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1、實驗二 細菌的芽孢染色一教學要求了解芽孢方法的原理，掌握操作技術。二實驗原理Afewgeneraofbacteria,suchasBacillusandClostridiumhavetheabilitytoproduceresistantsurvivalformstermedendospores.Unlikethereproductivesporesoffungiandplants,theseendosporesareresistanttoheat,drying,radiation,andvariouschemicaldisi
2、nfectants.Duetotheresistantnatureoftheendosporecoats,endosporesaredifficulttostain.Strongdyesandvigorousstainingconditionssuchasheatareneeded.Oncestained,however,endosporesareequallyhardtodecolorize.Sincefewbacterialgeneraproduceendospores,theendosporestainisagooddi
3、agnostictestforspeciesofBacillusandClostridium.三材料與器材菌種：蠟樣芽孢桿菌約2d營養(yǎng)瓊脂斜面培養(yǎng)物，球形芽孢桿菌(B.sphaericus)1-2d營養(yǎng)瓊脂斜面培養(yǎng)物。染色劑：5％孔雀綠水溶液，10.5％番紅水溶液。小試管，滴管，燒杯，載玻片，顯微鏡等。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.四操作步驟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
