Thermo Fisher Scientific Pathatrix 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates User guide

Brand: Thermo Fisher Scientific

Model: Pathatrix 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Type: User guide

USER GUIDE

For testing of Food and Environmental samples only.

Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked

to Selective Agar Plates

For use with the Pathatrix® Auto Instrument

Catalog Number APE500SDP

Publication Number MAN0007091

Revision 12 October 2012

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

DISCLAIMER

LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED,

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

ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR

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® 10-Pooling E. coli O157:H7 Kit 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® 10-Pooling E. coli O157:H7 Kit –

Same-Day–Enrichment Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

■CHAPTER 2 Pathatrix® 10-Pooling E. coli O157:H7 Kit –

Overnight-Enrichment Format . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Procedural guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

Sample preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

Sample pooling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

Samples and consumable loading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

Sample unloading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

Detection – direct plating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Retesting of individual samples after “Positive Pooled Sample” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

Test result interpretation and classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

4Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Contents

■APPENDIX A Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Product overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Description of target microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Audience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Sampling protocol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Kit sensitivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24

Operating conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

AOAC Performance Tested Methodssm Certification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

■APPENDIX B Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

Related materials from Life Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

■APPENDIX C Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Chemical safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

Specific chemical handling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Biological hazard safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

Documentation and Support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Obtaining SDSs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Obtaining Certificates of Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Obtaining support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Limited product warranty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Product Information

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

AppendixC,“Safety”onpage27.

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®10‐Pooling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®10‐PoolingE.coliO157:H7KitLinkedtoSelectiveAgarPlates,

presumptiveresultscanbeobtained,priortoconfirmation,within26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24foradditionalbackgroundinformation.

6Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Product Information

Kit contents

ThePathatrix®10‐PoolingE.coliO157:H7Kit(Cat.no.APE500SDP)containsenough

consumablecomponentsandPathatrix®paramagneticbeadstoprocess500 samples

(50Cartridgeruns).

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® 10-Pooling E. coli O157:H7 Kit 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

® 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, page 18, or Appendix A on page 24 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® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Product Information

Materials not included in the kit

Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Pathatrix® 10-Pooling 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 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 15)

10 Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Chapter1 Pathatrix® 10-Pooling E. coli O157:H7 Kit – Same-Day–Enrichment Format

Sample preparation

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7hours.

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 E. coli O157:H7 Kit Linked to Selective Agar Plates

Chapter1 Pathatrix® 10-Pooling E. coli O157:H7 Kit – Same-Day–Enrichment Format

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10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,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 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 E. coli O157:H7 Kit Linked to Selective Agar Plates

Chapter1 Pathatrix® 10-Pooling 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

Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Chapter1 Pathatrix® 10-Pooling 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

14 Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Chapter1 Pathatrix® 10-Pooling 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.

Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Chapter1 Pathatrix® 10-Pooling 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. Allowtheplatestodryforapproximately10minutestheninvertandincubateat

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

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

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

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30]).

Ifapositiveresultisobtainedfromthepooledsample,the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thatrequireretesting,shouldberewarmedto42±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.

Test result interpretation and classification

ThePathatrix®10‐Pooling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®10‐PoolingE.coliO157:H7Kit(same‐day–enrichmentformat)

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

confirmation,within26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)

16 Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Chapter1 Pathatrix® 10-Pooling E. coli O157:H7 Kit – Same-Day–Enrichment Format

Test result interpretation and classification

Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Pathatrix® 10-Pooling 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 preparation (page 18)

Sample pooling (page 19)

Samples and consumable loading (page 20)

Sample unloading (page 22)

Detection – direct plating (page 23)

Retesting of individual samples after “Positive Pooled Sample” (page 23)

Test result interpretation and classification (page 23)

18 Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Chapter2 Pathatrix® 10-Pooling E. coli O157:H7 Kit – Overnight-Enrichment Format

Sample preparation

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.

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 E. coli O157:H7 Kit Linked to Selective Agar Plates

Chapter2 Pathatrix® 10-Pooling E. coli O157:H7 Kit – Overnight-Enrichment Format

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10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,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 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)

20 Pathatrix® 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Chapter2 Pathatrix® 10-Pooling 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

Page is loading ...

Thermo Fisher Scientific Pathatrix 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates User guide

Brand: Thermo Fisher Scientific

Model: Pathatrix 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates

Type: User guide

Download PDF

report

Thermo Fisher Scientific Pathatrix 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates User guide

Thermo Fisher Scientific Pathatrix 10-Pooling E. coli O157:H7 Kit Linked to Selective Agar Plates User guide

Related papers

Other documents