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 (121 C [ 250 F]), 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 170 C [ 340 F]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.
–_ 0.45 m filters removes most bacteria,
0.20 m more inclusive, viruses need 0.01 m.
–_ Useful for sterilizing liquids (enzymes, vaccines) 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 1.05 kg/ cm2 ,121.3 ℃,20 min灭菌.
灭菌所需时间到后,切断电源,让灭菌锅内温度自然下降,当压力表的压力降至0时,打开排气阀,旋松螺栓,打开盖子,取出灭菌物品.如果压力未降到0时,打开排气阀,就会因锅内压力突然下降,使容器内的培养基由于内外压力不平衡而冲出烧瓶口或试管口,造成棉塞沾染培养基而发生污染.

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