细菌在培养基上的培养与分离技术要领、方法和实验
发布时间：
2022-11-16
作者：
国内培养基供应商
内容讲解摘要：细菌培养是将细菌接种到培养基内，并在适当的环境内，使细菌生长和繁殖。
分离培养是指从标本中培养出细菌或者从混有多种细菌的标本中将各个菌种分别同时培养出来。
一、基本条件：细菌实验室；无菌实验室；基本设备和器具；细菌实验室。
标本接种、培养、分离、鉴定及药敏试验等工作都要在此完成。
1.细菌室必须安装严密的门窗，以防室内环境受到外界的污染。且室内禁用风扇，避免细菌的播散。
2.细菌室必须安装供空气消毒的紫外线灯，置于操作台上面lm处，每天开始工作前照射20min。对其消毒效果要定期检查，及时更换失效的灯管。
3.室内应备有消毒剂，用于试验中发生菌液洒溅时的及时消毒处理。同时还应备有供工作人员浸手用的盛有消毒剂的水盆、肥皂及自来水源等。
4.室内操作台须每日用消毒剂擦洗，地面至少一周用消毒剂擦洗l次。
5.对接收的标本、无菌器具、用过的物品等应明显分开并放在指定位置。同时要对用过的物品及时进行灭菌处理。
6.细菌室根据当地的气温特点，安装空调机，以适合细菌实验工作。同时室内应设置必要的消防设备。
无菌实验室是细菌实验室内用于无菌操作的小室，其内部装饰、消毒条件要求更严格。
1.无菌室应完全封闭，人员出入应有两道门，其间应隔有缓冲区。
2.用前应以紫外线消毒30min，定期用乳酸或甲醛熏蒸，彻底消毒。
3.在无菌室中一般仅限于分装无菌的培养基及传染性强的细菌的接种，不进行有菌标本的分离及其他操作。
4.无菌室内应仅限操作人员进入，而且进入无菌室应着隔离衣和专用鞋，操作时戴口罩，随时保证室内的无菌状态。
5.条件有限的实验室，可用超净工作台代替无菌实验室进行相应的操作。超净工作台应选择垂直气流通风方式。
6.无菌室应配备空调设备，保证不因室温而影响工作。
（三）基本设备和器具：温箱、C02培养箱、厌氧培养设备；显微镜；高压蒸气灭菌器、干烤箱；冰箱和冷藏柜；接种器具，包括接种环和接种针；pH计；火焰灯或酒精灯；平皿、试管、吸管等玻璃器皿，以及离心机、天平等。
二、细菌的接种与分离技术
选择好合适的培养基，根据待检标本的来源、培养目的及所使用培养基的性状，采用不同的接种方法。
平板划线分离法:连续划线分离法，主要用于杂菌不多的标本。分区划线分离法，适用于杂菌量较多的标本。
斜面接种法，用于单个菌落的纯培养、保存菌种或观察细菌的某些特性。
液体接种法（避免气溶胶的产生），多用于一些液体生化试验管的接种。
穿刺接种法，此法主要用于半固体培养基、明胶及双糖管的接种。
倾注平板法，测定牛乳、饮水和尿液等标本细菌数时常用此方法。
涂布接种法，常用于纸片法药物敏感性测定，也可用于被检标本中的细菌计数。
三、细菌培养的方法：需氧培养法；二氧化碳培养法；厌氧培养法
（一）需氧培养法：临床细菌室最常用的培养方法，适于一般需氧和兼性厌氧菌的培养。
置于35℃温箱中孵育18～24h。
（二）二氧化碳培养法：有些细菌初次分离培养时须置5%～l0%C02环境才能生长良好。
1.二氧化碳培养箱：是一台特制的培养箱，既能调节CO2的含量，又能调节所需的温度。CO2从钢瓶通过培养箱的CO2运送管进入培养箱内，调节好所需CO2浓度自动控制器后，将接种好的培养基直接放入培养箱中培养即可。适于大型实验室应用。
2.烛缸法：将已接种好的培养基置干燥器内，并放入点燃的蜡烛。干燥器盖的边缘涂上凡士林，盖上盖子，烛光经几分钟后自行熄灭，此时干燥器内CO2含量约占5%～10%，然后将干燥器放入35℃温箱内培养。培养时间一般为18～24h，少数菌种需培养3～7d或更长。
3.化学法：按每升容积加入碳酸氢钠0.4g和浓盐酸0.35ml的比例，分别置于容器内。
（三）厌氧培养法
适用于专性厌氧菌和兼性厌氧菌的培养。
常用的厌氧培养法有：
①疱肉培养基法；②焦性没食子酸法；③厌氧罐法；④气袋法；⑤厌氧手套箱法。
四、细菌的生长现象：细菌在固体培养基上的生长现象；细菌在液体培养基中的生长现象；细菌在半固体培养基中的生长现象；细菌在固体培养基上的生长现象。
1.观察菌落；2.血琼脂上的溶血；3.气味；4.色素
菌落特征包括大小、形状、突起、边缘、颜色、表面、透明度和粘度等。
根据细菌菌落表面特征不同，可将菌落分为三型：
光滑型（S型）菌落；粗糙型（R型）菌落；粘液型（M型）菌落
2.血琼脂上的溶血：
α溶血：又叫草绿色溶血，菌落周围血培养基变为绿色环状。
β溶血：又称完全溶血，菌落周围形成一个完全清晰透明的环。
γ溶血：即不溶血，菌落周围的培养基没有变化；红细胞没有溶解或无缺损。
双环：上层溶血环，内层为β溶血，外层为α溶血。如产气荚膜梭菌。
3.气味：通过某些细菌在平皿培养基上代谢活动产生的气味，有助于细菌的鉴定。
如铜绿假单胞菌（生姜气味）、变形杆菌（巧克力烧焦气味）、厌氧梭菌（腐败的恶臭味）、放线菌（泥土味）等。
（二）细菌在液体培养基中的生长现象
浑浊生长：大多数细菌。
沉淀生长：少数链状排列的细菌如链球菌、炭疽芽胞杆菌。
菌膜生长（表面生长）：专性需氧菌如枯草芽胞杆菌、结核分枝杆菌和铜绿假单胞菌。
（三）细菌在半固体培养基中的生长现象
半固体培养基用于观察细菌的动力。
有鞭毛的细菌除了在穿刺接种的穿刺线上生长外，在穿刺线的两侧均可见羽毛状或云雾状浑浊生长，为动力阳性。
无鞭毛的细菌只沿穿刺线呈明显的线状生长，为动力试验阴性。
五、细菌L型的检查：表现为形态多形性、染色不确定性、可滤过性、渗透压敏感性，生化反应减弱特性以及对β-内酰胺类和其他作用细胞壁抗生素的抵抗性。
1.标本采集
应尽量采集无杂菌污染的组织或体液标本。
胸水、腹水及尿液标本，应加20%蔗糖无菌溶液，以保持高渗；血液标本应接种高渗肉汤增菌培养。
2.培养方法——
（1）L型检查程序：
将标本接种到高渗肉汤增菌培养1～7天，然后转种L型平板和血平板37℃培养2～7天。
典型菌落为“荷包蛋”样，但从患者标本中新分离的L型菌落常不典型，多呈颗粒型菌落，涂片染色为多形性。
（2）检验报告：
①血平板无菌生长，L型平板有菌落生长，可报告检出细菌L型；
②血平板中菌落细小，不易刮下。涂片检查细菌呈多形性，细胞壁缺损，L型平板中有L型菌落，报告检出L型；
③血平板及L型平板均有菌落生长。涂片有原菌及L型两种形态特征，可报告细菌型及L型同时存在，并分别作药敏试验以供临床用药参考。
Culture and isolation of bacteria
Abstract: Bacterial culture is to inoculate bacteria into the medium, and in the appropriate environment, so that bacteria grow and reproduce.
Isolation and culture refers to the cultivation of bacteria from specimens or the simultaneous cultivation of bacteria from a mixture of bacteria.
I. Basic Conditions: bacteria laboratory; aseptic laboratory; basic equipment and apparatus; bacteriological laboratory.
The work of specimen inoculation, culture, isolation, identification and drug sensitivity test should be completed here.
1. bacteriology room must install tight doors and windows to prevent indoor environment from being polluted by outside. In addition, the fan is forbidden in the room to avoid the spread of bacteria.
2. Bacteria room must be equipped with ultraviolet lamp for air disinfection, placed on the operation table above the lm, 20 minutes a day before starting work. The disinfection effect should be checked regularly, and the invalid lamp tube should be replaced in time.
3. disinfectants should be provided in the room for timely disinfection during spillage of bacteria. At the same time, there should be a water bath, soap and water for disinfectant.
4. the indoor operating platform must be scrubbed daily with disinfectant. The floor should be scrubbed l times with disinfectant for at least one week.
5. the received specimens, aseptic appliances, used articles and so on should be clearly separated and placed in the designated position. At the same time, we must sterilize the used articles in time.
6. the bacteriology room installed air conditioners according to the local air temperature characteristics, so as to be suitable for bacteriological experiment work. At the same time, the necessary fire-fighting equipment should be set up in the room.
Aseptic laboratory is a sterile room used in bacteriological laboratory, and its interior decoration and disinfection requirements are stricter.
1. asepsis room should be completely closed. There should be two doors in which the buffer zone should be separated.
2. 30min should be sterilized by ultraviolet light before being fumigated with lactic acid or formaldehyde regularly and thoroughly sterilized.
3. In the sterile room, the sterile medium and the inoculation of infectious bacteria are generally limited, and the isolation of bacterial specimens and other operations are not carried out.
4. the aseptic room should be limited to the operator, and enter the asepsis room with the isolation clothes and special shoes, wear a mask when operating, and keep the aseptic state of the room at any time.
5. in limited laboratory, ultra clean workbench can be used instead of aseptic Laboratory for corresponding operation. The ultra clean workbench should choose vertical airflow ventilation mode.
6. asepsis room should be equipped with air conditioning equipment to ensure that it does not affect the work due to room temperature.
(three) basic equipment and appliances: temperature box, C02 incubator, anaerobic culture equipment; microscope; high pressure steam sterilizer, dry oven; refrigerator and refrigerator; inoculation equipment, including inoculation ring and inoculation needle; pH meter; flame lamp or alcohol lamp; plate, test tube, pipe, etc., and centrifuge, balance and so on.
Two, the technique of inoculation and isolation of bacteria
Choose a suitable medium, according to the source of the specimen to be examined, the purpose of culture and the characteristics of the medium used, using different methods of grafting.
Plate streak separation method: continuous line separation method, mainly used for not many specimens. The zoning method is suitable for the specimens with high quantity of bacteria.
The method of oblique inoculation is used for pure culture of single colonies, preservation of bacteria or observation of certain characteristics of bacteria.
The liquid inoculation method (avoiding aerosol generation) is used for the inoculation of some liquid biochemical test tubes.
The inoculation method is mainly used for inoculation of semisolid medium, gelatin and disaccharide tube.
This method is often used to determine the number of bacteria in milk, drinking water and urine.
The coating inoculation method is commonly used for the determination of drug sensitivity by paper method, and can also be used for counting bacteria in the specimens examined.
Three. Methods of bacterial culture: aerobic culture; carbon dioxide culture; anaerobic culture.
(1) aerobic culture: the most commonly used culture method in clinical bacteriology room is suitable for the cultivation of general aerobic and facultative anaerobe.
It was incubated for 18 ~ 24h at 35 centigrade temperature box.
(two) carbon dioxide culture: when some bacteria first isolated and cultured, they must be placed in a 5% to l0%C02 environment to grow well.
1. carbon dioxide incubator: it is a special incubator, which not only regulates the content of CO2, but also adjusts the required temperature. CO2 comes into the incubator from the CO2 transport tube through the incubator and regulates the required CO2 concentration automatic controller. The inoculated medium is directly put into the incubator to be cultured. Suitable for large laboratory applications.
2. candle barrel method: put the inoculated medium into the dryer and put the lighted candle. The edge of the dryer cover is coated with Vaseline, covered with cover, and the candlelight is extinguished by itself after a few minutes. At this time, the CO2 content in the dryer is about 5% ~ 10%, and then the dryer is put into the temperature box of 35 C. The incubation time is generally 18 to 24h, and a few strains need to grow 3 to 7d or longer.
3. chemical method: add the ratio of sodium bicarbonate 0.4g and concentrated hydrochloric acid 0.35ml to the container according to the volume of each litre.
(three) anaerobic culture
It is suitable for the cultivation of obligate anaerobe and facultative anaerobe.
The commonly used anaerobic culture methods are:
(1) blister culture medium; (2) pyrogallol method; (3) anaerobic tank method; (4) air bag method; and anaerobic glove box method.
Four. Growth of bacteria: growth of bacteria on solid medium; growth of bacteria in liquid medium; growth of bacteria in semisolid medium; growth of bacteria on solid medium.
1. observe colony; 2. blood agar hemolysis; 3. odour; 4. pigment.
Colony characteristics include size, shape, protuberance, edge, color, surface, diaphaneity and viscosity.
According to the different surface characteristics of bacterial colonies, the colonies can be divided into three types:
Smooth type (S) colonies; rough (type R) colonies; mucoid (type M) colonies.
Hemolysis on 2. blood agar:
Alpha hemolysis: also called grass green hemolysis, the blood culture medium around the colony becomes green ring.
Beta hemolysis: also known as complete hemolysis, forming a completely clear and transparent ring around the colony.
Gamma hemolysis: that is, no hemolysis, no change in the medium around the colony, and no dissolution or no defect in the red blood cells.
Double ring: upper layer hemolytic ring, inner layer is beta hemolysis, outer layer is alpha hemolysis. Such as Clostridium perfringens.
3. odour: the odor produced by certain bacteria on the plate culture medium helps to identify bacteria.
Such as Pseudomonas aeruginosa (Pseudomonas aeruginosa), proteus (chocolate burning smell), anaerobic Clostridium (odour odor of corruption), actinomycetes (soil flavor) and so on.
(two) growth of bacteria in liquid medium
Turbid growth: most bacteria.
Precipitation growth: a few chained bacteria, such as Streptococcus and Bacillus anthracis.
Membrane growth (surface growth): obligate aerobic bacteria such as Bacillus subtilis, Mycobacterium tuberculosis and Pseudomonas aeruginosa.
(three) growth of bacteria in semisolid medium
The semisolid medium is used to observe the dynamics of bacteria.
In addition to the growth of flagellated bacteria on the puncture line, there are feathery or cloudy growth on both sides of the puncture line, which is dynamic positive.
Flagellate bacteria only grow along the puncture line in a clear linear manner, which is negative for the dynamic test.
Five. The examination of bacterial type L: morphological polymorphism, dyed uncertainty, filtration, osmotic sensitivity, attenuation of biochemical reaction and resistance to beta lactam and other action cell wall antibiotics.
1. specimen collection
We should try to collect specimens of tissues or body fluids that are not contaminated by heterozygous bacteria.
The samples of pleural effusion, ascites and urine should be treated with 20% sucrose aseptic solution to maintain hyperosmotic, and the blood samples should be inoculated with hyperosmotic broth to increase bacterial culture.
2. culture method --
(1) type L inspection procedure:
The specimens were inoculated with hypertonic meat soup for 1~7 days, then transferred to L plate and blood plate for 37 days for 2~7 days.
Typical colonies are "pooled egg" like, but the newly isolated L-type colonies from patients'specimens are often atypical, mostly granular and polymorphic.
(2) inspection report:
1. Aseptic growth of blood plate, colony growth of type L plates, bacterial L type can be reported.
2. In the blood plate, the colony is small and it is not easy to scrape down. Smears showed that the bacteria were polymorphic and cell wall defective, and L type colonies were found in the L type plates. L type was reported.
The growth of bacterial colonies was found in both the blood plate and the L type. There are two morphological characteristics of bacteria and L-forms on the smears, which can report the coexistence of bacteria and L-forms, and make drug susceptibility tests for clinical reference.