AimIncasesofgrosscontaminationthenakedeyemayidentifythepresenceofbacteriaandfungi.However,itisnecessarytodetectlow-levelinfectionsbyincubationofcellculturesand/ortheirproductsinmicroBIOLOGicalbroth.Equallythesesterilitytestscanbeusedtoconfirmtheabsenceofbacteriaandfungifromthepreparationwhichisimportantwhenpreparingcellbanksorcellcultureproducts.

Materials

• SoybeanCaseinDigest(TryptoneSoyaBroth,TSB)(15mlaliquots)(Prod.No.S1674)TSBPowder(Prod.No.T8907)
• FluidThioglycollateMedium(20mlaliquots)(TGM)(Prod.No.F4797)
• BacillussubtilisNCTC*
• CandidaalbicansNCTC*
• ClostridiumsporogenesNCTC*

Equipment

• Personalprotectiveequipment(latexmedicalgloves,laboratorycoat,safetyglasses)
• Waterbathsetto37ºC
• MicrobiologicalsafetycABInetatappropriatecontainmentlevel
• Centrifuge
• Incubatorsetat32ºC
• Incubatorsetat22ºC

ClickhereforFigure12.FlowSchemeforBacteriaandFungiTesting

Procedure

1. Culturecelllineintheabsenceofantibioticsfor2passagespriortotesting.
2. BringattachedcellsintosUSPensionwiththeuseofacellscraper.Suspensioncelllinesmaybetesteddirectly.
3. Inoculate2xThioglycollateMedium(TGM)(Prod.No.F4797)and2xTryptoneSoyabroth(TSB)(Prod.No.T8907)with1.5mltestsample.
4. Inoculate2(TGM)and2(TSB)with0.1mlC.albicans(containing100colonyformingunits,cfu).
5. Inoculate2(TGM)and2(TSB)with0.1mlB.subtilis(containing100cfu).
6. Inoculate1TGMwith0.1mlC.sporogenes(containing100cfu).
7. Leave2(TGM)and2(TSB)un-inoculatedasnegativecontrols.
8. Incubatebrothsasfollows:
   • ForTSB,incubateonebrothofeachpairat32ºCtheotherat22ºCfor14days
   • ForTGM,incubateonebrothofeachpairat32ºCtheotherat22ºCfor14days
   • FortheTGMinoculatedwithC.sporogenesincubateat32ºCfor14days
9. ExamineTestandControlbrothsforturbidityafter14days.

CriteriaforaValidResultAllpositivecontrolbrothsshowevidenceofbacteriaandfungiwithin14daysofincubationandthenegativecontrolbrothsshownoevidenceofbacteriaandfungi.

CriteriaforaPositiveResultTestbrothscontainingbacteriaorfungishowturbidity.

CriteriaforaNegativeResultTestbrothsshouldbeclearandshownoevidenceofturbidity.

Notes

1. Thepositivecontrolsshouldbehandledinalaboratoryremotefromthemaintissueculturelaboratory.
2. Controlorganisms(Bacillussubtilis,ClostridiumsporogenesandCandidaalbicans)arealsoavailablefromtheNationalCollectionofTypeCultures(NCTC),UK*.
3. Thistestprocedureshouldbecarriedoutinamicrobiologylaboratoryawayfromthecellculturelaboratory.