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1、Electronmicroscopy.Exponential-phaseculturesofdifferentM.smegmatisstrainswereharvested,washedthreetimeswith0.15Mcacodylatebuffer,treatedwith1%osmiumtetroxidefor1hatroomtemperature,centrifuged,andthecollectedcellswerefixedwith0.15Mcacodylatebuffercontaini
2、ng2%glutaraldehydefor2hatroomtemperature.Cellswereagaincollectedaftercentrifugationandfixedovernightwith0.15Mcacodylatebuffercontaining1%osmiumtetroxideat4℃.Thecellsweredehydratedusinggradedethanol(10%,30%,50%,70%and100%),mountedonmicroscopestubsandsputt
3、ercoatedonthesputtercoater.ThesampleswerethenexaminedunderaLeicaS440scanningelectronmicroscope.2.5.Scanningelectronmicroscope(SEM)ForSEManalysis,cellswerefixedwith2.5%(v/v)glutaraldehydefor2–3h.Thefixedcellswerewashedwith0.1Mphosphate-bufferedsaline(PBS)
4、(pH7.3)forthreetimes(10mineach).Subsequently,ethanolconcentrationgradient(v/v)of50%,70%,80%,90%and100%wasusedtodehydratethefixedcellsinasequentialway.Finally,theresultedcellsampleswerefurtherdehydratedbytwomore100%ethanoltreatments.Afterthat,tertiarybuty
5、lalcoholmixedwithethanolinaratioof1:1andpuretertiarybutylalcoholwasappliedtoachievemetathesisofethanolinthecells.Atlast,cellsweremixedintertiarybutylalcoholandlyophilizedforimaging.2.4.2.ElectronmicroscopyanalysisCellswereharvestedbycentrifugationat5000g
6、for3min,washedthreetimeswithPBS(phosphatebufferpH7.2).Subsequently,thecellswerefixedwith2%glutaraldehyde(pH7.2)atroomtemperaturefor2handwashedagainasabove.Thecellswerepreparedforscanningortransmissionelectronmicroscopyasdescribedpreviously(Denneretal.,19
7、94).真空扫描式电子显微镜JSM-6360LV扫描电子显微镜03040702-0020S-3400N二、扫描电子显微镜扫描电子显微镜（scanningelectronmicroscope，SEM）于20世纪60年代问世，用来观察标本的表面结构。其工作原理是用一束极细的电子束扫描样品，在样品表面激发出次级电子，次级电子的多少与电子束入射角有关，也就是说与样品的表面结构有关，次级电子由探测体收集，并在那里被闪烁器转变为光信号，再经光电倍增管和放大器转变为电信号来控制荧光屏上电子束的强度，显示出与电子束同步
8、的扫描图像。图像为立体形象，反映了标本的表面结构。为了使标本表面发射出次级电子，标本在固定、脱水后，要喷涂上一层重金属微粒，重金属在电子束的轰击下发出次级电子信号。要有s-4800扫描电镜看细胞形态，谁知道怎么处理样品啊？是发酵液中的菌体学固定液，滤纸分离，酒精梯度脱水，干燥，电镜实验室送检。扫描电镜样品前处理弧菌的电镜实验，曾经做过一次，按照下面的步骤做的，但是效果不好，细菌拍下来不立体。将菌液以3000r/min转速离心10min后，弃
