冷库蒸发器棋盘试验优化噬菌体与大肠埃希菌的最佳感染复数 王杰;李璐璐;胡明;骆延波;齐静;刘玉庆;张庆
推进式搅拌桨
【摘 要】目的 利用棋盘法测定噬菌体与大肠埃希菌在不同浓度比条件下的杀菌作用,优化传统的最佳感染复数试验.方法 先利用96孔培养板纵向10倍梯度稀释噬菌体,再利用新的96孔板横向10倍梯度稀释大肠埃希菌,最后用排分别从稀释好的噬菌体孔板和稀释好的菌液孔板中吸取100μL混合液转移到同一个新孔板中混合培养.用96点阵检测板沾取过夜培养的混合液分别接种于LB固体琼脂检测板与LB宿主菌半固体琼脂检测板上,37℃培养14h.结果 受噬菌体密度和宿主菌的数量的限制,多重性感染(multiplicity of infection,MOI)基本上在109～1010PFU/mL范围内波动,这种差异并没有本质的区别.棋盘试验3种方法都一致显示大肠埃希菌浓度低于106CFU/mL,噬菌体效价高于104pFU/mL时,呈现稳定的杀菌效果;固体检测板结果显示在呈现稳定的杀菌效果的区域内外,也有不同生长程度的菌落;半固体检测板均出现噬菌斑,表明两者作用结束后都有噬菌体存留,并生长成为充分的噬菌斑,但MOI测定时效价在一个数量级内有差异.结论 MOI试验着眼于噬菌体裂解固定数量的宿主菌的能力,棋盘试验则是着眼于对宿主菌的裂解杀灭活性;利用96孔液体培养板棋盘法可以很好的确定噬菌体与大肠埃希菌作用有效范围和最低噬菌体使用浓度,可以为杀灭不同浓度宿主大肠埃希菌提供噬菌体效价参考.一般情况下细菌的浓度最高达到109～1010CFU/mL,考虑到实际细菌的感染量不会太高的情况,采用105PFU/mL噬菌体能起到可靠的杀菌作用.%Objective The bactericidal effect of phage and Escherichia coli under the different concentration ratio was determined by using the checkerboard method,and the traditional optimal infection test was optimized.Methods The phage was diluted 10 times with a 96-well culture plate,and then diluted with a new 96-well plate 10 times gradient li.Finally,100μL of the mixture was transferred from the diluted phage plate and the diluted wells to separate the wells in the same new orifice.The mixture was incubated on a LB solid agar plate and a LB host bacteria semi-solid agar test plate respectively with a 96 dot matrix test plate,at 37℃.Results Probably because of being limited by the phage density and the number of host bacteria,multiplicity of infection (MOI) is basically in the range of 109～1010PFU/mL fluctuations.This difference is not significant.The three methods of the board test showed that the li was less than 106CFU/mL,and the titer was higher than 1&PFU/mL.The results of the solid test plate showed that there were colonies with differe
nt growth degrees in the area where the stable bactericidal effect is presented;the phage plaques were found in the semi-solid test plates,indicating that both phages remained and grew into plaque,but the titer was different in an order of magnitude when MOI was measured.Conclusion The MOI trial was focused on the ability of phage lysis to fix a fixed amount of host bacteria,and the chessboard test was focused on the killing activity of the host bacteria;The board test was focused on the killing activity of the host bacteria.The effective range and minimum phage concentration of phage li were determined by the 96-well liquid culture plate method,which could li phage titers for the killing of different li.Under normal circumstances,the concentration of bacteria is up to 109～1010CFU/mL.Given that the actual bacterial infection will not be too high,the use of 105PFU/mL phage can produce a reliable bactericidal effect.
【期刊名称】《中国抗生素杂志》
【年(卷),期】2017(042)009
【总页数】5页(P766-770)
【关键词】大肠埃希菌;噬菌体;棋盘试验;最佳感染复数
【作 者】王杰;李璐璐;胡明;骆延波;齐静;刘玉庆;张庆
【作者单位】山东大学生命科学学院,济南250100;山东省农业科学院畜牧兽医研究所,济南250100;山东省农业科学院畜牧兽医研究所,济南250100;山东省农业科学院畜牧兽医研究所,济南250100;山东省农业科学院畜牧兽医研究所,济南250100;山东省农业科学院畜牧兽医研究所,济南250100;山东省农业科学院畜牧兽医研究所,济南250100;山东省农业科学院畜牧兽医研究所,济南250100
边坡滑模施工【正文语种】中 文
【中图分类】Q939.48;R978.1
Abstract Objective The bactericidal effect of phage and Escherichia coli under the different concentration ratio was determined by using the checkerboard method, and the traditional optimal infection test was optimized.Methods The phage was diluted 10 times with a 96-well culture plate, and then diluted with a new 96-well plate 10 times gradient dil
电动液控闸阀ution of E. coli. Finally, 100μL of the mixture was transferred from the diluted phage plate and the diluted wells to separate the wells in the same new ori fi ce. The mixture was incubated on a LB solid agar plate and a LB host bacteria semi-solid agar test plate respectively with a 96 dot matrix test plate, at 37°C.Results Probably because of being limited by the phage density and the number of host bacteria, multiplicity of infection (MOI) is basically in the range of 109～1010PFU/mL fluctuations. This difference is not significant. The three methods of the board test showed that the concentration of E. coli was less than 106CFU/mL, and the titer was higher than 104PFU/mL. The results of the solid test plate showed that there were colonies with different growth degrees in the area where the stable bactericidal effectis presented; the phage plaques were found in the semi-solid test plates, indicating that both phages remained and grew into plaque, but the titer was different in an order of magnitude when MOI was measured. Conclusion The MOI trial was focused on the ability of phage lysis to fi x a fi xed amount of host bacteria, and the chessboard test was focused on the killing activity of the host bacteria; The board test was focused on the killing activity of the host bacteria. The effective range and minim
um phage concentration of phage and E. coli were determined by the 96-well liquid culture plate method, which could provide E. coli phage titers for the killing of different concentrations of E. coli. Under normal circumstances, the concentration of bacteria is up to 109～1010CFU/mL. Given that the actual bacterial infection will not be too high, the use of 105PFU/mL phage can produce a reliable bactericidal effect.
Key words Escherichia coli; Phage; Chessboard test; Optimal multiplicity of infection
致病性大肠埃希菌导致集约化饲养的家禽较高的发病率和死亡率，给养禽业造成巨大的经济损失[1-3]。采用抗生素和预防导致普遍而严重的抗药性[4]，加之新抗生素研发难度的增加，噬菌体又重新受到了研究人员的关注[5]：针对相应的致病菌具有较强的特异性，却不会破坏正常的菌[6]；有很强地宿主依赖性，会随着宿主菌的清除而死亡，不会被残留在体内和感染动物[7]；复制和筛选方便，成本低。但是噬菌体的方法仍然不够成熟，噬菌体与细菌作用的最佳浓度比和绝对数量是关键参数，通常用最佳感染复数(optimal multiplicity of infection, OMOI)表征，即将处于对数生长期的宿主菌用新鲜的LB液体培养液洗涤并调整为1.0×108CFU/mL，感染复数(multiplicity of infection, MOI)，即噬菌体与宿主
菌数量的比值分别按照0.001、0.01、0.1、1和10等体积混匀，37℃，160r/min震荡培养5h。混合培养物10000r/min，离心1min得到裂解液，测噬菌体效价，产生最高效价的感染复数即为OMOI。这类似药敏试验的经验性作法，当动物体内宿主菌浓度不是108CFU/mL时则不易判断OMOI。本文设计棋盘试验，全面和动态地量化分析噬菌体与细菌作用的浓度之间的关系[8-10]，以便为噬菌体提供依据。
大肠埃希菌YSF(实验室保存)、大肠埃希菌噬菌体vB_EcolS_YSF(实验室保存)、固体LB培养基、半固体LB培养基、液体LB培养基、麦康凯固体培养基、MHB培养基、SM缓冲液(20mm Tris-HCL, 10mm MgSO4, 10mm CaCl2, 100mm NaCl, pH7.5)、分光光度计、0.22μm滤器，排、96孔板，琼脂检测板等。
噬菌体vB_EcolS_YSF的效价可达到109PFU/mL，在60℃保温1h后噬菌体效价仍可达到107PFU/mL以上，最适生长温度在37℃左右，在pH值4～11内仍能保持较高的裂解活性，最适pH值在7.0左右；噬菌体的最佳感染复数一般为0.1～1，感染宿主菌的潜伏期一般在在20min左右。
将保存的大肠埃希菌YSF划线麦接种康凯固体培养基，37℃培养14h。然后挑取麦康凯板
上的玫红单菌落划线固体LB培养基，37℃培养14h。挑取固体LB培养基上的单菌落于液体LB中37℃摇菌8h，培养至对数生长期，经过106、107、108和109倍稀释后，分别涂LB固体平板(每个浓度涂布3个平板)，37℃静置培养14h，菌落计数。重复培养，将原菌液稀释到109CFU/mL备用。
内胎硫化机保存好的甘油vB_EcolS_YSF用SM缓冲液作10倍梯度稀释，取稀释液100μL与宿主YSF菌液100μL混合均匀，加入到4mL融化的半固体0.6%LB培养基中，充分混匀后立即倒入固体LB平板上，旋转平皿使混合液分布均匀，平置凝固后，放入37℃恒温箱培养4～6h。挑取单一的噬菌斑于500μL的SM缓冲液中，4℃静置2～4h，使噬菌体从半固体培养基中释放入SM缓冲液中。
取300μL的YSF菌液与100μL的vB_EcolS_YSF储存液加于300mL液体LB培养基中37℃，200r/min摇14h，然后将混合液12000r/min离心10min，用0.22μm微孔滤膜过滤除菌上清液。
将过滤好的vB_EcolS_YSF上清液依次进行10倍稀释，用SM缓冲液10-1～10-7梯度稀释上清液，取100μL稀释液加900μL SM缓冲液，振荡混匀后加入100μL对数期的菌悬液，混匀，
电容耐压测试仪加入45℃温浴的4mL半固体LB培养基中，混匀，倒入固体LB平板上，平置凝固后37℃恒温培养4～6h，观察噬菌斑的生长情况，每个稀释度做3组重复，计数噬菌斑个数，计算噬菌体效价。
噬菌体效价(PFU/mL)=平均噬菌斑数×稀释倍数×10
将噬菌体样液稀释到109PFU/mL备用。
将处于对数生长期的宿主菌用新鲜的LB液体培养液洗涤并调整为1.0×108CFU/mL，噬菌体与宿主菌数量的比值分别按照0.001、0.01、0.1、1和10等体积1mL混匀，37℃，160r/min震荡培养5h。混合培养物10000r/min，离心1min得到裂解液，每个MOI值做3组重复，测噬菌体效价，产生最高效价的感染复数即为OMIOI。
此实验使用96孔板，采用10倍稀释的方法，分别对噬菌体和细菌进行稀释。在96孔板中依次横向10倍梯度稀释大肠埃希菌YSF至8个梯度。纵向10倍梯度稀释噬菌体vB_EcolS_YSF至8个梯度。最后4列分别为只有大肠埃希菌YSF的对照和只有噬菌体vB_EcolS_YSF不同梯度的对照。
先利用一个96孔板横向10倍梯度稀释大肠埃希菌YSF，稀释8个梯度，再利用一个新的96板孔纵向10倍梯度稀释噬菌体vB_EcolS_YSF，稀释8个梯度，最后用排分别从稀释好的菌液YSF孔板和稀释好的噬菌体vB_EcolS_YSF孔板中吸取100μL混合液转移到同一个新孔板中，使得每个孔中各有100μL的菌液和100μL的噬菌体，混合均匀，向第9列与第10列孔板中加入200μL的菌液，向第11列与第12列孔板中加入200μL的噬菌体样液，37℃培养14h，每次做2组重复。
用96点阵板将过夜培养后的混合液沾取到有菌的半固体LB检测板与无菌的固体LB检测板上，37℃培养14h。做2组重复，观察噬菌体斑与菌落的生长情况。
过夜振荡培养的菌液经菌落计数为2.2×1010CFU/mL，如图1所示。原菌液稀释10倍后至109CFU/mL备用。
稀释了106倍的噬菌体板有噬菌斑出现如图2所示，分别为325、345和321个。平均为330个噬菌斑，则此噬菌体富集液的效价为：330×106×10=3.3×109PFU/mL。
