详情

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、*ATCC 6538、*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*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