文档介绍：实验一培养基的配制与灭菌
一教学要求
通过配制牛肉膏蛋白胨等培养基,掌握配制培养基的一般方法和步骤。
了解采用的灭菌方法和原理,掌握高压蒸汽灭菌技术。
二实验原理
1、Media Types
Solid Media,semi-solid Media, liquid Media .
Enriched Media, Selective Media, Differential Media, combination selective and differential media
2 Sterilization – destruction of all forms of microbial life, including endospores.
Physical Methods
—Moist heat
Autoclaving
•   Action - protein denaturation.
•   Very effective method of sterilization; at about 15 psi(Ib/in2 ) of pressure (121ºC [ 250ºF]), all vegetative cells and their endospores are killed in about 15 min.
—Dry heat
Direct flaming
•   example: Inoculation loop
Hot-air sterilization
•   Baking in oven
•   Very effective method of sterilization, but requires temperature of 170ºC [ 340 ºF]for about 2 hr.
—Filtration
–  Separation of bacteria from suspending liquid.
–  Removes microbes by passage of a liquid or gas through a screen like material. Most filters consist of diatomaceous earth, cellulose acetate or nitrocellulose.
–  m filters removes most bacteria,
m more inclusive, viruses need m.
–  Useful for sterilizing liquids (enzymes, ines) that are destroyed by heat.
三实验材料与器材
培养基:牛肉膏、蛋白胨、琼脂、可溶性淀粉、葡萄糖、孟加拉红、链霉素、1 mol/L NaOH、 1 mol/L HCl、KNO3 、K2HPO4·3H2O MgSO4·7H2O FeSO4·7H2O.
器材:玻璃器皿、天平、pH试纸、灭菌锅等
四操作步骤
称量→溶解→调pH *→(过滤*)→分装*→
加塞*→包扎→高压蒸汽灭菌
高压蒸汽灭菌灭菌操作步骤
首先将内层灭菌桶取出,再向外层锅内加入适量的水,使水面与三角搁架相平为宜。
放回灭菌桶,并装入待灭菌物品。注意不要装得太挤,以免防碍蒸汽流通而影响灭菌效果。三角烧瓶与试管口端均不要与桶壁接触,以免冷凝水淋湿包口的纸而透入棉塞。
加盖,并将盖上的排气软管插入内层灭菌桶的排气槽内。再以两两对称的方式同时旋紧相对的两个螺栓,使螺栓松紧一致,勿使漏气。
开启电源加热,并同时打开排气阀,使水沸腾以排除锅内的冷空气。待冷空气完全排尽后,关上排气阀,让锅内的温度随蒸汽压力增加而逐渐上升。当锅内压力升到所需压力时,控制热源,维持压力至所需时间。本实验用 kg/ cm2 , ℃,20 min灭菌。