试验3土壤中细菌总数的测定-平板计数.PPT

实验三 土壤中细菌总数的测定 一 教学要求 通过土壤中细菌总数的测定，了解微生物数量的测定方法，掌握平板菌落计数的原理和方法 二 实验原理 1、测定微生物生长的方法 Direct methods Direct counting，Coulter counter Indirect methods Viable Cell methods，Membrane filtration ， turbidity methods，most probable number(MPN) ,Detect chemical products or metabolic activity Alternative methods for viable cell counts 2、The plate count (viable count) The number of bacteria in a given sample is usually too great to be counted directly. However, if the sample is serially diluted and then plated out on an agar surface in such a manner that single isolated bacteria form visible isolated colonies, the number of colonies can be used as a measure of the number of viable (living) cells in that known dilution. 三 材料与器材 无菌培养皿12套、1 mL无菌移液管10 支、土壤样品、天平等。 四 操作步骤 无菌平皿编号→制备土壤稀释液→ 倾注平板→ 培养(48h) → 计数 五 注意事项 倒平板时要注意控制培养基的温度。 稀释操作时，要尽量减少样品稀释误差，而且每个稀释度的菌液应各用一支无菌吸管。 为使菌落能在平板上均匀分布，样品液加入平皿后，应尽快倾注培养基并旋转混匀。 六 思考题 要测定某种液体饮料中的活细菌总数，除了采用平板菌落计数法外，还可采用哪些方法？ * * Colony-Forming Units (CFUs) However, keep in mind that if the organism normally forms multiple cell arrangements, such as chains, the colony-forming unit may consist of a chain of bacteria rather than a single bacterium. In addition, some of the bacteria may be clumped together. Therefore, when doing the plate count technique, we generally say we are determining the number of Colony-Forming Units (CFUs) in that known dilution. By extrapolation, this number can in turn be used to calculate the number of CFUs in the original sample. calculate the number of CFUs in the original sample number of CFUs per ml of sample = number of colonies (30-300 plate) X the dilution factor of the plate counted For a more accurate count it is advisable to plate each dilution in duplicate or triplicate and then find an average count.