細(xì)菌培養(yǎng)方法

細(xì)菌培養(yǎng)方法

1儀器：

K罩細(xì)菌過(guò)濾效率檢測(cè)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(tái)、菌落計(jì)數(shù)器

1. 試劑及材料：

蒸餾水、75%酒精、*ATCC 6538、*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(gè)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(shí)，將液體培養(yǎng)基逐個(gè)倒25ml左右入無(wú)菌培養(yǎng)皿中，操作應(yīng)在超潔凈工作臺(tái)上進(jìn)行，以酒精燈的火焰周圍作為無(wú)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(zhǎng)的培養(yǎng)基舍棄不用，無(wú)雜菌生長(zhǎng)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*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(wú)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(jí)稀釋10倍，稀釋至10^-7(根據(jù)菌種實(shí)際情況來(lái)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(gè)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(shí)間（24±2）h。培養(yǎng)結(jié)束后用菌落計(jì)數(shù)器計(jì)數(shù)，根據(jù)稀釋倍數(shù)確定原始菌液的濃度。

計(jì)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(shí)驗(yàn)結(jié)束后逐級(jí)取出培養(yǎng)皿并蓋上皿蓋，標(biāo)記后倒置放入恒溫培養(yǎng)箱中，37℃培養(yǎng)24-48h.試驗(yàn)中，陽(yáng)性對(duì)照組應(yīng)進(jìn)行至少兩次實(shí)驗(yàn)，終陽(yáng)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(jì)數(shù)器進(jìn)行計(jì)數(shù)，并將每一級(jí)菌落數(shù)對(duì)應(yīng)陽(yáng)性孔轉(zhuǎn)換表進(jìn)行轉(zhuǎn)換，轉(zhuǎn)換后的數(shù)值求和，即得出菌落總數(shù)，陽(yáng)性質(zhì)控值范圍應(yīng)在（2200±500）cfu之間。

細(xì)菌過(guò)濾效率計(jì)算公式如下：

BFE=(C-T)/C*

式中：C為陽(yáng)性質(zhì)控平均值，T為實(shí)驗(yàn)組菌落總和