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細(xì)菌培養(yǎng)方法

  • 發(fā)布日期:2020-08-06      瀏覽次數(shù):3356
    • 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、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(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(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

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