黄山学院生物实验步骤

黄山学院生物实验步骤 黄山学院生命与环境学院 实验1: 显微镜的使用及细菌形态的观察(3学时) 目的要求 (1) 熟悉普通光学显微镜的构造及各部分的功能。 (2) 学习并掌握油镜的原理和使用方法。 1. 基本原理 显微镜由机械装置和光学系统两大部分组成。 , 油镜物镜的基本原理 微生物学研究用的显微镜的物镜通常有低倍物镜(16mm，10×)、高倍物镜(4mm，40—45×)和油镜(1(8 mm，95—100×)三种。油镜通常标有黑圈或红圈，也有的以“OI字样表示，它是三者中放大倍数最大的。根据使用不同放大倍数的目镜，可使被检物体放大1000—2 000多倍。油镜的焦距和工作距离(标本在焦点上看得最清晰时，物镜与样品之间的距离)最短，光圈则开得最大，因此，在使用油镜观察时，镜头离标本十分近，需特别小心。 使用时，油镜与其他物镜的不同是载玻片与物镜之间不是隔一层空气，而是隔一层油质，称为油浸系。这种油常选用香柏油，因香柏油的折射率n=1(52，与玻璃相同。当光线通过载玻片后，可直接通过香柏油进入物镜而不发生折射。如果玻片与物镜之间的介质为空气，则称为干燥系，当光线通过玻片后，受到折射发生散射现象，进入物镜的光线显然减少，这样就减低了视野的照明度。 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 利用油镜不但能增加照明度，更主要的是能增加数值孔径，因为显微镜的放大效能是由其数值孔径决定的。所谓数值孔径，即光线投射到物镜上的最大角度(称为镜口角)的一半正弦，乘上玻片与物镜间介质的折射率所得的乘积，可用下列公式表示: NA,n?sinα 式中 NA=数值孔径; n=介质折射率; α=最大入射角的半数，即镜口角的半数。 因此，光线投射到物镜的角度愈大，显微镜的效能就愈大(图?-5)，该角度的大小决定于物镜的直径和焦距。同时，α的理论限度为90?，sin90?=1，故以空气为介质时(n=1)，数值孔径不能超过1，如以香柏油为介质时，则n增大，其数值孔径也随之增大。如光线入射角为120?，其半数的正弦为sin60?=0(87，则: 以空气为介质时: NA=1×0(87=0(87 以水为介质时: NA=1(33×0(87=1(15 以香柏油为介质时: NA=1(52×0(87=1(32 显微镜的分辨力是指显微镜能够辨别两点之间最小距离的能力。它与物镜的数值孔径成正比，与光波长度成反比。因此，物镜的数值孔径愈大，光波波长越短，则显微镜的分辨力愈大，被检物体的细微结构也愈能明晰地区别出来。因此，一个高的分辨力意味着一个小的可分辨距离，这两个因素是成反比关系的，通常有人把分辨力说成是多少微米或纳米，这实际上是把分辨力和最小分辨距离混淆起来了。显微镜的分辨力是用可分辨的最小距离来表示的。 式中λ=光波波长。 我们肉眼所能感受的光波平均长度为0(55μm，假如数值孔径为0(65的高倍物镜，它能辨别两点之间的距离为0(42μm。而在0(42μm以下的两点之间的距离就分辨不出，即使用倍数更大的目镜，使显微镜的总放大率增加，也仍然分辨不出。只有改用数值孔径更大的物镜，增加其分辨力才行。例如用数值孔径为1(25的油镜时，能辨别两点之间的 因此，我们可以看出，假如采用放大率为40倍的高倍物镜(NA=0(65)，和放大率为24倍的目镜，虽然总放大率为960倍，但其分辨的最小距离只有0(42μm。假如采用放大率为90倍的油镜(NA=1(25)，和放大率为9倍的目镜，虽然总的放大率为810倍，但却能分辨出0(22μm间的距离。 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 2. 材料及器材 显微镜、擦镜纸、二甲苯、香柏油、各种细菌微生物制片标本、吸水纸、纱布、瓷盘、酒精灯、接种工具、打火机、试剂瓶、牙签等。 3. 方法与步骤 (1) 观察前的准备 A. 置显微镜于平稳的实验台上，镜座距实验台边沿约3—4cm。镜检者姿势要端正，一般用左眼观察，右眼便于绘图或记录，两眼必须同时睁开，以减少疲劳，亦可练习左右眼均能观察。 B. 调节光源，对光时应避免直射光源，因直射光源影响物像的清晰，损坏光源装置和镜头，并刺激眼睛。如阴暗天气，可用日光灯或显微镜灯照明。 调节光源时，先将光圈完全开放，升高聚光镜至与载物台同样高，否则使用油镜时光线较暗。然后转下低倍镜观察光源强弱，调节反光镜，光线较强的天然光源宜用平面镜;光线较弱的天然光源或人工光源宜用凹面镜。在对光时，要使全视野内为均匀的明亮度。凡检查染色标本时，光线应强;检查未染色标本时，光线不宜太强。可通过扩大或缩小光圈、升降聚光器、旋转反光镜调节光线。 (2) 低倍镜观察 找目的物 (3) 高倍镜观察 将高倍镜转至正下方，在转换物镜时，需用眼睛在侧面观察，避免镜头与玻片相撞。然后由目镜观察，并仔细调节光圈，使光线的明亮度适宜，同时用粗调节器慢慢升起镜筒至物像出现后，再用细调节器调节至物像清晰为止，找到最适宜观察的部位后，将此部位移至视野中心，准备用油镜观察。 (4) 油镜观察 A. 用粗调节器将镜筒提起约2cm，将油镜转至正下方。 B. 在玻片标本的镜检部位滴上一滴香柏油。 C. 从侧面注视，用粗调节器将镜筒小心地降下，使油镜浸在香柏油中，其镜头几乎与标本相接，应特别注意不能压在标本上，更不可用力过猛，否则不仅压碎玻片，也会损坏镜头。 D. 从接目镜内观察，进一步调节光线，使光线明亮，再用粗调节器将镜筒徐徐上升，直至视野出现物像为止，然后用细调节器校正焦距。如油镜已离开油面而仍未见物像，必须再从侧面观察，将油镜降下，重复操作至物像看清为止。 E. 用同样的方法观察枯草芽孢杆菌染色标本。 F. 观察完毕，上旋镜筒。先用擦镜纸拭去镜头上的油，然后用擦镜纸蘸少许二甲苯(香柏油溶于二甲苯)擦去镜头上残留油迹，最后再用干净擦镜纸擦去残留的二甲苯。切忌用手或其他纸擦镜头，以免损坏镜头。用绸布擦净显微镜的金属部件。 G. 将各部分还原，反光镜垂直于镜座，将接物镜转成八字形，再向下旋。同时把聚光镜降下，以免接物镜与聚光镜发生碰撞危险。 4. 实验作业 (1) 分别绘出你在低倍镜、高倍镜和油镜下观察到的金黄色葡萄球菌及枯草芽孢杆菌的状态，包括在三种情况下视野中的变化，同时注明物镜放大倍数和总放大率。 (2) 在使用高倍镜和油镜进行调焦时，应将镜筒徐徐上升还是下降,为什么, (3) 用油镜观察时，为什么要在载玻片上滴加香柏油, bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 实验2: 细菌的单染色与革兰氏染色(3学时) 1. 目的要求 (1) 学习微生物涂片、染色的基本技术，掌握细菌的单染色方法及革兰氏染色方法。 (2) 巩固显微镜的使用方法。 2. 基本原理 所谓单染色法是利用单一染料对细菌进行染色的一种方法。此法操作简便，适用于菌体一般形态的观察。 在中性、碱性或弱酸性溶液中，细菌细胞通常带负电荷，所以常用碱性染料进行染色。碱性染料并不是碱，和其他染料一样是一种盐，电离时染料离子带正电，易与带负电荷的细菌结合而使细菌着色。例如，美蓝(亚甲蓝)实际上是氯化亚甲蓝盐(缩写为MBC)，它可被电离成正、负离子。 带正电荷的染料离子可使细菌细胞染成蓝色。常用的碱性染料除美蓝外，还有结晶紫、碱性复红、番红(又称沙黄)等。 细菌体积小，较透明，如未经染色常不易识别，而经着色后，与背景形成鲜明的对比，使易于在显微镜下进行观察。 革兰氏染色反应是细菌分类和鉴定的重要性状。它是1884年由丹麦医师Gram创立的。革兰氏染色法不仅能观察到细菌的形态而且还可将所有细菌区分为两大类:染色反应呈蓝紫色的称为革兰氏阳性细菌，用G+表示;染色反应呈红色(复染颜色)的称为革兰氏阴性细菌，用G-表示。细菌对于革兰氏染色的不同反应，是由于它们细胞壁的成分和结构不同而造成的。革兰氏阳性细菌的细胞壁主要是由肽聚糖形成的网状结构组成的，在染色过程中，当用乙醇处理时，由于脱水而引起网状结构中的孔径变小，通透性降低，使结晶紫-碘复合物被保留在细胞内而不易脱色，因此，呈现蓝紫色;革兰氏阴性细菌的细胞壁中肽聚糖含量低，而脂类物质含量高，当用乙醇处理时，脂类物质溶解，细胞壁的通透性增加，使结晶紫-碘复合物易被乙醇抽出而脱色，然后又被染上了复染液(番红)的颜色，因此呈现红色。 3、材料及器材 (1) 载玻片，接种环，双层瓶，生理盐水，嗜碱性美蓝染色液，石炭酸复红染色液;显微镜、擦镜纸、二甲苯、香柏油、吸水纸、纱布、瓷盘、酒精灯、接种工具、打火机、试剂瓶、牙签等、培养箱、灭菌锅、革兰氏染色液。 (2) 白萄球菌，枯草芽孢杆菌菌种、大肠杆菌菌种、金黄色葡萄球菌菌种、乳酸菌种 4 方法与步骤 A、单染色法 (1)涂片 取两块干净的载玻片，各滴一小滴生理盐水于载玻片中央，用无菌操作，分别挑取白萄球菌和枯草芽孢杆菌于二载玻片的水滴中(每一种菌制一片)，调匀并涂成薄膜。注意滴生理盐水时不宜过多，涂片必须均匀。 (2)干燥 于室温中自然干燥。 (3)固定 涂片面向上，于火焰上通过2—3次，使细胞质凝固，以固定细菌的形态，并使其不易脱落。但不能在火焰上烤，否则细菌形态将毁坏。 (4) 染色 放标本于水平位置，滴加染色液于涂片薄膜上，染色时间长短随不同染色液而定。吕氏碱性美蓝染色液约染2—3分钟，石炭酸复红染色液约染1—2分钟。 (5) 水洗 染色时间到后，用自来水冲洗，直至冲下之水无色时为止。注意冲洗水流不宜过急，过大，水由玻片上端流下，避免直接冲在涂片处。冲洗后，将标本晾干或用吹风bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 机吹干，待完全干燥后才可置油镜下观察。 (6) 镜检 按实验一作程序进行显微镜观察。 B、革兰氏染色 (1) 涂片将培养14—16小时的枯草芽孢杆菌和培养24小时的大肠杆菌分别作涂片(注意涂片切不可过于浓厚)，干燥、固定。固定时通过火焰1—2次即可，不可过热，以载玻片不烫手为宜。 (2) 初染 加草酸铵结晶紫一滴，约一分钟，水洗。… (3) 媒染 滴加碘液冲去残水，并覆盖约一分钟，水洗。… (4) 脱色 将载玻片上面的水甩净，并衬以白背景，用95,酒精滴洗至流出酒精刚刚不出现紫色时为止，约20—30秒钟，立即用水冲净酒精。 (5) 复染 用番红液染1—2分钟，水洗。 (6) 镜检 干燥后，置油镜观察。革兰氏阴性菌呈红色，革兰氏阳性菌呈紫色。以分散开的细菌的革兰氏染色反应为准，过于密集的细菌，常常呈假阳性。 (7) 同法在一载玻片上以大肠杆菌与枯草芽孢杆菌混合制片，作革兰氏染色对比。 革兰氏染色的关键在于严格掌握酒精脱色程度，如脱色过度，则阳性菌可被误染为阴性菌;而脱色不够时，阴性菌可被误染为阳性菌。此外，菌龄也影响染色结果，如阳性菌培养时间过长，或已死亡及部分菌自行溶解了，都常呈阴性反应。 5、实验作业 (1) 在你所作的革兰氏染色制片中，大肠杆菌和枯草芽孢杆菌各染成何色,它们是革兰氏阴性菌还是革兰氏阳性菌, (2) 作革兰氏染色涂片为什么不能过于浓厚,其染色成败的关键一步是什么, (3) 当你对一株未知菌进行革兰氏染色时，怎样能确证你的染色技术操作正确，结 果可靠, (4) 绘图 实验3:细菌芽孢染色及鞭毛的观察(3学时) 1. 目的要求 学习并掌握芽孢染色法。 观察细菌的鞭毛 2. 基本原理 芽孢染色法是利用细菌的芽孢和菌体对染料的亲合力不同的原理，用不同染料进行着色，使芽孢和菌体呈不同的颜色而便于区别。芽孢壁厚、透性低，着色、脱色均较困难，因此，当先用一弱碱性染料，如孔雀绿或碱性品红在加热条件下进行染色时，此染料不仅可以进入菌体，而且也可以进入芽孢，进入菌体的染料可经水洗脱色，而进入芽孢的染料则难以透出，若再用复染液(如番红液)或衬托溶液(如黑色素溶液)处理，则菌体和芽孢易于区分。 3. 材料及器材 (1) 枯草芽孢杆菌，蜡状芽孢杆菌，苏云金芽孢杆菌、鞭毛菌装片 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary (2) 孔雀绿染液，番红水溶液，苯酚品红溶液，黑色素溶液等。载玻片，接种环，双层瓶，生理盐水，嗜碱性美蓝染色液，石炭酸复红染色液;显微镜、擦镜纸、二甲苯、香柏油、吸水纸、纱布、瓷盘、酒精灯、接种工具、打火机、试剂瓶、牙签等、培养箱、灭菌锅、革兰氏染色液。 4. 方法与步骤 方法1、 (1) 将培养24小时左右的枯草芽孢杆菌或其他芽孢杆菌，作涂片、干燥、固定。 (2) 滴加3—5滴孔雀绿染液于已固定的涂片上。 (3) 用木夹夹住载玻片在火焰上加热，使染液冒蒸汽但勿沸腾，切忌使染液蒸干，必要时可添加少许染液。加热时间从染液冒蒸汽时开始计算约4—5分钟。这一步也可不加热，改用饱和的孔雀绿水溶液(约7(6,)染10分钟。 (4) 倾去染液，待玻片冷却后水洗至孔雀绿不再褪色为止。 (5) 用番红水溶液复染1分钟，水洗。 (6) 待干燥后，置油镜观察，芽孢呈绿色，菌体呈红色。 观察细菌的鞭毛装片。 5. 实验作业 结果 绘图表示你观察到的两种芽孢杆菌的芽孢在形状、大小、着生位置上有什么不同, 绘鞭毛菌图 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 实验4: 酵母菌的形态观察及显微计数(3学时) 1. 目的要求 (1) 观察酵母菌的细胞形态及出芽生殖方式。 (2) 学习掌握区分酵母菌死、活细胞的染色方法。 (3)学习微生物细胞计数的方法 2. 基本原理 酵母菌是多形的、不运动的单细胞微生物，细胞核与细胞质已有明显的分化，菌体比细菌大。繁殖方式也较复杂，无性繁殖主要是出芽生殖，仅裂殖酵母属是以分裂方式繁殖;有性繁殖是通过接合产生子囊孢子。本实验通过用美蓝染色制成水浸片，和水-碘水浸片来观察生活的酵母形态和出芽生殖方式。美蓝是一种无毒性染料，它的氧化型是蓝色的，而还原型是无色的，用它来对酵母的活细胞进行染色，由于细胞中新陈代谢的作用，使细胞内具有较强的还原能力，能使美蓝从蓝色的氧化型变为无色的还原型，所以酵母的活细胞无色，而对于死细胞或代谢缓慢的老细胞，则因它们无此还原能力或还原能力极弱，而被美蓝染成蓝色或淡蓝色。因此，用美蓝水浸片不仅可观察酵母的形态，还可以区分死、活细胞。但美蓝的浓度、作用时间等均有影响，应加注意。 通过计数已知微小容积内细菌，可换算成每ml含菌量。， 利用血球计数板在显微镜下直接计数，是一种常用的微生物计数方法。此法的优点是直观、快速。将经过适当稀释的菌悬液(或孢子悬液)放在血球计数板载玻片与盖玻片之间的计数室中，在显微镜下进行计数。由于计数室的容积是一定的( 0(1mm2)，所以可以根据在显微镜下观察到的微生物数目来换算成单位体积内的微生物总数目。由于此法计得的是活菌体和死菌体的总和，故又称为总菌计数法。 血球计数板，通常是一块特制的载玻片，其上由四条槽构成三个平台。每个方格网共分九个大方格，中间的大方格即为计数室， 计数室的刻度一般有两种规格， 16小方格×25中方格=400小方格;25小方格×16中方格=400小方格; 每一个大方格边长为1mm，则每一大方格的面积为1mm2，盖上盖玻片后，载玻片与盖玻片之间的高度为0(1mm，所以计数室的容积为0(1mm3。 在计数时，通常数五个中方格的总菌数，然后求得每个中方格的平均值，再乘上16或25，就得出一个大方格中的总菌数，然后再换算成1ml菌液中的总菌数。 下面以一个大方格有25个中方格的计数板为例进行计算:设五个中方格中总菌数为A，菌液稀释倍数为B，那么，一个大方格中的总菌数 因1ml=1cm3=1000mm3， =50000A?B(个) bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 3、材料及器材 (1) 酿酒酵母或卡尔酵母、干酵母 (2) 0(05,、0(1,吕氏碱性美蓝染液，革兰氏染色用的碘液、血球计数板 (3) 显微镜，载玻片，接种环，双层瓶，生理盐水，、擦镜纸、二甲苯、香柏油、吸水纸、纱布、瓷盘、酒精灯、接种工具、打火机、试剂瓶、牙签等、培养箱、灭菌锅、冰箱、超净工作台、盖玻片等 4、方法与步骤 (1) 在载玻片中央加一滴0(2,吕氏碱性美蓝染液;再滴一滴酵母菌液，盖盖玻片; (2) 将制好的水浸片放置3分钟后镜检。先用低倍镜观察，然后换用高倍镜观察酿酒酵母的形态和出芽情况，同时可以根据是否染上颜色来区别死、活细胞。 (3) 染色半小时后，再观察一下死细胞数是否增加。 (4) 用0(05,吕氏碱性美蓝染液重复上述的操作。 酵母菌的计数: 洁净计数板 加盖玻片 滴加菌液 低倍镜找计数室 高倍镜计数 5、实验作业 (1) 绘图说明你所观察到的酵母菌的形态特征。 (2) 说明观察到的吕氏碱性美蓝染液浓度和作用时间对死活细胞数的影响。 实验5: 放线菌、霉菌的形态观察(3学时) 1. 目的要求 掌握观察放线菌、霉菌形态的基本方法，并观察其形态特征。 2. 基本原理 霉菌菌丝较粗大，细胞易收缩变形，而且孢子很容易飞散，所以制标本时常用乳酸石炭酸棉bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 蓝染色液。此染色液制成的霉菌标本片其特点是:(a)细胞不变形;(b)具有杀菌防腐作用，且不易干燥，能保持较长时间;(c)溶液本身呈蓝色，有一定染色效果。 霉菌自然生长状态下的形态，常用载玻片观察，此法是接种霉菌孢子于载玻片上的适宜培养基上，培养后用显微镜观察。此外，为了得到清晰、完整、保持自然状态的霉菌形态还可利用玻璃纸透析培养法进行观察。此法是利用玻璃纸的半透膜特性及透光性，将霉菌生长在覆盖于琼脂培养基表面的玻璃纸上，然后将长菌的玻璃纸剪取一小片，贴放在载玻片上用显微镜观察。 放线菌的插片培养法:放线菌的菌丝沿盖玻片生长，可清楚观察菌丝呈管状，孢子丝的形态 • • • 3、材料及器材 (1) 曲霉，青霉，根霉，毛霉、5406放线菌培养物及装片 (2) 乳酸石炭酸棉蓝染色液，查氏培养基平板，马铃薯培养基;显微镜，载玻片，接种环，双层瓶，生理盐水，、擦镜纸、二甲苯、香柏油、吸水纸、纱布、瓷盘、酒精灯、接种工具、打火机、试剂瓶、牙签等、培养箱、灭菌锅、冰箱、超净工作台、盖玻片等 4、方法与步骤 放线菌的观察: 在载玻片上滴一滴香柏油-----------抽出培养好的盖玻片----------檫净背面---------放在玻片上-----------低倍镜下找一条白白的粉线-------------------高倍镜下观察。 在高倍镜下观察放线菌装片。 霉菌的观察 于洁净载玻片上，滴一滴乳酸石炭酸棉蓝染色液-------用解剖针从霉菌菌落的边缘处取小量带有孢子的菌丝置染色液中，再细心地将菌丝挑散开------小心地盖上盖玻片，注意不要产生气泡------------置显微镜下先用低倍镜观察，必要时再换高倍镜。 在低倍镜或高倍镜下观察霉菌装片。 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 5、 实验作业 (1) 绘图说明你所观察到的霉菌形态特征、放线菌的形态 (2) 比较细菌、放线菌、酵母菌和霉菌形态上的异同 实验6: 微生物培养基的配制和灭菌(3学时) 1. 目的要求 3、 了解培养基的配制原理和方法，掌握其配制过程。 4、 了解几种灭菌方法，掌握干热灭菌法和加压蒸汽灭菌法的原理及其使用方法。 5、 熟悉分离、培养微生物前的有关准备工作及操作方法。 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 2. 基本原理 培养基是人工地将多种物质按各种微生物生长的需要配制而成的一种混合营养基质，用以培养或分离各种微生物。因此，营养基质应当有微生物所能利用的营养成分(包括碳源、氮源、能源、无机盐、生长因素)和水。根据微生物的种类和实验目的不同，培养基也有不同的种类和配制方法。 3. 材料及器材 (1) 药品:牛肉膏、蛋白胨、氯化钠、琼脂、马铃薯、蔗糖;可溶性淀粉、K2HPO4、KNO3、MgSO4•7H2O、FeSO4•7H2O等。 (2) 高压蒸汽灭菌器、恒温干热灭菌箱、细菌过滤器、紫外线杀菌灯、其它:天平、牛角匙、电炉、1N HCl、1N NaOH、pH试纸、刻度搪瓷杯、量筒、漏斗、漏斗架、玻棒、带玻璃珠的三角瓶、带棉塞的无菌试管、无棉塞的空试管、培养皿、吸管、各种包装纸、防水纸、绳索、棉花、标签、试管架、扎线、橡皮筋、记号笔、纱布等 4. 方法与步骤 (1) 培养基的配制: A. 培养基的种类 据组成成分可分为: 合成培养基:由各种纯化学物质按一定比例配制而成。 半合成培养基:有一部分纯化学物质和另一部分天然物质配制而成。 天然培养基:利用天然来源的有机物配制而成。 从培养基的物理状态可分为: 液体培养基:不加凝固剂的液体状态培养基。 固体培养基:在液体培养基中加入2%左右的凝固剂的固体状态的培养基或农副产品培养基。 半固体培养基:在液体培养基中加入0.5-1%凝固剂而成的半固体状培养基。 常用凝固剂为琼脂(其次为明胶) B. 培养基的配制方法和步骤 称量:按照配方正确称取各种原料放于搪瓷杯中 溶化:在搪瓷杯中加入所需水量，玻棒搅匀，加热溶解 调pH值:用1N NaOH或1N HCl调pH，用pH试纸对照 加琼脂溶化:加热过程中要不断搅拌，可适当补水 分装:注意不要污染棉塞 包扎成捆，挂上标签，注明何种培养基 灭菌备用:培养基灭菌后必须在370C下恒温培养24h，确定无菌生长，方可使用 (2) 分离培养微生物常用器皿的准备: bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 清洗一些玻璃仪器:如三角烧瓶、试管、培养皿、吸管等 棉塞的制作及包装培养皿和吸管等 (3) 培养基和玻璃器材的灭菌方法 恒温干燥灭菌法步骤如下: 将要灭菌的物品放入干燥箱内，堆积时要留有空隙，勿使接触器壁、铁壳，关闭箱门。 接通电源，把位于箱顶的通气孔打开，使湿空气能逸出，至箱内温度达到100?时关闭。 调节温度控制器旋钮，直至箱内温度达到所需温度为止，观察温度是否恒定。若不稳定，再行调节，稳定后不可再拨动旋钮和通气孔，保持160~170?，2h。 灭菌结束后切断电源，待箱内温度降至60?时才能打开箱门取出灭菌物品。同时，应将温度调节旋钮跳到零点，并打开通气孔。 加压蒸汽灭菌法其步骤如下: 接通电源，进行加热。 排除高压锅内的冷空气，可将排气阀打开，待排出大气后关闭排气阀。 当压力达15bl/in2(即1.05kg/cm2)时，此时灭菌器内的温度为1210C，维持20min。对热不稳定的培养基如含有葡萄糖、氨基酸等物时，应适当降低压力，延长时间。 灭菌时间一到，切断电源，待压力降至零时，才能打开排气阀，然后打开灭菌器盖，取出物品。 (4) 培养基和玻璃器材的灭菌方法 间歇灭菌法: 待灭菌的物品放在灭菌器或蒸笼里，每天蒸煮1次，每次煮沸1h，连续3d重复进行。在每两次蒸煮之间，将物品(指培养基)放在370C恒温条件下培养过夜，这样可以使每次蒸煮后未杀死残留的芽孢萌发成营养体，以便下次蒸煮时杀灭。 过滤除菌法: 不能用加热灭菌的液体物质(如维生素、血清)，一般可用细菌过滤器进行除菌。 紫外线灭菌法: 一般30W灯管，9m3空间，距地面2m每次打开紫外线照射0.5h，就使室内空气灭菌。若照射紫外线时先喷洒石炭酸等化学消毒剂，可增强灭菌效果。紫外线虽有较强的杀菌力，但穿透力弱，即使一薄层玻璃或水层就能将大部分紫外线滤除，因此只适用于空气及表面杀菌。 化学药剂消毒灭菌: 微生物实验室中常用的化学杀菌剂有升汞、甲醛、高锰酸钾、酒精、碘酒、龙胆紫、石炭酸、漂白粉、新洁尔灭、煤酚皂溶液，它们有的是杀菌剂，有的是抑菌剂。 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 5. 实验作业 (1) 固体培养基加琼脂后加热溶化时要注意哪些问 题 快递公司问题件快递公司问题件货款处理关于圆的周长面积重点题型关于解方程组的题及答案关于南海问题 , (2) 培养基中加琼脂的作用是什么, (3) 干热灭菌、高压蒸汽灭菌和间歇灭菌的场合有何不同, (4) 如何检查培养基灭菌是否彻底, bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 实验7: 土壤微生物的纯化与平板菌落计数 (综合性实验: 6学时) 1. 目的要求 (1)了解不同微生物培养在斜面上和液体、半固体培养基中的特征。 (2)进一步熟练和掌握微生物的无菌操作接种技术。 2. 基本原理 微生物的培养特征是指微生物培养在培养基上所表现出的群体形态和生长情况。一般可用斜面、液体和半固体培养基来检验不同微生物的培养特征。它们培养在斜面培养基上，可以呈丝线状、刺毛状、串珠状、疏展状、树枝状或假根状。生长在液体培养基内，可以呈混浊、絮状、粘液状、形成菌膜、上层清晰而底部显沉淀状。穿刺培养在半固体培养基中，可以沿接种线向四周蔓延;或仅沿线生长;也可上层生长得好，甚至连成一片，底部很少生长;或底部长得好，上层甚至不生长。利用微生物的培养特征，可以作为它们的种类鉴定和识别纯培养是否污染的参考。 检验微生物的培养特征，或进行其他微生物学实验时，接种过程必须保证不被其他微生物所污染，为此，除工作环境要求尽可能地避免或减少杂菌污染外，熟练地掌握各种无菌操作接种技术是很重要的。 3. 材料及器材 (1) 肉膏蛋白胨斜面培养基，高氏1号培养基、马丁氏培养基、苯酚、孟加拉红、 (2) 放大镜、计数器、涂布棒、接种环，接种针，移液器、无菌吸管，无菌平皿，电子秤、三角瓶、显微镜，载玻片，接种环，双层瓶，生理盐水，、擦镜纸、二甲苯、香柏油、吸水纸、纱布、瓷盘、酒精灯、接种工具、打火机、试剂瓶、牙签等、培养箱、灭菌锅、冰箱、超净工作台、盖玻片等 方法与步骤 ?接种的操作方法 ?斜面接种 在肉膏蛋白胨斜面试管上，用记号笔写上将接种的菌名、日期和接种者。 点燃酒精灯或煤气灯。 将菌种试管和待接种的斜面试管，用大拇指和食指、中指、无名指握在左手中，并将中指夹在两试管之间，使斜面向上，成水平状态。在火焰边用右手松动试管塞，以利于接种时拔出。 右手拿接种环通过火焰烧灼灭菌，在火焰边用右手的手掌边缘和小指，小指和无名指分别夹持棉塞(或试管帽)，将其取出，并迅速烧灼管口。 将灭菌的接种环伸入菌种试管内，先将环接触试管内壁或未长菌的培养基，达到冷却的目的，然后再挑取少许菌苔。将接种环退出菌种试管，迅速伸入待接种的斜面试管，用环在斜面上自试管底部向上端轻轻地划直线，勿将培养基划破，也不要使环接触管壁或管口。 接种环退出斜面试管，再用火焰烧管口，并在火焰边将试管塞塞上。将接种环逐渐接近火焰，再烧灼。如果接种环上沾的菌体较多时，应先将环在火焰边烤干，然后烧灼，以免未烧死的菌种飞溅出污染环境，接种病原菌时更要注意此点。 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary ?液体培养基接种 向肉膏蛋白胨液体培养基中接种少量菌体时，其操作步骤基本与斜面接种时相同，不同之处是挑取菌体的接种环放入液体培养基后，应在液体表面处的管内壁上轻轻磨擦，使菌体从环上脱落，混进液体培养基，塞好试管塞后，摇动液体，使菌体在液体中分布均匀，或用试管振荡器混匀。 向液体培养基中接种量大或要求定量接种时，可将无菌水或液体培养基注入菌种试管，用接种环将菌苔刮下，再将菌种悬液以无菌吸管定量吸出加入，或直接倒入液体培养基。如果菌种为液体培养物，则可用无菌吸管定量吸出加入或直接倒入液体培养基。整个接种过程都要求无菌操作。 穿刺接种 ?穿刺接种 用接种针挑取菌种(针必须挺直)，自培养基的中心垂直地刺入半固体培养基中，直至接近管底，但不要穿透，然后沿原穿刺线将针拔出，塞上试管塞，烧灼接种针。 试验者应反复练习无菌操作接种技术，直至较熟练地掌握。 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary ?将已接种的斜面、液体和半固体培养基放置28—30?温箱，培养2—3天后取出观察结果。 ?分离的操作方法 ?平板划线分离法: 倒平板:将融化的琼脂培养基冷却至45?左右，在酒精灯火焰旁，以右手的无名指及小指夹持棉塞，左手打开无菌培养皿的盖的一边，右手持三角瓶向平皿里注入10-15ml培养基。将培养皿稍加旋转摇动后，置于水平位置待凝。 划线分离:在酒精灯火焰上灼烧接种环，待其冷却后，以无菌操作取一环分离菌液。划线时，琼脂平板可放在台面上也可以持在手中。左手握琼脂平板，在火焰上方稍抬起皿盖，右手持接种环伸入皿内，在平板上第一个区域沿“Z”字形来回划线。划线时，使接种环与平板表面成30,40?角轻轻接触，以腕力使接种环在琼脂表面作轻快地滑动，勿划破表面。灼烧接种环，待其冷却后，将手中平皿旋转约70?角，用接种环在划过的第一区域接触一下，然后在第二区域进行划线，并依次对第三和第四区域进行划线。划线完毕后，在平皿底用记号笔注明样品名称、日期、姓名(或学号)，将整个平皿倒置放入28,30?恒温培养箱中培养。18,24小时后观察并记录单菌落的生长和分布情况。 ?倾注平板法: 编号:取6支盛有4.5ml无菌水的试管排列于试管架上，依次标上10,1，10,2，10,3，10,4，10,5，10,6字样。 稀释:以1ml无菌吸管按无菌操作从样品中吸取0.5ml菌液于10,1试管中，然后用另一吸管在10,1试管中吹吸三次，使其混合均匀，制成10,1稀释液。再用此吸管从10,1管中吸取0.5ml稀释液注入10,2管中，依次制成10,2，10,3，10,4，10,5，10bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary ,6稀释液。 加样:用1ml无菌吸管分别吸取10,4，10,5，10,6稀释液1ml注入已编好号的10,4，10,5，10,6号无菌培养皿中。 倾注平板:将融化后冷至45?左右的琼脂培养基，向加有稀释液的各培养皿中分别倒入10,15ml，迅速旋转培养皿，使培养基和稀释液充分混合，水平放置，待其凝固后，倒置于28,30?恒温箱中培养。18,24h后，观察并记录各平板上菌落生长和分布情况。哪个稀释度最合适, ?涂布平板法: 平板制备:制备三套无菌平板，并分别写上10,4，10,5，10,6。 稀释:同倾注平板法。 加样:用无菌吸管分别吸取10,4，10,5，10,6稀释液0.2ml对号注入编好号的琼脂平板中。 涂布:用无菌棒在各平板表面进行均匀涂布。待涂布的菌液干后，将培养皿倒置于28,30?恒温培养箱中培养。18,24小时后观察并记录单菌落的生长和分布情况。 4. 实验作业 ?为什么要把培养皿倒置培养, ?记录你所做的试验中各种微生物在斜面上的培养特征。 ?用各种方法接种时，怎样才能更好地保证菌种不被污染, (4)描述三大类微生物的菌落特征、及微生物的种类。 (5)计算每g土壤各类微生物的数量。 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 实验8:微生物的生理生化特性反应 (大分子水解实验)(3学时) 1、目的要求 掌握细菌坚定中常用的生化反应及其原理。 2、基本原理 由于各种微生物的新陈代谢类型不同，因此对各种物质利用后所产生的代谢产物也不同，我们可以用化学反应来测定微生物的代谢产物，这种反应称之为生理生化反应。生理生化反应常用来鉴别在形态或其他方面不易区别的微生物。 3、材料及器材 ?菌种:大肠杆菌、产气杆菌、变形杆菌。 ?培养基:葡萄糖发酵培养基、乳糖发酵培养基、淀粉水解培养基、明胶水解培养基。 2. 接种用具:接种环(针、钩)，酒精灯，镊子，消毒棉球，打火机，标签贴纸，废物杯，一次性口罩，纸巾，油性笔、水浴锅、厌氧罐、带帽试管、摇床、试管架 3. . 配置培养基的工具:1套 4、方法步骤: 明胶液化实验 (1)以穿刺接种法分别接种枯草杆菌和大肠杆菌于明胶培养基中。 (2)接种后置于20?恒温培养箱，培养2-5d。 (3)观察结果时，注意培养基有无液化现象。(以“+”、“—”表示有无液化现象) 注意: 如果细菌在20?时不能生长，则必须培养在所需的最适温度下，观察结果时需将试管从恒温箱里取出，置于冰浴中，才能观察液化程度。 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 实验9: 理化因素对微生物的影响(3学时) 1. 目的要求 了解紫外线、化学试剂、抗生素的杀菌作用 2. 基本原理 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 各种外界因素如紫外线、化学试剂、抗生素对微生物的生长有抑制甚至杀灭作用。紫外线具有杀菌能力，但穿透力不强。许多化学试剂能使蛋白质变性、妨碍代谢中某些重要酶的活力或损毁细胞膜等。抗生素能选择性的妨碍细菌代谢过程中某一或几个环节。 3. 材料及器材 ?菌种:大肠杆菌、葡萄球菌 ?培养基:营养琼脂培养基 ?试剂:碘伏、过氧乙酸，、龙胆紫、84消毒液、青霉素、庆大霉素、HgCL2 ?其它:星形黑色亮光纸片、无菌滤纸片、镊子、无菌棉签、紫外灯、接种箱 4. 方法与步骤 ?紫外线 ?用棉签蘸取菌液致密接种于营养琼脂平板上。 ?把镊子在火焰上迅速通过2,3次以杀灭其表面杂菌，镊取“H”或“五角形”黑纸片贴于培养基的中央。 ?打开平皿盖，至于紫外灯管下直接照射30min。 ?揭去黑纸片，把纸片丢弃于消毒缸内。盖好皿盖，28,37?培养18,24h后观察结果。可见到培养基上出现与黑纸片形状相同的“H”或“五角形”菌苔，其余部分没有细菌生长。 ?化学试剂 ?用棉签蘸取菌液致密接种于营养琼脂平板上。 ?用无菌镊子夹取4个圆形无菌滤纸片，分别浸于碘伏、过氧乙酸、龙胆紫、84消毒液内，再一一取出，分别放在已接有细菌的平板上，各纸片间距离大小大致相等。 ?28,37?培养18,24h后观察各消毒纸片周围有无抑菌圈，并比较抑菌圈大小。 ?抗生素 ?用棉签分别蘸取两种菌液接种于营养琼脂平板上，一个平板接种一种菌。 ?用无菌镊子夹取2个圆形无菌滤纸片，分别浸于青霉素、庆大霉素内，再一一取出，分别放在已接有细菌的平板上，各纸片间距离大小大致相等。 ?28,37?培养18,24h后观察结果，测量抑菌圈直径。 5. 实验作业 记录抑菌圈大小 青霉素 链霉素 碘酒 过氧乙酸 龙胆紫 HgCI2 大肠杆菌 mm mm mm mm mm mm 葡萄球菌 mm mm mm mm mm mm bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 实验10、水中细菌总数的测定 (3学时) (一)实验目的 (1)了解和学习水中细菌总数和大肠菌群的测定原理和测定意义。 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary (2)学习和掌握用稀释平板计数法测定水中细菌总数的方法。 (3)学习和掌握水中大肠菌群的检测方法。 (二)实验原理 水是微生物广泛分布的天然环境。各种天然水中常含有一定数量的微生物。水中微生物的主要来源有:水中的水生性微生物(如光合藻类)、来自土壤径流、降雨的外来菌群和来自下水道的污染物和人畜的排泄物等。水中的病原菌主要来源于人和动物的传染性排泄物。 水的微生物学的检验，特别是肠道细菌的检验，在保证饮水安全和控制传染病上有着重要意义，同时也是 评价 LEC评价法下载LEC评价法下载评价量规免费下载学院评价表文档下载学院评价表文档下载 水质状况的重要指标。国家饮用水 标准 excel标准偏差excel标准偏差函数exl标准差函数国标检验抽样标准表免费下载红头文件格式标准下载 规定 关于下班后关闭电源的规定党章中关于入党时间的规定公务员考核规定下载规定办法文件下载宁波关于闷顶的规定 ，饮用水中大肠菌群数每升中不超过3个，细菌总数每mL不超过100个。 所谓细菌总数是指1mL或1g检样中所含细菌菌落的总数，所用的方法是稀释平板计数法，由于计算的是平板上形成的菌落(colony-forming unit，cfu)数，故其单位应是cfu/g(mL)。它反映的是检样中活菌的数量。 所谓大肠菌群，是指在37?24h内能发酵乳糖产酸、产气的兼性厌氧的革兰氏阴性无芽胞杆菌的总称，主要由肠杆菌科中四个属内的细菌组成，即埃希氏杆菌属、柠檬酸杆菌属、克雷伯氏菌属和肠杆菌属。 水的大肠菌群数是指100mL水检样内含有的大肠菌群实际数值，以大肠菌群最近似数(MPN)表示。在正常情况下，肠道中主要有大肠菌群、粪链球菌和厌氧芽胞杆菌等多种细菌。这些细菌都可随人畜排泄物进入水源，由于大肠菌群在肠道内数量最多，所以，水源中大肠菌群的数量，是直接反映水源被人畜排泄物污染的一项重要指标。目前，国际上已公认大肠菌群的存在是粪便污染的指标。因而对饮用水必须进行大肠菌群的检查。 水中大肠菌群的检验方法，常用多管发酵法和滤膜法。多管发酵法可运用于各种水样的检验，但操作繁琐，需要时间长。滤膜法仅适用于自来水和深井水，操作简单、快速，但不适用于杂质较多、易于阻塞滤孔的水样。 (三)实验器材 (1) 菌落总数的测定: 1)培养基:牛肉膏蛋白胨琼脂培养基，无菌生理盐水。 2)器材:灭菌三角瓶，灭菌的具塞三角瓶，灭菌平皿，灭菌吸管，灭菌试管等。 (2)大肠菌群的测定; 1)培养基: ?乳糖胆盐蛋白胨培养基: 蛋白胨20g，猪胆盐(或牛、羊胆盐)5g，乳糖10g，0.04%溴甲酚紫水溶液25mL，水1000mL，pH7.4。 制法:将蛋白胨、胆盐从乳糖溶于水中，校正pH，加入指示剂，分装，每瓶50mL或每管5mL，，并倒置放入一个杜氏小管，l15?灭菌15min。 双倍或三倍乳糖胆盐蛋白胨培养基:除水以外，其余成分加倍或取三倍用量。 ?伊红美蓝琼脂培养基: 蛋白胨10g，乳糖10g， K2HP04 2g， 2,伊红水溶液20Ml，0.65,美蓝水溶液lOmL，琼脂17g，水1000mL，pH7.1。 制法:将蛋白胨、磷酸盐和琼脂溶于水中，校正pH后分装(121?灭菌15min备用。临用时加入乳糖并熔化琼脂，冷至50-55?，加入伊红和美蓝溶液，摇匀，倾注平板。 ?乳糖发酵管: 除不加胆盐外(其余同乳糖胆盐蛋白胨培养基。 2)器材:灭菌三角瓶，灭菌的具塞三角瓶，灭菌平皿，灭菌吸管，灭菌试管等。 四)实验方法 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary (1)水样的采集: 1)自来水:先将自来水龙头用酒精灯火焰灼烧灭菌，再开放水龙头使水流5min，以灭菌三角瓶接取水样以备分析。 2)池水、河水、湖水等地面水源水:在距岸边5m处，取距水面10-15cm的深层水样，先将灭菌的具塞三角瓶，瓶口向下浸入水中，然后翻转过来，除去玻璃塞，水即流入瓶中，盛满后，将瓶塞盖好，再从水中取出。如果不能在2h内检测的，需放入冰箱中保存。 (2)细菌总数的测定: 1)水样稀释及培养: ?按无菌操作法，将水样作10倍系列稀释: ?根据对水样污染情况的估计，选择2-3个适宜稀释度(饮用水如自来水、深井水等，一般选择1、1:10两种浓度;水源水如河水等，比较清洁的可选择1:10、1:100、1:1000三种稀释度;污染水—被选择1:100、1:1000、1:10000三种稀释度)，吸取1mL稀释液于灭菌平皿内，每个稀释度作3个重复。 ?将熔化后保温度45?的牛肉膏蛋白胨琼脂培养基倒平皿，每皿约15mL，并趁热转动平皿混合均匀。 ?待琼脂凝固后，将平皿倒置于37?培养箱内培养24?1h后取出，计算平皿内菌落数目，乘以稀释倍数，即得1mL水样中所含的细菌菌落总数。 2)计算方法: 作平板计数时，可用肉眼观察，必要时用放大镜检查，以防遗漏。在记下各平板的菌落数后，求出同稀释度的各平板平均菌落数。 3)计数的报告: ?平板菌落数的选择: 选取菌落数在30-300之间的平板作为菌落总数测定标准。一个稀释度使用两个重复时，应选取两个平板的平均数。如果一个平板有较大片状菌落生长时，则不宜采用，而应以无片状菌落生长的平板计数作为该稀释度的菌数。若片状菌落不到平板的一半，而其余一半中菌落分布又很均匀，可计算半个平板后乘2以代表整个平板的菌落数。 ?稀释度的选择: a. 应选择平均菌落数在30-300之间的稀释度，乘以该稀释倍数报告之(表1例1)。 b(若有两个稀释度，其生长的菌落数均在30-300之间，则视二者之比如何来决定。若其比值小于2，应报告其平均数;若比值大于2，则报告其中较小的数字(l例2、例3)。 c(若所有稀释度的平均菌落均大于300，则应按稀释倍数最低的平均菌落数乘以稀释倍数报告之(l例4)。 d(若所有稀释度的平均菌落数均小于30、则应按稀释倍数最低的平均菌落数乘以稀释倍数报告之(1例5)。 e. 若所有稀释度均无菌落生长，则以小于1乘以最低稀释倍数报告之(表1例6)。 f. 若所有稀释度的平均菌落数均不在30-300之间，则以最接近30或300的平均菌落数乘以该稀释倍数报告之(表l例7)。 ?细菌总数的报告: 细菌的菌落数在l00以内时，按其实有数报告;大于100时，用二位有效数字，在二位有效数字后面的数字，以四舍五入方法修约。为了缩短数字后面的0的个数，可用10的指数来表示，如表1“报告方式”一栏所示。 表 1 稀释度的选择及细菌数报告方式 稀释度及菌落数 两稀释度菌落总数/(cfu/g报告方式(菌落总数) 之比 或cfu/mL) /(cfu/mL或cfu/g) 10-1 10-2 10-3 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 16000或多不可计 1 164 20 - 16400 1.6×104 38000或多不可计 2 295 46 1.6 37750 3.8×104 27000或多不可计 3 271 60 2.2 27100 2.7×104 310000 多不可计 多不可计 或3.1×4 313 - 313000 105 270或 5 27 11 5 - 270 ,10 ,10 6 0 0 0 - 31000或多不可计 7 305 12 - 30500 3.1×104 • 思考题: • 1、从自来水的细菌总数看，是否符合饮用标准, • 2、国家对自来水有一标准，各地能否自行设计测定条件，为什么, 附录I 染色液的配制 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 一、吕氏(Loeffler)碱性美蓝染液 A液:美蓝(methylene blue) 0(6g 95%酒精 30ml B液:KOH 0(01g 蒸馏水 100ml 分别配制A液和B液，配好后混合即可。 二、齐氏(Ziehl)石炭酸复红染色液 A液:碱性复红(basic fuchsin) 0(3g 95%酒精 10ml B液:石炭酸 5(0g 蒸馏水 95ml 将碱性复红在研钵中研磨后，逐渐加入95%酒精，继续研磨使其溶解，配成A液。 将石炭酸溶解于水中，配成B液。 混合A液及B液即成。通常可将此混合液稀释5—10倍使用，稀释液易变质失效，一 次不宜多配。 三、革兰氏(Gram)染色液 1(草酸铵结晶紫染液 A液:结晶紫(crystal violet) 2g 95%酒精 20ml B液:草酸铵(ammonium oxalate) 0(8g 蒸馏水 80ml 混合A、B二液，静置48小时后使用。 2(卢戈氏(Lugol)碘液 碘片 1(0g 碘化钾 2(0g 蒸馏水 300ml 先将碘化钾溶解在少量水中，再将碘片溶解在碘化钾溶液中，待碘全溶后，加足水分。 3(95%的酒精溶液。 4(番红复染液 番红(safranine O) 2(5g 95%酒精 100ml 取上述配好的番红酒精溶液10ml与80ml蒸馏水混匀即成。 四、芽孢染色液 1(孔雀绿染液 孔雀绿(malachite green) 5g 蒸馏水 100ml 2(番红水溶液 番红 0(5g 蒸馏水 100ml 3(苯酚品红溶液 碱性品红 11g 无水酒精 100ml 制法取上述溶液10ml与100ml5%的苯酚溶液混合，过滤备用。 4(黑色素(nigrosin)溶液 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 水溶性黑色素 10g 蒸馏水 100ml 称取10g黑色素溶于100ml蒸馏水中，置沸水浴中30分钟后，滤纸过滤 二次，补加水到100ml，加0(5ml甲醛，备用。 五、荚膜染色液 1(黑色素水溶液 黑色素 5g 蒸馏水 100ml 福尔马林(40%甲醛) 0(5ml 将黑色素在蒸馏水中煮沸5分钟，然后加入福尔马林作防腐剂。 2(番红染液 与革兰氏染液中番红复染液相同。 六、鞭毛染色液 A液:单宁酸 5g FeCl3 1(5g 蒸馏水 100ml 福尔马林(15%) 2(0ml NaOH(1%) 1(0ml 配好后，当日使用，次日效果差，第三日则不宜使用。 B液:AgNO3 2g 蒸馏水 100ml 待AgNO3溶解后，取出10ml备用，向其余的90mlAgNO3中滴入浓NH4ON，使之成为很浓厚的悬浮液，再继续滴加NH4OH，直到新形成的沉淀又重新刚刚溶解为止。再将备用的10mlAgNO3慢慢滴入，则出现薄雾，但轻轻摇动后，薄雾状沉淀又消失，再滴入AgNO3，直到摇动后仍呈现轻微而稳定的薄雾状沉淀为止。如所呈雾不重，此染剂可使用一周，如雾重，则银盐沉淀出，不宜使用。 七、富尔根氏核染色液 1(席夫氏(Schiff)试剂 将1g碱性复红加入200ml煮沸的蒸馏水中，振荡5分钟，冷至50?左右过滤，再加入1mol/L HC120ml，摇匀。待冷至25?时，加Na2S2O5(偏重亚硫酸钠)3g，摇匀后装在棕色瓶中，用黑纸包好，放置暗处过夜，此时试剂应为淡黄色(如为粉红色则不能用)，再加中性活性炭过滤，滤液振荡1分钟后，再过滤，将此滤液置冷暗处备用(注意:过滤需在避光条件下进行)。 在整个操作过程中所用的一切器皿都需十分洁净、干燥，以消除还原性物质。 2(Schandium固定液 A液:饱和升汞水溶液 50ml升汞水溶液加95%酒精25ml混合即得。 B液:冰醋酸 取A液9毫升+B液1毫升，混匀后加热至60?。 3(亚硫酸水溶液 10%偏重亚硫酸钠水溶液5ml，1mol/LHCl5ml，加蒸馏水100ml混合即得。 八、Bouin氏固定液 苦味酸饱和水溶液 75ml 福尔马林(40%甲醛) 25ml bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 冰醋酸 5ml 1g苦味酸可制成75ml饱和水溶液。 先将苦味酸溶解成水溶液，然后再加入福尔马林和冰醋酸摇匀即成。 九、乳酸石炭酸棉蓝染色液 石炭酸 10g 乳酸(比重1(21) 10ml 甘油 20ml 蒸馏水 10ml 棉蓝0(02g 将石炭酸加在蒸馏水中加热溶解，然后加入乳酸和甘油，最后加入棉蓝，使其溶解即成。 十、瑞氏(Wright)染色液 瑞氏染料粉末 0(3g 甘油 3ml 甲醇 97ml 将染料粉末置于干燥的乳缽内研磨，先加甘油，后加甲醇，放玻璃瓶中过夜，过滤即可。 十一、美蓝(Levowitz-Weber)染液 在52ml 95%酒精和44ml四氯乙烷的三角烧瓶中，慢慢加入0.6g氯化美蓝，旋摇三角 烧瓶，使其溶解。放5—10?下，12—24小时，然后加入4ml冰醋酸。用质量好的滤纸过滤。贮存于清洁的密闭容器内。 附录? 试剂和溶液的配制 一、3%酸性酒精溶液 浓盐酸 3ml 95%酒精 97ml 二、中性红指示剂 04g 中性红 0( 95%乙醇 28ml 蒸馏水 72ml 中性红pH6(8—8，颜色由红变黄，常用浓度为0(04%。 三、淀粉水解试验用碘液(卢戈氏碘液) 碘片 1g 碘化钾 2g 蒸馏水 300ml 先将碘化钾溶解在少量水中，再将碘片溶解在碘化钾溶液中，待碘全溶后，加足水分即 成。 四、溴甲酚紫指示剂 溴甲酚紫 0(04g 0(01mol/L NaOH 7(4ml 蒸馏水 92(6ml 溴甲酚紫pH5(2—6(8，颜色由黄变紫，常用浓度为0(04%。 五、溴麝香草酚蓝指示剂 溴麝香草酚蓝 0(04g 0(01mol/L NaOH 6(4ml 蒸馏水 93(6ml 溴麝香草酚蓝pH6(0—7(6，颜色由黄变蓝，常用浓度为0(04%。 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 六、甲基红试剂 甲基红(Methyl red) 0(04g 95%酒精 60ml 蒸馏水 40ml 先将甲基红溶于95%酒精中，然后加入蒸馏水即可。 七、V(P(试剂 1(5%α-萘酚无水酒精溶液 α-萘酚 5g 无水乙醇 100ml 2(40%KOH溶液 KOH 40g 蒸馏水 100ml 八、吲哚试剂 对二甲基氨基苯甲醛 2g 95%乙醇 190ml 浓盐酸 40ml 九、格里斯氏(Griess)试剂 A液:对氨基苯磺酸 0(5g 10%稀醋酸 150ml B液:α-萘胺 0(1g 蒸馏水 20ml 10%稀醋酸 150ml 十、二苯胺试剂 5g溶于100ml浓硫酸中，用20ml蒸馏水稀释。 二苯胺0( 十一、阿氏(Alsever)血液保存液 柠檬酸三钠?2H2O 8g 柠檬酸 0(5g 无水葡萄糖 18(7g NaCl 4(2g 蒸馏水 1000ml 将各成分溶解于蒸馏水后，用滤纸过滤，分装，0(56—0(70kg/cm2(8—10磅/英寸2)， 灭菌20分钟。冰箱保存备用。 十二、肝素溶液 取一支含12500单位的注射用肝素溶液，用生理盐水稀释500倍，即成为每毫升含25 单位的肝素溶液。作白细胞吞噬试验用。大约12(5单位肝素可凝1毫升全血。 十三、pH8(6离子强度0(075mol/L巴比妥缓冲液 巴比妥 2(76g 巴比妥钠 15(45g 蒸馏水 1000ml 十四、1%离子琼脂 琼脂粉 1g 巴比妥缓冲液 50ml 蒸馏水 50ml 1%硫柳汞 1滴 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 称取琼脂粉1g先加至50ml蒸馏水中，于沸水浴中加热溶解，然后加入50ml巴比妥缓冲液，再滴加1滴1%硫柳汞溶液防腐，分装试管内，放冰箱中备用。 十五、其他细胞悬液的配制 1(1%鸡红细胞悬液 取鸡翼下静脉血或心脏血，注入含灭菌阿氏液的玻璃瓶内，使血与阿氏液的比例为1:5，放冰箱中保存2—4周。临用前取出适量鸡血，用无菌生理盐水洗涤，离心，倾去生理盐水，如此反复洗涤三次，最后一次离心使成积压红细胞，然后用生理盐水配成1%。供吞噬试验用。 2(白色葡萄球菌菌液 白色葡萄球菌接种于肉汤培养基中，37?温箱培养12小时左右，置水浴中加热100?，10分钟杀死细菌，用无菌生理盐水配制成每毫升含6亿个细胞，分装于小瓶内，置冰箱保存备用。 附录3 培养基 1、牛肉膏琼脂培养基 牛肉膏0.3克 ，蛋白胨1.0克，氯化钠 0.5克，琼脂 1.5克， 水 100毫升 在烧杯内加水100毫升，放入牛肉膏、蛋白胨和氯化钠，用蜡笔在烧杯外作上记号后，放在火上加热。待烧杯内各组分溶解后，加入琼脂，不断搅拌以免粘底。等琼脂完全溶解后补足失水，用10%盐酸或10%的氢氧化钠调整pH值到7.2,7.6,分装在各个试管里，加棉花塞，用高压蒸汽灭菌30分钟。 、 淀粉琼脂培养基(高氏培养基) 2 可溶性淀粉 2克 硝酸钾 0.1克 磷酸氢二钾 0.05克 氯化钠 0.05克 硫酸镁 0.05克 硫酸亚铁 0.001克 琼脂 2克 水 1000毫升 先把淀粉放在烧杯里，用5毫升水调成糊状后，倒入95毫升水，搅匀后加入其他药品，使它溶解。在烧杯外做好记号，加热到煮沸时加入琼脂，不停搅拌，待琼脂完全溶解后，补足失水。调整pH值到7.2,7.4，分装后灭菌，备用。 3、马铃薯糖琼脂培养基 把马铃薯洗净去皮，取200克切成小块，加水1000毫升，煮沸半小时后，补足水分。在滤液中加入10克琼脂，煮沸溶解后加糖20克(用于培养霉菌的加入蔗糖，用于培养酵母菌的加入葡萄糖)，补足水分，分装，灭菌，备用。 把这培养基的pH值调到7.2,7.4，配方中的糖，如用葡萄糖还可用来培养放线菌和芽孢杆菌。 4、酵母菌富集培养基 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary 葡萄糖5% 尿素0.1% 硫化铵0.1% 磷酸二氢钾0.25% 磷酸氢二钠0.05% 七水合硫酸镁0.1% 七水合硫酸铁0.01% 酵母膏0.05% 孟加拉红0.003% pH4.5 5、 Ashby无氮培养基 富集好养自生固氮菌 甘露醇1% 磷酸二氢钾0.02% 七水合硫酸镁0.02% 氯化钠0.02% 二水合硫酸钙0.01% 碳酸钙0.5% 6、鉴别培养基 EMB培养基，常用于鉴别E.coli 蛋白胨 10g 乳糖5g 蔗糖5g 磷酸氢二钾2g 伊红Y 0.4g 美蓝0.065g 蒸馏水1000g pH7.2 7、糖发酵培养基 牛肉膏 5g 蛋白胨 10g 氯化钠 3g 磷酸氢二钠(Na2HPO4•12H2O) 2g 0.2%滇麝香草酚蓝溶液 12mL 蒸馏水 1000mL pH7.4 制法: 1葡萄糖发酵管按上述成分配好后，按0.5%加入葡萄糖，(或乳糖)，分装于有一个倒置小管的小试管内，121?高压灭菌15min。 2其他各种糖发酵管可按上述成分配好后，分装每瓶100mL，121?高压灭菌15min。另将各种糖类分别配好10%溶液，同时高压灭菌。将5mL糖溶液加入于100mL培养基内，以无菌操作分装小试管。 8、淀粉水解培养基 蛋白胨 10g 牛肉膏 5g Nacl 5g 可溶性淀粉 2g 琼脂 20g 蒸馏水 1000ml 9、明胶培养基 牛肉膏蛋白胨液 100ml 明胶12——18g 10、马丁氏培养基 葡萄糖 10g 蛋白胨 5g KH2PO4 1g MgSO4 0.5g 孟加拉红 1/3000 bridge, in the response from the crowd was great, and Wang soon expanded to 2 battalions. Has developed more than 40 party members on the party organization in the army and local people. Only political training Department on the development of the Zhuang Xueying, Xu Jiasheng, Huiqin Xu, Wang Qun, many party members. Wang Department of development of the KMT was deeply disturbed by activities suffered seems scary or unusual or, rating and no. The same month, by Shen Liqun Wang, demanding administrative office supplies in the South, head of the Bureau took the opportunity to appoint Wang Yi titles and be incorporated as a third mission for security in the South. Party organizations of Wang's actions to discourage invalid. End of the year, Wang Hesong first battalion and regimental headquarters to liyang and consolidation, and soon, Regiment, battalion, all political leaders have been replaced, military cadres assigned deputies, the entire force controlled by the Jiangsu national security headquarters. Presence in the tomb of the second battalion, according to the next higher party organization for instructions, led by Zheng Communist party organizations in order to organize people, arousing the masses against Japan, in organizations "Book Club" youth reading progress newspaper, on the current situation, stimulate Japanese enthusiasm. In order to solve the reading books issue, Shao working through relationship, from the Shanghai underground secrets back to some banned books, including the protracted war and the popular philosophy and the theory of new democracy, and so on; there are many magazines, such as the woman crafts such as, set up an underground 300 people, 13 employees of the school. 1938 three lunar new year in early June (Gregorian calendar, June 30), the Japanese "sweeps" to Yan Zhen, South Street elementary school was the Japanese burned, robbed more than things, on fire. Bei Jie, the former girls ' school were Japanese invaders burned down. The next day, the Japanese education center and the gymnasium built in 20 of the people [in the tomb of the stadium (now the tongluo secondary