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1、CAMS/PUMC细菌学实习细菌学实习-II多途径细菌采样培养及初步多途径细菌采样培养及初步鉴定鉴定CAMS/PUMC实验材料实验材料q咽拭子咽拭子 1支支/人人q大号普通培养皿大号普通培养皿 2块块/人人q小号羊血培养皿小号羊血培养皿 2块块/人人q10ml无菌肉汤管无菌肉汤管 1支支/人人q载玻片载玻片 1片片/人人q A、B菌菌 3套套/桌桌qNS 1份份/人人q滤纸滤纸 1张张/人人q无菌棉棒无菌棉棒CAMS/PUMC实验目的实验目的q了解各种培养基的制备了解各种培养基的制备q掌握机体常见部位及环境中的细菌采样、掌握机体常见部位及环境中的细菌采样、培养及初步鉴定培养及初步鉴定q掌握革兰
2、氏染色方法及结果判定掌握革兰氏染色方法及结果判定CAMS/PUMC实验内容实验内容q机体不同部位的细菌采样、培养机体不同部位的细菌采样、培养v咽部正常菌群的采样、培养咽部正常菌群的采样、培养v未消毒手的细菌采样培养未消毒手的细菌采样培养CAMS/PUMC实验内容实验内容q环境中细菌采样培养环境中细菌采样培养v常用生活用品的细菌采样培养常用生活用品的细菌采样培养手机手机流通纸币流通纸币v抗菌洗浴用品抑菌功效的检测抗菌洗浴用品抑菌功效的检测CAMS/PUMCq紫外线穿透能力试验紫外线穿透能力试验CAMS/PUMC实验内容实验内容qA、B菌菌鉴定（鉴定（Gram stain）v报告内容报告内容染色结
3、果染色结果细菌形态细菌形态CAMS/PUMC细菌涂片制备程序细菌涂片制备程序q取一干净玻片，火焰上方缓缓通过三次去取一干净玻片，火焰上方缓缓通过三次去油油q蜡笔画线将玻片分割为蜡笔画线将玻片分割为A、B两区域两区域q接种环取接种环取NS分别滴入分别滴入A、B两区域各一滴两区域各一滴q分别取少许分别取少许A、B菌菌 ，与，与NS充分混匀成菌充分混匀成菌液，风干后形成菌膜液，风干后形成菌膜q固定（火焰上方缓缓通过三次）固定（火焰上方缓缓通过三次）q革兰氏染色革兰氏染色q滤纸吸干水分，油镜观察滤纸吸干水分，油镜观察CAMS/PUMCABCAMS/PUMC Heat fixingCAMS/PUMCSm
4、ear preparation CAMS/PUMCCAMS/PUMCcrystal violet stainCAMS/PUMCwash with water CAMS/PUMCStain with grams iodine solution CAMS/PUMCDecolorize by adding 95% alcohol CAMS/PUMC Counterstain with Safranin CAMS/PUMCCAMS/PUMCG+菌菌G-菌菌CAMS/PUMCCAMS/PUMCCAMS/PUMCCAMS/PUMC细菌接种法细菌接种法q平行划线法平行划线法q分区划线法分区划线法q密集接种法
5、密集接种法CAMS/PUMC平行划线法平行划线法CAMS/PUMC分区划线法分区划线法CAMS/PUMCbackCAMS/PUMCCAMS/PUMC培养基（培养基（Culture medium）vSubstances used for artificial cultivation of bacteria. CAMS/PUMCNutrient requirements of bacterial growth q waterq Carbon source q Nitrogen source q Minerals q Growth factorsv organic compounds such as
6、 amino acids, purines嘌呤嘌呤, pyrimidines嘧啶嘧啶, and vitamins that a cell must have for growth but cannot synthesize itself. CAMS/PUMCClassification of culture mediumvAccording to basic ingredients:Minimal essential growth medium基础培养基基础培养基enriched medium营养培养基营养培养基selective medium选择培养基选择培养基differential me
7、dium鉴别培养基鉴别培养基CAMS/PUMCvAccording to physical conditionliquid medium液体培养基液体培养基solid medium固体培养基固体培养基 1.52.5% of agar semisolid medium半固体培养基半固体培养基0.30.5% of agar CAMS/PUMC Phenomena of bacterial growth细菌在培养基中的生长情况细菌在培养基中的生长情况qIn liquid medium： surface growth pellicle表面生表面生长长, uniformly turbid均匀混浊生长均匀
8、混浊生长, sediment in bottom沉淀生长沉淀生长 CAMS/PUMC菌膜菌膜菌沉淀菌沉淀均匀浑浊均匀浑浊对照对照CAMS/PUMCCAMS/PUMC抗菌用品抑菌功效试验步骤抗菌用品抑菌功效试验步骤q将同一人双前臂暴露将同一人双前臂暴露q自来水冲洗自来水冲洗数秒数秒q分别用舒肤佳及力士香皂，自上而下涂抹前臂分别用舒肤佳及力士香皂，自上而下涂抹前臂三三次次q自来水冲洗数秒自来水冲洗数秒q将琼脂棒贴在双前臂皮肤表面将琼脂棒贴在双前臂皮肤表面5分钟分钟q放入无菌培养皿中放入无菌培养皿中q37 培养培养24小时小时q通过菌落数的观察判断抑菌效果通过菌落数的观察判断抑菌效果咽部正常菌群的采
9、样、培养咽部正常菌群的采样、培养咽拭子擦拭咽部，平行划线法接种于咽拭子擦拭咽部，平行划线法接种于羊血平皿羊血平皿CAMS/PUMCCAMS/PUMC未消毒手的细菌采样培养未消毒手的细菌采样培养将将10 ml液体培养基加入无菌手套内充液体培养基加入无菌手套内充分洗手分洗手混匀后吸出混匀后吸出用棉棒进行密集接种用棉棒进行密集接种CAMS/PUMC密集接种密集接种q用无菌棉棒儿蘸取菌液用无菌棉棒儿蘸取菌液q将多余菌液在液面上方管壁内轻轻旋转挤将多余菌液在液面上方管壁内轻轻旋转挤出，出，涂布在琼脂平板整个平面三次，每次涂布在琼脂平板整个平面三次，每次旋转旋转60度，最后沿平板内缘涂抹一周，以度，最后沿平板内缘涂抹一周，以取得均匀接种取得均匀接种紫外线穿透能力试验紫外线穿透能力试验q密集接种法接种细菌密集接种法接种细菌q将不同形状的纸片贴至培养基表面将不同形状的纸片贴至培养基表面q紫外线照射紫外线照射2020分钟分钟q将纸片焚烧灭菌将纸片焚烧灭菌CAMS/PUMCCAMS/PUMCq采样结束后，将培养皿放入铁丝筐，统一采样结束后，将培养皿放入铁丝筐，统一置培养箱置培养箱 37 18-2437 18-24小时小时CAMS/PUMCIn semisolid medium无动力 有动力
