细菌的培养法.doc

实验一 细菌的培养法 一般细菌均可用人工方法进行培养，使其生长繁殖，以便进一步观察和研究它们的各种生物学特性。为了获得良好的细菌培养物，在分离培养细菌时，除采用适宜的培养基并考虑到其他的培养条件（如温度、湿度、酸碱度、气体等）之外，掌握各种分离培养和接种的基本技术，也是重要环节。 在土壤、水、空气、以及人和动、植物体中，不同种类的细菌混杂地生活在一起。若要研究其中某种细菌，就必须先将各种细菌进行分离，以得到只含有这一种细菌的纯培养。分离培养细菌的方法有多种，平板划线法即是其中之一。该法主要是借划线而将混杂的细菌在琼脂平板表面分散开，使单个细菌能固定在某一点，生长繁殖后形成单个的细菌集团（即菌落）以达到分离纯种的目的。 当细菌分离成纯种后，常需要接种到有关的培养基中，以测试其各种生物学性状。一般可用斜面、液体和半固体培养基来检验细菌的培养特征，因此接种方法可相应的分为三种。 斜面培养基接种法 常用于细菌的大量繁殖，保存菌种，或观察其某些生化特性。琼脂斜面、尿素培养基、双糖铁培养基、柠檬酸盐培养基等具有斜面外形的固体培养基均可用此法接种。 液体培养基接种法 可用于观察细菌不同的生长状况，有的呈均匀混浊，有的呈沉淀生长，还有的在液体表面形成菌膜；另外还可以供测定细菌生化特性之用。凡是肉汤、葡萄糖蛋白胨水以及各种单糖发酵管等液体培养基均用此法接种。穿刺接种法 常用于半固体琼脂培养基、醋酸铅培养基、双糖铁培养基等的接种，前者用于测定细菌的动力，后二者则用于观察细菌的生化反应。目的要求 1学习和掌握细菌分离和培养的各种基本技术。 2进一步熟悉和掌握微生物的无菌操作技术。材料 1菌种和培养基平板划线接种法斜面培养基接种法液体培养基接种法穿刺接种法菌种葡萄球菌和大肠杆菌的混合菌液大肠杆菌培养物大肠杆菌培养物枯草杆菌培养物变形杆菌培养物痢疾杆菌培养物培养基*普通琼脂平板琼脂斜面培养基普通肉汤培养基半固体琼脂培养基 *各培养基的组成及配置方法参见书后附录。 2接种环、接种针、酒精灯、记号笔、试管架等方法1 平板划线接种法 （1）标记 在培养皿底玻璃上，用记号笔注明接种的菌名、接种者姓名、班级、日期等。 （2）灭菌接种环 点燃酒精灯，右手以持笔式握持接种环并放置火焰中烧灼灭菌，如图1-1，先将接种环的接种丝部分置于火焰中，待金属丝烧红并蔓延至金属环端，再直接烧灼金属环直至烧红，然后由金属环至金属杆方向快速通过火焰，随后再反方向通过火焰，如此23次。图1-1 接种环的灭菌操作然后将接种环移开火焰，待其冷却。注意，接种环不能距离火焰过远，一般应在距火焰10cm范围之内（视此范围为无菌环境）；灭菌后的接种环不能再碰及他物。 （3）取菌种 左手持装有葡萄球菌和大肠杆菌混合菌液的试管，用持有接种环的右手手掌及小指拔取试管棉塞，将试管管口迅速通过火焰23次进行灭菌。 将已灭菌且已冷却的接种环伸入菌种管中，取一接种环的混合菌液，然后退出菌种管。将菌种管管口再次通过火焰23次灭菌，塞好棉塞，放至原来的位置。 （4）分离划线接种细菌（见图3-1） 左手持琼脂平板培养基（将皿盖反放在桌上酒精灯附近），尽量使之直立以免空气中的细菌落入其中，并靠近火焰。右手持接种环在琼脂平板上端来回划线，涂成一细菌薄膜（约占平板面的1/10），视为一区。划线时使接种环与平板面成3040度角，以腕力在平板表面行轻而快地来回滑动动作。切记，接种环不应嵌进培养基内，避免将琼脂表面戳破。 旋转琼脂平板90度。烧灼接种环，以杀灭环上的残留细菌，将接种环触及培养基表面以使其冷却。灭菌接种环通过薄膜处作连续平行划线（约占平板1/5），此为二区。注意接种环只通过薄膜12次，以获取薄膜处少量的细菌。 旋转琼脂平板90度。烧灼接种环灭菌并使之冷却。将灭菌接种环接二区连续平行划线（约占平板1/4），此为三区。接种环只通过二区12次以获得少量细菌。 旋转琼脂平板90度。接种环不必再灭菌，接三区连续平行划线，划满平板其余部分，此为四区。 注意各区接种线间尽量互不交接，以达到细菌逐渐稀释的目的。 （5）划线完毕，将琼脂平板放进皿盖，将培养皿倒放（这样可避免培养过程中凝结水自皿盖滴下，冲散菌落），送进37温箱培养。 （6）培养1824小时后将培养皿取出。观察琼脂平板表面生长的各种菌落，注意其大小、形状、边缘、表面结构、透明度、颜色等性状。 1 2 3 4图1-2 四区划线接种法2 斜面培养基接种法 （1）用记号笔在待接种的培养管上写明标记。 （2）点燃酒精灯。 （3）左手拇指、食指、中指及无名指分别握持菌种与待接种的培养基管，使菌种管位于左侧，斜面部均应向上，勿成水平，以免凝结水浸润培养基表面，甚至沾湿棉塞。 （4）以右手拇指和食指捏持转动两管棉塞，以便在接种时易于拔取。 （5）右手持接种环，在火焰上烧灼灭菌。 （6）以右手手掌及小指，小指及无名指分别拔取并夹持两管棉塞，将两管管口灭菌。 （7）将已灭菌且已冷却的接种环伸入菌种管内，从斜面上轻轻挑取少量菌苔退出菌种管（注意，一要防止取菌过多；二要防止弄破培养基表面）。再伸进待接种的培养基管，如图3-2进行斜面培养基接种，从斜面底部轻轻向顶端弯曲划线，不要触破培养基表面。沾有细菌的接种环进出试管时不应触及到试管内壁和试管口。 （8）接种完毕，灭菌两试管的管口，塞好棉塞，并放至原来的位置上。重新烧灼接种环，灭菌后放回试管架上。接种好的试管放37温箱培养，1824小时后观察生长情况。图1-3 斜面培养基接种法3 液体培养基接种法 （1）用记号笔在待接种培养基上写明标记。 （2）如斜面培养基的接种方法一样，握持菌种管及待接种的肉汤管。（3）接种环灭菌冷却后，伸入菌种管取少量细菌再伸入肉汤管内，在接近液面管壁处轻轻研磨，蘸取少量肉汤调和，使菌混于肉汤中（见图3-3）。塞好试管棉塞后，摇动液体，使细菌在液体中均匀分布。 （4）接种完毕，将接种环灭菌后放回试管架上。肉汤管放37温箱培养，1824小时后观察生长情况。图1-4 液体培养基接种法4 穿刺接种法（1）用记号笔在待接种培养基上写明标记。（2）如斜面培养基接种法，握持菌种管及待接种的半固体琼脂培养基。（3）右手持接种针，灭菌冷却后，以针挑取菌苔，如图3-4垂直刺入半固体琼脂培养基的中心，刺达近管底部，但不必及于管底，然后循原路退出。（4）接种完毕，接种针重行灭菌后放至试管架上，塞好棉塞，37培养1824小时后取出观察细菌的生长情况。图1-5 穿刺接种法 实验二 细菌的形态与结构 各种细菌在一定环境条件下，有相对恒定的形态与结构。了解细菌的形态与结构是鉴别细菌的重要方法之一。此外，对分析细菌的致病性和免疫发生的机理等方面，也有一定的意义。目的要求 观察细菌的基本形态和一些细菌的特殊结构。一、细菌的基本形态 细菌在适宜的生长繁殖条件下所显示的形态可分为三大类：球菌、杆菌和螺形菌。不同的细菌又可表现出不同的排列方式，在细菌的鉴别上有一定的参考价值。材料1 球菌示教片 葡萄球菌、链球菌和肺炎链球菌2 杆菌示教片 大肠杆菌和炭疽杆菌3 螺形菌示教片 霍乱弧菌方法1 使用显微镜的油镜观察球菌、杆菌和螺形菌的示教片。2 注意各菌的形状、大小、排列方式等特点（注意：细菌染成什么颜色不是本实验要求，染色方法将在以后实验中实际操作）。结果记录实验结果葡萄球菌链球菌肺炎链球菌大肠杆菌炭疽杆菌霍乱弧菌细菌形状细菌排列方式二、细菌的特殊结构 细菌的特殊结构仅为某些细菌所具有，而且特殊结构的形成受到一定条件的限制。虽然特殊结构不是细菌生存所必须的，但他们的存在将赋予细菌一定的功能，在致病性、抗原性、以及对细菌的鉴别上都有一定的意义。材料1 破伤风梭菌示教片（示芽胞）2 肺炎链球菌示教片（示荚膜）3 变形杆菌示教片（示鞭毛）方法1 使用显微镜的油镜，观察细菌的芽胞、荚膜和鞭毛的示教片。2 注意芽胞在菌体上的位置和大小；荚膜的大小及其与菌体的关系；注意鞭毛形态、数量及其位置。将所观察到的细菌特殊结构的形态特点记录于下表中。结果记录 细菌破伤风梭菌肺炎链球菌变形杆菌特殊结构实验三 革兰染色法 活的细菌为无色半透明状，在普通光学显微镜下不易观察清晰。若用染色的方法可使菌体表面及内部结构着色与背景形成鲜明对比，可在显微镜下清晰地观察其形态。 细菌的染色方法很多，其中最为广泛使用的一种鉴别染色法是由丹麦医生Christian Gram于1884年创建的革兰（Gram）染色法。利用此法可将细菌分为革兰阳性细菌和革兰阴性细菌两大类。革兰阳性菌因细胞壁中含有大量的肽聚糖和磷壁酸，可保留结晶紫与碘的复合物，并不被酒精脱色而显示为蓝紫色。革兰阴性菌因其细胞壁中肽聚糖含量少，脂类物质含量高而被酒精脱色，经复染呈红色。目的要求 了解革兰染色的原理，掌握革兰染色的方法。材料1 菌种 葡萄球菌、大肠杆菌培养物2 染液 结晶紫染液、卢戈（Rugol）碘液、95%酒精和沙黄染液3 其他 玻片、接种环、酒精灯、无菌生理盐水等方法 1涂片 取洁净玻片1张，作好标记后置实验台上。 点燃酒精灯，右手以持笔式握持接种环并放置火焰中烧灼灭菌。用灭菌的接种环取无菌生理盐水2环，分别置于玻片左右两处。然后左手持培养物琼脂平板；右手仍以持笔式将接种环再次放在火焰上灭菌，待接种环冷却后，挑取单一金黄色葡萄球菌菌落适量培养物，将培养物琼脂平板放回皿盖。然后将挑取的细菌混合于其中一处的盐水中，涂抹均匀使成一层薄膜（若检材是液体，则不必加盐水），薄膜涂抹的面积约1.52.0cm2。按上法制备大肠杆菌涂膜。 2于室温中自然干燥。 3固定 涂片在酒精灯火焰上快速通过34次以固定细菌，并使其不易从玻片上脱落。注意不要将涂片直接放在火焰上烤，以免破坏细菌结构。4 染色 （1）初染 在涂膜上滴加结晶紫染液12滴，染1分钟，水冲洗，并轻轻倾去玻片上的积水。 （2）媒染 加卢戈碘液12滴，染1分钟，水冲洗。将表面积水甩干。 （3）脱色 用95%酒精数滴滴于玻片上，频频摇晃以脱色，半分钟左右，立即用水冲洗（若涂膜较厚，可延长脱色时间；必须随时观察，以免脱色过度）。 （4）复染 最后滴加沙黄染液12滴，复染1分钟后用水冲洗。最后用吸水纸吸干。 5用显微镜的油镜观察染色结果，将实验结果记录于下表中。结果记录 细菌染色性形态排列方式葡萄球菌大肠杆菌实验四 抗酸染色法 结核分枝杆菌是引起结核病的病原体，菌体细长略弯曲，有分枝生长趋势，因其细胞壁含有大量脂质，一般不易着色，若经加温或延长染色时间或提高染液浓度而着色后能抵抗盐酸酒精的脱色，故又称抗酸杆菌。目的要求 学习抗酸染色方法。材料 1开放型肺结核病人痰标本涂片和枯草杆菌涂片 2抗酸染色剂（石炭酸复红染液、3盐酸酒精、碱性美蓝染液），载玻片方法 1. 染色在已固定的涂片上滴加石炭酸复红液数滴，染色810分钟，水冲洗。滴加3盐酸酒精脱色，至复红的颜色不再继续脱下为止，时间为半分钟至1分钟，水冲洗。滴加碱性美蓝液23滴，1分钟后水冲洗。用吸水纸把玻片吸干，油镜检查。 2. 抗酸染色标本镜检 结核杆菌为抗酸染色阳性，在蓝色背景下可见染成红色的细长或略带弯曲的杆菌，有分枝生长趋向，有时菌体内可含浓染颗粒，呈念珠状。Experiment 1 Handling and Examining CulturesIntroduction: To obtain information about bacteria biologic characteristics, it is necessary to observe microorganisms in culture. If we are to cultivate them successfully in the laboratory, we must provide them with suitable nutrients, such as protein components, carbohydrates, minerals, vitamins, and moisture in the right composition. This mixture is called a culture medium. It may be prepared in liquid form, as a broth, or solidified with agar. Agar medium may be used in tubes as a solid column or as slants. They are also commonly used in Petri dishes or plates. Solid medium is essential for the isolation and separation of bacteria growing together in a specimen. If a mixture of bacteria is spread across the surface of a plated agar medium, individual organisms will multiply at individual sites until a visible aggregate called a colony forms. One colony of a single species can then be separated from the rest and transferred to another medium, where it will grow as a pure culture, and can be studied as such. The appearance of colony can be very distinctive for individual species, such as color, density, consistency, surface texture, shape, and size of colonies. They can provide clues for the identification of an organism, although final identification can not be made by morphology alone. In liquid medium, some bacteria may grow diffusely, producing uniform clouding. Others may look very granular in broth. Observation of such features can also be helpful in recognizing types of organisms. There is another kind of culture medium called semisolid medium. It is used to detect motility of bacteria. Non-motile bacteria will grow only along the line of inoculation, whereas motile bacteria will migrate throughout the medium. In this experiment, you will learn how to make aseptic transfers of pure culture and to examine them for important gross features.Materials:1.Culture Medium and Bacterial Strains:Culture MediumBacteria StrainsPlatea mixture of S.aureus & E.coliLiquid MediumE.coli, B.subtitisSlantE.coliSemisolid MediumS.dysenteriae, Proteus2.Inoculating loop, Inoculating needle, Alcohol lamp, Matches, Marking pencil.Methods: A. Liquid Medium Inoculation: Procedures:1. Take up the loop by the handle and hold it as holding a pencil, loop down. Put the wire in the flame of the alcohol lamp (see Fig.1-1) until it turns red. Remove the loop from the flame and hold it until it becomes cool (Do not put it down or touch it to anything).Fig.1-1 Flaming the loop 2. Pick up the slant culture of E. coli ( or B. Subtilis) with your left hand, and use the little finger of the loop hand to remove the cotton plug off the culture tube. Keep your little finger curled around this cotton plug. Note: Dont place it on the table.3. Pass the neck of the open tube rapidly through the flame two or three times. This flaming sterilizes the air in and immediately around the mouth of the tube.4. Insert the loop into the open tube (holding both horizontally). Touch the loop to the growth on the slant and remove a loopful of culture. Note: Dont dig the loop into the agar. (see Fig. 1-2) Fig. 1-2. Get some culture out of a slant. Note: the tube is held horizontally. 5. Withdraw the loop slowly and steadily, being careful not to touch it to the mouth of the tube. Keep it steadily, and do not touch anything while you replace the tube closure. Put the tube back in the rack. 6. Use the other hand to pick up a tube of sterile nutrient broth, remove the tube closure. Flame the neck of the tube; insert the loop into the tube and into the broth. Gently rub the loop against the wall of the tube. 7. Withdraw the loop, flame the tube neck, replace the closure, put the tube back in the rack. Then carefully flame the loop, holding it first in the coolest part of the flame (yellow), then in the hot blue cone until it glows. When the wire becomes cool, the loop can be placed on the bench top. 8. Label the tube you have just inoculated with your name, the name of the microorganism, and the date.9. Incubate the tubes overnight at 37. B. Slant Inoculation: Procedures: 1. Flame the loop. 2. Pick up the slant culture of E. coli, open it, flame, and take some growth with the sterile loop. 3. Pick up a nutrient agar slant. Open and flame it as above. 4. Introduce the loop into the tube. Streak the surface of the plate from bottom to top. Then withdraw the loop from the tube without touching its inner surface. 5. Flame, close, and replace the inoculated tube in the rack, then sterilize the loop as above. 6. Label the freshly inoculated tube and then incubate it overnight at 37. C. Semisolid Medium Inoculation:Procedures: 1. Flame the inoculating needle. 2. Pick up the slant culture of S.dysenteriae (or Proteus), open and flame it, then take some growth with the sterile needle. 3. Pick up a tube. Open and flame it as above. 4. Stab the needle into the semisolid medium without touching the bottom of the tube, then retrace the way back. 5. Flame, close and replace the inoculated tube in the rack.6. Label the tube and incubate it overnight at 37.D. Streak Plate: A pure culture contains a single kind of microorganism. The pure culture of bacteria alone can be widely used in the following studies: morphologic; cultural; physiological or biochemical; pathogenic; serologic; genetic; and bacteriophage typing. A pure culture can be obtained from mixture culture or clinical specimens by plating. In this experiment you will prepare a streak plate with four-area streaking pattern. (see Fig. 1-3) 1.With a sterile loop, transfer 2.Rotate the plate 90. Streak a loopful of culture to a corner from the first quadrant into of the first quadrant. Spread the the second quadrant. This is culture over 1/10 of the plate streak area 2. It accounts for surface. This is streak area 1. 1/5 of the plate. 3.Flame the loop and rotate the 4.Rotate the plate 90again and plate 90. Repeat the streaking continue the streaking from from the second quadrant into the streak area 3 into the rest area third quadrant. This is streak of the plate. This is streakarea 3. It occupies 1/4 of the area 4. plate.Fig. 1-3 Method to obtain pure culture Procedures: 1. Label plate. 2. Streak the plate according to the steps above. 3. Incubate the plate overnight in an inverted position at 37.After incubation, isolated colonies should be seen in the last area of the streaked plate.Experiment 2 Observation Morphology of BacteriaMaterials and Equipment:1.Smears: Basic shape of the Bacteria: 1) Cocci Staphylococci, Streptococci, Streptococcus pneumoniae 2) Bacilli Escherichia coli, Bacillus subtilis 3) Spirilla Vibrio choleraSpecial Structures of the bacteria 1) Capsule Streptococcus pneumoniae, Bacillus anthrax 2) Spore (or Endospore) Clostridium tetani 3) Flagella Proteus2.Instrament:Light Microscope (oil-immersion lens)Experiment 3 Gram StainIntroduction: Gram stain has become one of the most useful tools of microbiology. In the diagnostic laboratory, it is used not only for the study of microorganisms in cultures, but it is also often applied to smears made directly from clinical specimens. Direct, Gram-stained smears are read promptly to determine the relative number and morphology of bacteria in the specimen. This information is often of value to the physician in planning the patients treatment before culture results are available. It is also of value to the microbiologist, who can plan his/her culture procedures on the basis of his/her knowledge of the bacterial forms he/she has seen in the specimen. Purpose of this experiment is to learn the Gram Stain technique and to understand its value in the study of bacterial morphology.Gram Stain Reagents:ReagentFunctionCrystal violetPrimary stain: stains all bacteria blue to purple.Grams iodineMordant: enhances reaction between cell wall and primary stain.Ethyl alcohol oracetoneDecolorization: Gram-positive bacteria retain the primary stain because of the peptidoglycan and teichoic acid cross links. Gram-negative bacteria lose the primary stain because of the large amount of lipopolysaccharide in the cell wall. Safranin orcarbolfuchsinCounterstain: no effect on Gram-positive bacteria, stain Gram-negative bacteria pink to red.Materials: 1. Plate culture of Staphylococcus aureus and Escherichia coli 2. Crystal violet solution 3. Iodine solution 4. Ethyl alcohol, 95% 5. Safranine solution 6. Normal saline 7. Slides and bibulous paper 8. Marking pencil and label paper 9. Alcohol lampProcedures: 1. Prepare a smear: Emulsify a colony in normal saline on a slide to make a thin layer of smear. Air dry and then pass the smear over flame for three or four times (Note: dont overheat it). On the underside of each slide make a penciled code mark so that you can identify the slide after staining. 2. Stain each smear by the following procedures: 1) Overlay smear with crystal violet solution. Allow it to stand for one minute. 2) Gently wash with tap water. 3) Flood with iodine solution. Leave it for one minute. 4) Gently wash with tap water. 5) Decolorize with alcohol (95%) for about half a minute until no color being washed off. This is a most critical step. Be careful not to overdecolorize it, as many Gram-positive bacteria may lose the primary stain and thus