細(xì)菌培養(yǎng)方法

細(xì)菌培養(yǎng)方法

1儀器：

K罩細(xì)菌過濾效率檢測儀、高壓蒸汽滅菌器、電子天平、生化培養(yǎng)箱相互配合、軌道式振蕩器質量、超潔凈工作臺迎難而上、菌落計數(shù)器

1. 試劑及材料：

蒸餾水結構、75%酒精相結合、金黃色葡萄球菌ATCC 6538攜手共進、胰蛋白酶大豆瓊脂（TSA)實力增強、胰蛋白胨大豆肉湯培養(yǎng)基（TSB）、蛋白胨水擴大公共數據、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的錐形瓶設計標準，稱取16g TSA,溶于400ml水中深度，包扎后放入高壓蒸汽滅菌器中滅菌（121℃-123℃，20min）經過，滅菌結(jié)束后取出帶來全新智能，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)皿中核心技術體系，操作應(yīng)在超潔凈工作臺上進(jìn)行自主研發，以酒精燈的火焰周圍作為無菌區(qū)域力度，避免雜菌污染。

待培養(yǎng)基冷卻凝固后預判，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)基舍棄不用調解製度，無雜菌生長的取出備用聽得懂，盡量現(xiàn)用現(xiàn)做，如不能盡快使用協調機製，應(yīng)密封放在4℃冰箱內(nèi)冷藏，可保存3-4天有效性。                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                                               

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）中高質量發展，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形勢，混勻攻堅克難，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）高效節能，然后分別取10^-5相關、10^-6、10^-7菌液各0.1ml接種到備TSA培養(yǎng)基上基地，每個稀釋稀釋倍數(shù)做少4組平行影響力範圍，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）約定管轄，放入生化培養(yǎng)箱中培養(yǎng)雙向互動，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)新創新即將到來，根據(jù)稀釋倍數(shù)確定原始菌液的濃度生產效率。

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設計能力。

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

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

實驗結(jié)束后逐級取出培養(yǎng)皿并蓋上皿蓋系統穩定性，標(biāo)記后倒置放入恒溫培養(yǎng)箱中背景下，37℃培養(yǎng)24-48h.試驗中，陽性對照組應(yīng)進(jìn)行至少兩次實驗科技實力，終陽性質(zhì)控結(jié)果取平均值開展試點。

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

細(xì)菌過濾效率計算公式如下：

BFE=(C-T)/C*

式中：C為陽性質(zhì)控平均值在此基礎上，T為實驗組菌落總和