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细菌培养方法

  • 发布日期:2020-08-06      浏览次数:841
    • 细菌培养方法

      1仪器:

      K罩细菌过滤效率检测仪、高压蒸汽灭菌器、电子天平、生化培养箱、轨道式振荡器、超洁净工作台、菌落计数器

      1. 试剂及材料:

      蒸馏水、75%酒精、金黄色葡萄球菌ATCC 6538、胰蛋白酶大豆琼脂(TSA)、胰蛋白胨大豆肉汤培养基(TSB)、蛋白胨水、酒精灯、90mm平皿、500ml锥形瓶

      1. TSA培养基的配制

      取一个干净的500ml的锥形瓶,称取16g TSA,溶于400ml水中,包扎后放入高压蒸汽灭菌器中灭菌(121℃-123℃,20min),灭菌结束后取出,待其冷却至50℃-60℃时,将液体培养基逐个倒25ml左右入无菌培养皿中,操作应在超洁净工作台上进行,以酒精灯的火焰周围作为无菌区域,避免杂菌污染。

      待培养基冷却凝固后,将其倒置放入恒温培养箱中,37℃条件下培养24h,将有菌落生长的培养基舍弃不用,无杂菌生长的取出备用,尽量现用现做,如不能尽快使用,应密封放在4℃冰箱内冷藏,可保存3-4天。                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               

      1. 菌悬液的制备

      将金黄色葡萄球菌ATCC6538接种在已灭菌好的100mlTSB(胰蛋白胨大豆肉汤液体培养基)中,在37℃震荡培养24h。

      用无菌移液枪取出上述培养好的菌液1ml注入9ml一灭菌好的蛋白胨水中,混匀,一次逐级稀释10倍,稀释至10-7(根据菌种实际情况来判断需要稀释至多少),然后分别取10-5、10-6、10-7菌液各0.1ml接种到备TSA培养基上,每个稀释稀释倍数做少4组平行,用涂布棒沿同一方向涂抹均匀,(不要涂抹到培养皿的边缘上),放入生化培养箱中培养,培养温度(37±2)℃,培养时间(24±2)h。培养结束后用菌落计数器计数,根据稀释倍数确定原始菌液的浓度。

      计算公式:原始浓度=(A/V)*D

      A为培养皿上的平均菌落数

      V为接种液的体积

      D为稀释倍数

      按照上述方法确定原始菌液的浓度后,用1.5%的蛋白胨水将上述培养物稀释至约5*105cfu/ml。

      TSB的制备:称取3gTSB, 溶于100ml水中,放入高压蒸汽灭菌器中(121℃-123℃,20min)灭菌,冷却后备用。

      蛋白胨水的配制:称取1.5g蛋白胨溶于100ml水中,包扎,放入高压蒸汽灭菌器中(121℃-123℃,20min)灭菌,冷却后备用。

      实验结束后逐级取出培养皿并盖上皿盖,标记后倒置放入恒温培养箱中,37℃培养24-48h.试验中,阳性对照组应进行至少两次实验,终阳性质控结果取平均值。

      培养结束后,将所有培养皿取出,使用菌落计数器进行计数,并将每一级菌落数对应阳性孔转换表进行转换,转换后的数值求和,即得出菌落总数,阳性质控值范围应在(2200±500)cfu之间。

      细菌过滤效率计算公式如下:

      BFE=(C-T)/C*

      式中:C为阳性质控平均值,T为实验组菌落总和

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