AimDetectionofmycoplasmabycultureisthereferencemethodofdetectionandhasatheoreticallevelofdetectionof1colony-formingunit(cfu).Howevertherearesomestrainsofmycoplasmathatarenon-cultivable(certainstrainsofMycoplasmahyorhinis).Themethodissuitableforthedetectionofmycoplasmainbothcellculturesandcellculturereagentsandresultsareobtainedwithin4weeks.Mycoplasmacoloniesobservedonagarplateshavea‘friedegg’appearance(seeMycoplasmapneumoniae","");"cmimpressionsent="1">Figure14).

Materials

• 70%ethanolinwater(Prod.No.R8382)
• MycoplasmaPigAgarplates(in5cmpetridishes)
• MycoplasmaPigAgarbroths(in1.8mlaliquots)
• M.oraleNCTC*10112
• M.pneumoniaeNCTC*10119

Equipment

• Personalprotectiveequipment(sterilegloves,laboratorycoat,safetyvisor)
• Waterbathsetto37ºC
• MicroBIOLOGicalsafetycABInetatappropriatecontainmentlevel
• CO₂Incubatorsetat32ºC
• GasJar(Gallenkamp)
• GasPakAnaerobicSystem(Gallenkamp)
• GasPakCatalyst(Gallenkamp)
• GasPakAnaerobicIndicator(Gallenkamp)

Procedure

1. Inoculate2agarplateswith0.1mloftestsample.
2. Inoculate1agarplatewith100cfuM.pneumoniae.
3. Inoculate1agarplatewith100cfuM.orale.
4. Leave1agarplateun-inoculatedasanegativecontrol.
5. Inoculate1brothwith0.2mloftestsample.
6. Inoculate1brothwith100cfuM.pneumoniae.
7. Inoculate1brothwith100cfuM.orale.
8. Leave1agarplateun-inoculatedasanegativecontrol.
9. Incubateagarplatesanaerobicallyfor14daysat37ºCusingagasjarwithanaerobicgaspakandcatalyst.
10. Incubatebrothsaerobicallyfor14daysat37ºC.
11. Between3and7daysand10and14daysincubation,subculture0.1mloftestbrothontoanagarplateandincubateplateanaerobicallyasabove.
12. Observeagarplatesafter14daysincubationatx300magnificationusinganinvertedmicroscopeforthepresenceofmycoplasmacolonies(seeFigure14).

CriteriaforaValidResultAllpositivecontrolagarplatesandbrothsshowevidenceofmycoplasmabytypicalcolonyformationonagarplatesandusuallyacolorchangeinbroths.Allnegativecontrolagarplatesandbrothsshownoevidenceofmycoplasma.

CriteriaforaPositiveResultTestagarplatesinfectedwithmycoplasmashowtypicalcolonyformation.

CriteriaforaNegativeResultThetestagarplatesshownoevidenceofmycoplasma.

Notes

1. Mycoplasmacolonieshaveatypicalcolonyformationcommonlydescribedas“friedegg”(SeeFigure8)duetotheopaquegranularcentralzoneofgrowthpenetratingtheagarsurroundedbyaflattranslucentperipheralzoneonthesurface.HoweverinmanycasesonlythecontrolzonewillbevisIBLe.
2. Positivecontrolsmaybeincludedataconcentrationtogive100colony-formingunits.Thesecontrolsshouldobviouslybehandledinalaboratoryremotefromthemaintissueculturelaboratory.
3. Controlorganisms(M.pneumoniae,andM.orale)areavailablefromNationalCollectionofTypeCultures(UK).
4. MycoplasmapneumoniaeisapotentialpathogenandmustbehandledinaclassIImicrobiologicalsafetycabinetoperatingtoACDPCategory2Conditions.
5. Thistestprocedureshouldbecarriedoutinamicrobiologylaboratoryawayfromthecellculturelaboratory.