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蘇州守真儀器設備有限公司>技術文章>細菌培養方法

技術文章

細菌培養方法

閱讀:4794          發布時間:2020-8-6

細菌培養方法

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、生化培養箱、軌道式振蕩器、超潔凈工作臺、菌落計數器

  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基(TSB)、蛋白胨水、酒精燈、90mm平皿、500ml錐形瓶

  1. TSA培養基的配制

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),滅菌結束后取出,待其冷卻至50℃-60℃時,將液體培養基逐個倒25ml左右入無菌培養皿中,操作應在超潔凈工作臺上進行,以酒精燈的火焰周圍作為無菌區域,避免雜菌污染。

待培養基冷卻凝固后,將其倒置放入恒溫培養箱中,37℃條件下培養24h,將有菌落生長的培養基舍棄不用,無雜菌生長的取出備用,盡量現用現做,如不能盡快使用,應密封放在4℃冰箱內冷藏,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基)中,在37℃震蕩培養24h。

用無菌移液槍取出上述培養好的菌液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培養基上,每個稀釋稀釋倍數做少4組平行,用涂布棒沿同一方向涂抹均勻,(不要涂抹到培養皿的邊緣上),放入生化培養箱中培養,培養溫度(37±2)℃,培養時間(24±2)h。培養結束后用菌落計數器計數,根據稀釋倍數確定原始菌液的濃度。

計算公式:原始濃度=(A/V)*D

A為培養皿上的平均菌落數

V為接種液的體積

D為稀釋倍數

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。

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

培養結束后,將所有培養皿取出,使用菌落計數器進行計數,并將每一級菌落數對應陽性孔轉換表進行轉換,轉換后的數值求和,即得出菌落總數,陽性質控值范圍應在(2200±500)cfu之間。

細菌過濾效率計算公式如下:

BFE=(C-T)/C*

式中:C為陽性質控平均值,T為實驗組菌落總和

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