onpage24.

CAUTION! E.coliO157:H7isaBiosafetyLevel2(BSL‐2)organism.Caremustbetaken

whenhandlingsamplesthatmaycontainE.coliO157:H7.Laboratorypersonnelmust

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

forE.coliO157:H7.Laboratorypersonnelmustwearappropriateprotective

equipment,whichincludesbutisnotlimitedto:protectiveeyewear,faceshield,

clothing/labcoat,andgloves.Extremeprecautionsshouldbetakenwithcontaminated

sharpitems.Accesstothelaboratoryshouldbelimitedwhenworkisbeingconducted.

Wasteshouldbedisposedofincompliancewithlocalandnationallegislationas

appropriate.

About the kit

ThePathatrix®E.coliO157:H7Kitisasamplepreparationmethodforpresence/

absencetestingbasedonthedetectionofasfewas1–10cfu(colonyformingunits)/

25–375goffoodsample.

UsingthePathatrix®E.coliO157:H7Kit(IndividualSamples)LinkedtoSelectiveAgar

Plates,presumptiveresultscanbeobtained,priortoconfirmation,within24hours

(same‐day–enrichmentformat)or36hours(overnight‐enrichmentformat).

Apresumptive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:

•E.coliO157:H7Detectedin25–375g(samplematrices)

•E.coliO157:H7Notdetectedin25–375g(samplematrices)

SeeAppendixAonpage21foradditionalbackgroundinformation.

6Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Product Information

Kit contents

ThePathatrix®E.coliO157:H7Kit(Cat.no.APE50SD)containsenoughconsumable

componentsandPathatrix®paramagneticbeadstoprocess50 samples.

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

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

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

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).

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-E. coli O157:H7 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

Item Source

Equipment

Incubator, 37 ±1°C MLS

Incubator, 42 ±1°C MLS

Pathatrix® Auto Instrument Life Technologies Cat. no. PATHATRIXAUTO

Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Product Information

Materials not included in the kit

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

® 5-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. POOL510MLN

Sterile, disposable 10-µL loops MLS

Media

See page 10 and Appendix A on page 21 for recommendations about enrichment media

choice. Media is supplied by several manufacturers (e.g., Oxoid [product codes shown], BD,

Difco, and Merck) in a dehydrated form and should be prepared according to the

manufacturer's instructions.

Buffered peptone water

Buffered peptone water (ISO formulation)

(alternative)

Oxoid product code CM0509

Oxoid product code CM1049

Selective agar

For CT-SMAC plates:

• Sorbitol MacConkey agar

• C-T Supplement

• Oxoid product code CM0813B

• Oxoid product code SR0172E

BBL CHROMagar O157 BD Catalog # 214984

Reagents

PBS, 10X, pH 7.4 Life Technologies Cat. no. AM9624 or AM9625

Item Source

8Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Product Information

Materials not included in the kit

Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Pathatrix® E. coli O157:H7 Kit –

Same-Day–Enrichment Format

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 enrichment (page 10)

Sample preparation (page 10)

Samples and consumable loading (page 11)

Sample unloading (page 13)

Detection – direct plating (page 14)

Test result interpretation and classification (page 14)

10 Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Chapter1 Pathatrix® E. coli O157:H7 Kit – Same-Day–Enrichment Format

Sample enrichment

1. Weighthefoodsample(typically25–375g)intoanappropriatesterilebag.

•Forsamplesizesbetween25–55g,preparea1:10dilutionofthefoodsample

inpre‐warmed(42 ±1°C)BufferedPeptoneWater.Forexample,add25gof

foodsampleto225mLofprewarmedmedia.

•Samplesizesabove55gshouldbedilutedin500mLofpre‐warmed

BufferedPeptoneWater.

IMPORTANT! Itiscriticalthattheenrichmentmediaisprewarmedto42 ±1°C

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

immediatelybeplacedintheincubatorat42 ±1°C.

2. Homogenizethesamplebymassagingthesamplebetweenthefingersfor

10–15secondstodisperseanylargeclumpsofmaterial(NarangandCray,2006).

3. Incubateat42 ±1°Cforaminimumof5hours.

Sample preparation

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. Place50mLofyoursampleintheSampleVessel.

Note: Ifyoursampleishighlyparticulateand/orhasahighfatcontent,theuseof

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

straws(Cat.no.POOL510MLN)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refrigeratedafterenrichment,

samplesshouldberewarmedto37 ±1°Cpriortoremovalofaliquotsforanalysis.

Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Chapter1 Pathatrix® E. coli O157:H7 Kit – Same-Day–Enrichment Format

Samples and consumable loading

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

(approximately35mLof1XPBS).

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

sealedallthewayaround.

3. EnsurethePathatrix®paramagneticbeads(includedinthekit)arefully

resuspendedbyagitatingthebeadvial(forexample,byvortexingbeadvialor

inversionofsealedvial),andadd50μLofthePathatrix®paramagneticbead

suspensionintothespoutonthelidoftheSampleVessel.

4. Removethecapture‐phasekitfromthebagandorientwiththevalveplunger

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

Vessels.

Elution Vessel

Sterile 1X PBS (35 mL)

Sample Vessel Holder

(ATUBEHOLD)

Sample Vessel Spout

Sample Vessel

Capture Phase

Valve Plunger

12 Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Chapter1 Pathatrix® E. coli O157:H7 Kit – Same-Day–Enrichment Format

Samples and consumable loading

5. Holdingbothvessels,lifttheassembledvesselsandattachedcapture‐phasekit

outoftherack(alsoknownastheSampleVesselHolder).

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

upwards.

7. FirmlypushtheremainderofthekitintotheCartridge,ensuringthatthevalve,

thecapture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capture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 Button

Valve Plunger

Capture Phase

Sample Vessel

Elution Vessel

Cartridge Holder

(ACARTHOLD)

Sliding Magnet

Holder

Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Chapter1 Pathatrix® E. coli O157:H7 Kit – Same-Day–Enrichment Format

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,capwithaflatlid(providedinthekit),and

leaveintherackfor1minutetoallowcaptureofthePathatrix®paramagnetic

beads.

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

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

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

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

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

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

Note: ThePathatrix®paramagneticbeadsuspensionmayberetainedforlatertesting

ifnecessary.ThebeadsshouldbestoredAWAYfrommagnets(forexample,Pathatrix®

vesselholders)at5±3°Cforupto24hours.

14 Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Chapter1 Pathatrix® E. coli O157:H7 Kit – Same-Day–Enrichment Format

Detection – direct plating

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

opposedtospreadplating.

1. PipetallofthePathatrix®paramagneticbeadsuspensionontotheedgeofwell‐

driedselectiveagarplates(forexample,CefiximePotassiumTelluriteSorbitol‐

MacConkeyAgar[CT‐SMAC],CHROMagarO157)forisolationofthetarget.

Note: Retainanamountofbeadsuspensionyouwishtokeep.Dividethe

remainderofthebeadsuspensionintoequalamountsforstreakingonyour

selectiveagarplates.

2. Usingasterile10‐μLinoculationloop,streakfromthispooltogenerateisolated

colonies.

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

attherequiredtemperaturefor18–24hours.

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

atypicalE.coliO157:H7coloniesontheagarplatesused.Apresumptivepositive

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

biochemicalandserologicaltests(forexample,asdetailedinISO16654:2001orUSDA

MicrobiologyLaboratoryGuidebook[MLG]5.04asusedintheAOACResearch

Institutevalidationstudy[See“References”onpage27]).

Test result interpretation and classification

ThePathatrix®E.coliO157:H7Kitisdesignedasasamplepreparationmethodfor

presence/absencedetectionofE.coliO157:H7infoodmatrices.

UsingthePathatrix®E.coliO157:H7Kit(IndividualSamples;same‐day–enrichment

format)linkedtoselectiveagarplates,presumptiveresultscanbeobtained,priorto

confirmation,within24hours.

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

•E.coliO157:H7Detectedin25–375g(samplematrices)

•E.coliO157:H7Notdetectedin25–375g(samplematrices)

Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Pathatrix® E. coli O157:H7 Kit –

Overnight-Enrichment Format

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 enrichment (page 16)

Sample preparation (page 16)

Samples and consumable loading (page 17)

Sample unloading (page 19)

Detection – direct plating (page 20)

Test result interpretation and classification (page 20)

16 Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Chapter2 Pathatrix® E. coli O157:H7 Kit – Overnight-Enrichment Format

Sample enrichment

1. Weighthefoodsample(typically25–375g)intoanappropriatesterilebag.

•Forsamplesizesbetween25–55g,preparea1:10dilutionofthefoodsample

inpre‐warmed(42 ±1°C)BufferedPeptoneWater.Forexample,add25gof

foodsampleto225mLofprewarmedmedia.

•Samplesizesabove55gshouldbedilutedin500mLofpre‐warmed

BufferedPeptoneWater.

IMPORTANT! Itiscriticalthattheenrichmentmediaisprewarmedto42 ±1°C

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

immediatelybeplacedintheincubatorat42 ±1°C.

2. Homogenizethesamplebymassagingthesamplebetweenthefingersfor

10–15secondstodisperseanylargeclumpsofmaterial(NarangandCray,2006).

3. Incubateat42 ±1°Cforaminimumof16 hours,uptoamaximumof24hours.

Sample preparation

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. Place50mLofyoursampleintheSampleVessel.

Note: Ifyoursampleishighlyparticulateand/orhasahighfatcontent,theuseof

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

straws(Cat.no.POOL510MLN)isrecommended.Alternatively,Sewardplain

sterilebagswithinternalstrainers(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refrigeratedafterenrichment,

samplesshouldberewarmedto37 ±1°Cpriortoremovalofaliquotsforanalysis.

Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Chapter2 Pathatrix® E. coli O157:H7 Kit – Overnight-Enrichment Format

Samples and consumable loading

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

(approximately35mLof1XPBS).

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

sealedallthewayaround.

3. EnsurethePathatrix®paramagneticbeads(includedinthekit)arefully

resuspendedbyagitatingthebeadvial(forexample,byvortexingbeadvialor

inversionofsealedvial),andadd50μLofthePathatrix®paramagneticbead

suspensionintothespoutonthelidoftheSampleVessel.

4. Removethecapture‐phasekitfromthebagandorientwiththevalveplunger

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

Vessels.

Elution Vessel

Sterile 1X PBS (35 mL)

Sample Vessel Holder

(ATUBEHOLD)

Sample Vessel Spout

Sample Vessel

Capture Phase

Valve Plunger

18 Pathatrix® E. coli O157:H7 Kit (Individual Samples) Linked to Selective Agar Plates

Chapter2 Pathatrix® E. coli O157:H7 Kit – Overnight-Enrichment Format

Samples and consumable loading

5. Holdingbothvessels,lifttheassembledvesselsandattachedcapture‐phasekit

outoftherack(alsoknownastheSampleVesselHolder).

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

upwards.

7. FirmlypushtheremainderofthekitintotheCartridge,ensuringthatthevalve,

thecapture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capture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 Button

Valve Plunger

Capture Phase

Sample Vessel

Elution Vessel

Cartridge Holder

(ACARTHOLD)

Sliding Magnet

Holder