細菌培養(yǎng)方法

1儀器：

K罩細菌過濾效率檢測儀,、高壓蒸汽滅菌器,、電子天平、生化培養(yǎng)箱,、軌道式振蕩器,、超潔凈工作臺、菌落計數(shù)器

1. 試劑及材料：

蒸餾水,、75%酒精,、金黃色葡萄球菌ATCC 6538、胰蛋白酶大豆瓊脂（TSA),、胰蛋白胨大豆肉湯培養(yǎng)基（TSB）,、蛋白胨水、酒精燈,、90mm平皿,、500ml錐形瓶

1. TSA培養(yǎng)基的配制：

取一個干凈的500ml的錐形瓶，稱取16g TSA,溶于400ml水中,，包扎后放入高壓蒸汽滅菌器中滅菌（121℃-123℃,，20min），滅菌結(jié)束后取出，待其冷卻至50℃-60℃時,，將液體培養(yǎng)基逐個倒25ml左右入無菌培養(yǎng)皿中,，操作應(yīng)在超潔凈工作臺上進行，以酒精燈的火焰周圍作為無菌區(qū)域,，避免雜菌污染,。

待培養(yǎng)基冷卻凝固后，將其倒置放入恒溫培養(yǎng)箱中,，37℃條件下培養(yǎng)24h，將有菌落生長的培養(yǎng)基舍棄不用,，無雜菌生長的取出備用,，盡量現(xiàn)用現(xiàn)做，如不能盡快使用,，應(yīng)密封放在4℃冰箱內(nèi)冷藏,，可保存3-4天。                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               

1. 菌懸液的制備：

將金黃色葡萄球菌ATCC6538接種在已滅菌好的100mlTSB(胰蛋白胨大豆肉湯液體培養(yǎng)基）中,，在37℃震蕩培養(yǎng)24h,。

用無菌移液槍取出上述培養(yǎng)好的菌液1ml注入9ml一滅菌好的蛋白胨水中，混勻,，一次逐級稀釋10倍,，稀釋至10^-7(根據(jù)菌種實際情況來判斷需要稀釋至多少），然后分別取10^-5,、10^-6,、10^-7菌液各0.1ml接種到備TSA培養(yǎng)基上，每個稀釋稀釋倍數(shù)做少4組平行,，用涂布棒沿同一方向涂抹均勻,，（不要涂抹到培養(yǎng)皿的邊緣上），放入生化培養(yǎng)箱中培養(yǎng),，培養(yǎng)溫度(37±2)℃,培養(yǎng)時間（24±2）h,。培養(yǎng)結(jié)束后用菌落計數(shù)器計數(shù)，根據(jù)稀釋倍數(shù)確定原始菌液的濃度,。

計算公式：原始濃度=（A/V)*D

A為培養(yǎng)皿上的平均菌落數(shù)

V為接種液的體積

D為稀釋倍數(shù)

按照上述方法確定原始菌液的濃度后,，用1.5%的蛋白胨水將上述培養(yǎng)物稀釋至約5*10⁵cfu/ml。

TSB的制備：稱取3gTSB, 溶于100ml水中,，放入高壓蒸汽滅菌器中（121℃-123℃,，20min）滅菌，冷卻后備用,。

蛋白胨水的配制：稱取1.5g蛋白胨溶于100ml水中,，包扎，放入高壓蒸汽滅菌器中（121℃-123℃,，20min）滅菌,，冷卻后備用,。

實驗結(jié)束后逐級取出培養(yǎng)皿并蓋上皿蓋，標記后倒置放入恒溫培養(yǎng)箱中,，37℃培養(yǎng)24-48h.試驗中,，陽性對照組應(yīng)進行至少兩次實驗，終陽性質(zhì)控結(jié)果取平均值,。

培養(yǎng)結(jié)束后,，將所有培養(yǎng)皿取出，使用菌落計數(shù)器進行計數(shù),，并將每一級菌落數(shù)對應(yīng)陽性孔轉(zhuǎn)換表進行轉(zhuǎn)換,，轉(zhuǎn)換后的數(shù)值求和，即得出菌落總數(shù),，陽性質(zhì)控值范圍應(yīng)在（2200±500）cfu之間,。

細菌過濾效率計算公式如下：

BFE=(C-T)/C*

式中：C為陽性質(zhì)控平均值，T為實驗組菌落總和