AimDNAstainingmethodssuchasHoechststainingtechniquesarequickwithresultsavailablewithin24hours,whichcomparesfavorablywith4weeksfordetectionbyculture.HoweverthestainingofculturesdirectlywithaDNAstain,resultsinamuch-reducedsensitivity(~10₆cfu/ml).Thismaybeimprovedbyco-culturingthetestcelllineinthepresenceofanindicatorcelllinesuchasVero(Prod.No.84113001-1v1).Thisenrichmentstepresultsinasensitivityof104cfu/mlofculture.Thisstepalsoimprovessensitivitybyincreasingthesurfaceareauponwhichmycoplasmacanadhere.Likedetectionbyculture,DNAstainingmethodsaresuitableforthedetectionofmycoplasmafromcellculturesorcellculturereagents.

Materials

• Media–pre-warmedto37ºC(refertotheECACCCellLineDataSheetforthecorrectmedium)
• 70%ethanolinwater(Prod.No.R8382)
• Methanol(Prod.No.175)
• AceticAcidGlacial(Prod.No.A6283)
• Hoechst33258stainsolution(Prod.No.H6024)
• Verocells(Prod.No.84113001-1v1)
• Mountant(Autoclave22.2ml0.2Mcitricacidwith27.8ml0.2Mdisodiumphosphate.Add50mlglycerol.Filtersterilizeandstoreat4ºC)(Prod.No.M1289)
• MycoplasmahyorhinisNCTC*10112

Equipment

• Personalprotectiveequipment(sterilegloves,laboratorycoat,safetyvisor)
• Waterbathsetto37ºC
• MicroBIOLOGicalsafetycABInetofappropriatecontainmentlevel
• Centrifuge
• CO₂Incubatorsetat37ºC
• Microscope(uvEpi-Fluorescent.)
• 35mmplastictissueculturedishes(Prod.No.C6296)
• Multidish24well(Prod.No.M9655)
• Cellscraper
• Microscopeslidesand22mmcoverslips
• Aluminumfoil(Prod.No.Z185140)

ProcedureEquipment

1. Foreachsampleandcontrolsterilize2coverslipsinahotovenat180ºCfor2hoursorbyimmersingin70%ethanol(Prod.No.R8382)andflaminginablueBunsenflameuntiltheethanolhasevaporated.Alsosterilize2coverslipstouseasanegativecontrol.
2. Placethecoverslipsin35mmculturedishes(Prod.No.C6296)(1perdish).
3. Storeuntilneeded.
4. TopreparetheVero(Prod.No.84113001-1v1)indicatorcellsadd2x104cellsin2mlofantibiotic-freegrowthmediumtoeachtissueculturedish.
5. Incubateat37ºCin5%CO₂for2–24hrstoallowthecellstoadheretothecoverslips.
6. BringattachedtestcelllinesintosUSPensionusingacellscraper.Suspensioncelllinesmaybetesteddirectly.
7. Remove1mlofculturesupernatantfromduplicatedishesandadd1mloftestsampletoeach.Inoculate2disheswith100cfuM.hyorhinisand2with100cfuM.orale..org.uk
8. Leaveduplicatetissueculturedishesun-inoculatedasnegativecontrols.
9. Incubatedishesat37ºCin5%CO₂for1-3days.
10. After1dayobserveonedishfromeachpairforbacterialorfungalinfection.Ifcontaminateddiscardimmediately.Leavetheremainingdishofeachpairforafurther2days.
11. Fixcellstocover-slipbyaddingaminimumof2mloffreshlypreparedfixative(1:3glacialaceticacid:absolutemethanol)tothetissueculturedishandleavefor3to5minutes.
12. Decantusedfixativetotoxicwastebottle.Addanother2mlaliquotoffixativetocover-slipandleaveforafurther3to5min.Decantusedfixativetotoxicwaste.
13. Airdrycover-slipbyrestingitagainstthetissueculturedishfor30-120min.
14. Replacecover-slipindishandaddaminimumof2mlHoechststain(Prod.No.H6024).Leavefor5minutesshieldedfromdirectlightbyaluminumfoil(Prod.No.Z185140).
15. Decantusedandunusedstaintotoxicwaste.
16. Add1dropofmountanttoapre-labeledmicroscopeslideandplacecover-slip(cellsidedown)ontoslide.
17. Keepslidecoveredwithaluminumfoil(Prod.No.Z18514-0),allowingittosetforatleast15minat37ºCorfor30minatroomtemperature.
18. ObserveslideunderuvEpi-Fluorescenceatx1000.

Notes

1. DNAstainssuchasHoechststain(Prod.No.H6024)bindspecificallytoDNA.Inallculturescellnucleiwillfluoresce.Uncontaminatedcultureswillshowonlyfluorescentnucleiwhereasmycoplasmapositiveculturescontainsmallcocciorfilamentswhichmayormaynotbeadsorbedontothecells(seefigure16).
2. Hoechststainistoxicandshouldbehandledanddiscardedwithcare.
3. Culturedishesshouldbeplacedinasealedboxorculturedinlargepetridishestoreduceevaporation.
4. Positivesshouldobviouslybehandledinalaboratoryremotefromthemaintissueculturelaboratory.
5. Controlorganisms(M.hyorhinis)areavailablefromtheNationalCollectionofTypeCultures(UK).
6. Insomeinstancesresultsmaybedifficulttointerpretforthefollowingreasons:
   • Bacterial/yeast/fungalcontamination
   • Toomuchdebrisinthebackground
   • Brokennucleiascellsarealldead
   • Toofewornolivecells
7. Althoughthisprocedurerecommendsthesettingupofpositivecontrols,thismaynotnecessarilybefeasIBLenordesirableinacellculturefacilitywithlimitedresources.Ifpositivecontrolsaretobesetuptheyshouldbedonesoinaseparatelaboratoryfromthemaintissueculturefacility.IfthisisnotpossiblethenpositiveslidescanbepurchasedfromECACC.Ifpositivecontrolsarenotbeingusedthenitisstronglyrecommendedthatyougetanindependenttestinglaboratorytoperiodicallytestyourcelllines.