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1、实验二 细菌的芽孢染色一教学要求了解芽孢方法的原理，掌握操作技术。二实验原理Afewgeneraofbacteria,suchasBacillusandClostridiumhavetheabilitytoproduceresistantsurvivalformstermedendospores.Unlikethereproductivesporesoffungiandplants,theseendosporesareresistanttoheat,drying,radiation,andvariouschemicaldisi
2、nfectants.Duetotheresistantnatureoftheendosporecoats,endosporesaredifficulttostain.Strongdyesandvigorousstainingconditionssuchasheatareneeded.Oncestained,however,endosporesareequallyhardtodecolorize.Sincefewbacterialgeneraproduceendospores,theendosporestainisagooddi
3、agnostictestforspeciesofBacillusandClostridium.三材料与器材菌种：蜡样芽孢杆菌约2d营养琼脂斜面培养物，球形芽孢杆菌(B.sphaericus)1-2d营养琼脂斜面培养物。染色剂：5％孔雀绿水溶液，10.5％番红水溶液。小试管，滴管，烧杯，载玻片，显微镜等。1.Heat-fixasmearofBacillusmegateriumasfollows:a.Usingthedropperbottleofdistilledwaterfoundinyourstainingrack,place
4、asmalldropofwateronacleanslidebytouchingthedroppertotheslide.b.Asepticallyremoveasmallamountoftheculturefromtheedgeofthegrowthontheagarsurfaceandgenerouslymixitwiththedropofwateruntilthewaterturnscloudy.c.Burntheremainingbacteriaoffoftheloop.d.Usingtheloop,spreadthe
5、suspensionovertheentireslidetoformathinfilm.e.Allowthisthinsuspensiontocompletelyairdry.f.Passtheslide(film-sideup)throughtheflameofthebunsenburner3or4timestoheat-fix.四操作步骤2.Placeapieceofblottingpaperoverthesmearandsaturatewithmalachitegreen.3.Letthemalachitegreensi
6、tontheslideforoneminuteandproceedtothenextstep.4.Holdingtheslidewithforceps,carefullyheattheslideintheflameofabunsenburneruntilthestainjustbeginstosteam.Removetheslidefromtheheatuntilsteamingstops;thengentlyreheat.Continuesteamingthesmearinthismannerforfiveminutes.A
7、sthemalachitegreenevaporates,continuallyaddmore.Donotletthepaperdryout.5.Afterfiveminutesofsteaming,washtheexcessstainandblottingpaperofftheslidewithwater.Don'tforgettowashofanydyethatgotontothebottomoftheslide.6.Blottheslidedry.7.Nowfloodthesmearwithsafraninandstai
8、nforoneminute.8.Washofftheexcesssafraninwithwater,blotdry,andobserveusingoilimmersionmicroscopy.Withthisendosporestainingprocedure,endospo
