細(xì)菌在培養(yǎng)基上的培養(yǎng)與分離技術(shù)要領(lǐng)、方法和實(shí)驗(yàn)
發(fā)布時(shí)間：
2022-11-16
作者：
國(guó)內(nèi)培養(yǎng)基供應(yīng)商
內(nèi)容講解摘要：細(xì)菌培養(yǎng)是將細(xì)菌接種到培養(yǎng)基內(nèi)，并在適當(dāng)?shù)沫h(huán)境內(nèi)，使細(xì)菌生長(zhǎng)和繁殖。
分離培養(yǎng)是指從標(biāo)本中培養(yǎng)出細(xì)菌或者從混有多種細(xì)菌的標(biāo)本中將各個(gè)菌種分別同時(shí)培養(yǎng)出來(lái)。
一、基本條件：細(xì)菌實(shí)驗(yàn)室；無(wú)菌實(shí)驗(yàn)室；基本設(shè)備和器具；細(xì)菌實(shí)驗(yàn)室。
標(biāo)本接種、培養(yǎng)、分離、鑒定及藥敏試驗(yàn)等工作都要在此完成。
1.細(xì)菌室必須安裝嚴(yán)密的門(mén)窗，以防室內(nèi)環(huán)境受到外界的污染。且室內(nèi)禁用風(fēng)扇，避免細(xì)菌的播散。
2.細(xì)菌室必須安裝供空氣消毒的紫外線(xiàn)燈，置于操作臺(tái)上面lm處，每天開(kāi)始工作前照射20min。對(duì)其消毒效果要定期檢查，及時(shí)更換失效的燈管。
3.室內(nèi)應(yīng)備有消毒劑，用于試驗(yàn)中發(fā)生菌液灑濺時(shí)的及時(shí)消毒處理。同時(shí)還應(yīng)備有供工作人員浸手用的盛有消毒劑的水盆、肥皂及自來(lái)水源等。
4.室內(nèi)操作臺(tái)須每日用消毒劑擦洗，地面至少一周用消毒劑擦洗l次。
5.對(duì)接收的標(biāo)本、無(wú)菌器具、用過(guò)的物品等應(yīng)明顯分開(kāi)并放在指定位置。同時(shí)要對(duì)用過(guò)的物品及時(shí)進(jìn)行滅菌處理。
6.細(xì)菌室根據(jù)當(dāng)?shù)氐臍鉁靥攸c(diǎn)，安裝空調(diào)機(jī)，以適合細(xì)菌實(shí)驗(yàn)工作。同時(shí)室內(nèi)應(yīng)設(shè)置必要的消防設(shè)備。
無(wú)菌實(shí)驗(yàn)室是細(xì)菌實(shí)驗(yàn)室內(nèi)用于無(wú)菌操作的小室，其內(nèi)部裝飾、消毒條件要求更嚴(yán)格。
1.無(wú)菌室應(yīng)完全封閉，人員出入應(yīng)有兩道門(mén)，其間應(yīng)隔有緩沖區(qū)。
2.用前應(yīng)以紫外線(xiàn)消毒30min，定期用乳酸或甲醛熏蒸，徹底消毒。
3.在無(wú)菌室中一般僅限于分裝無(wú)菌的培養(yǎng)基及傳染性強(qiáng)的細(xì)菌的接種，不進(jìn)行有菌標(biāo)本的分離及其他操作。
4.無(wú)菌室內(nèi)應(yīng)僅限操作人員進(jìn)入，而且進(jìn)入無(wú)菌室應(yīng)著隔離衣和專(zhuān)用鞋，操作時(shí)戴口罩，隨時(shí)保證室內(nèi)的無(wú)菌狀態(tài)。
5.條件有限的實(shí)驗(yàn)室，可用超凈工作臺(tái)代替無(wú)菌實(shí)驗(yàn)室進(jìn)行相應(yīng)的操作。超凈工作臺(tái)應(yīng)選擇垂直氣流通風(fēng)方式。
6.無(wú)菌室應(yīng)配備空調(diào)設(shè)備，保證不因室溫而影響工作。
（三）基本設(shè)備和器具：溫箱、C02培養(yǎng)箱、厭氧培養(yǎng)設(shè)備；顯微鏡；高壓蒸氣滅菌器、干烤箱；冰箱和冷藏柜；接種器具，包括接種環(huán)和接種針；pH計(jì)；火焰燈或酒精燈；平皿、試管、吸管等玻璃器皿，以及離心機(jī)、天平等。
二、細(xì)菌的接種與分離技術(shù)
選擇好合適的培養(yǎng)基，根據(jù)待檢標(biāo)本的來(lái)源、培養(yǎng)目的及所使用培養(yǎng)基的性狀，采用不同的接種方法。
平板劃線(xiàn)分離法:連續(xù)劃線(xiàn)分離法，主要用于雜菌不多的標(biāo)本。分區(qū)劃線(xiàn)分離法，適用于雜菌量較多的標(biāo)本。
斜面接種法，用于單個(gè)菌落的純培養(yǎng)、保存菌種或觀(guān)察細(xì)菌的某些特性。
液體接種法（避免氣溶膠的產(chǎn)生），多用于一些液體生化試驗(yàn)管的接種。
穿刺接種法，此法主要用于半固體培養(yǎng)基、明膠及雙糖管的接種。
傾注平板法，測(cè)定牛乳、飲水和尿液等標(biāo)本細(xì)菌數(shù)時(shí)常用此方法。
涂布接種法，常用于紙片法藥物敏感性測(cè)定，也可用于被檢標(biāo)本中的細(xì)菌計(jì)數(shù)。
三、細(xì)菌培養(yǎng)的方法：需氧培養(yǎng)法；二氧化碳培養(yǎng)法；厭氧培養(yǎng)法
（一）需氧培養(yǎng)法：臨床細(xì)菌室最常用的培養(yǎng)方法，適于一般需氧和兼性厭氧菌的培養(yǎng)。
置于35℃溫箱中孵育18～24h。
（二）二氧化碳培養(yǎng)法：有些細(xì)菌初次分離培養(yǎng)時(shí)須置5%～l0%C02環(huán)境才能生長(zhǎng)良好。
1.二氧化碳培養(yǎng)箱：是一臺(tái)特制的培養(yǎng)箱，既能調(diào)節(jié)CO2的含量，又能調(diào)節(jié)所需的溫度。CO2從鋼瓶通過(guò)培養(yǎng)箱的CO2運(yùn)送管進(jìn)入培養(yǎng)箱內(nèi)，調(diào)節(jié)好所需CO2濃度自動(dòng)控制器后，將接種好的培養(yǎng)基直接放入培養(yǎng)箱中培養(yǎng)即可。適于大型實(shí)驗(yàn)室應(yīng)用。
2.燭缸法：將已接種好的培養(yǎng)基置干燥器內(nèi)，并放入點(diǎn)燃的蠟燭。干燥器蓋的邊緣涂上凡士林，蓋上蓋子，燭光經(jīng)幾分鐘后自行熄滅，此時(shí)干燥器內(nèi)CO2含量約占5%～10%，然后將干燥器放入35℃溫箱內(nèi)培養(yǎng)。培養(yǎng)時(shí)間一般為18～24h，少數(shù)菌種需培養(yǎng)3～7d或更長(zhǎng)。
3.化學(xué)法：按每升容積加入碳酸氫鈉0.4g和濃鹽酸0.35ml的比例，分別置于容器內(nèi)。
（三）厭氧培養(yǎng)法
適用于專(zhuān)性厭氧菌和兼性厭氧菌的培養(yǎng)。
常用的厭氧培養(yǎng)法有：
①皰肉培養(yǎng)基法；②焦性沒(méi)食子酸法；③厭氧罐法；④氣袋法；⑤厭氧手套箱法。
四、細(xì)菌的生長(zhǎng)現(xiàn)象：細(xì)菌在固體培養(yǎng)基上的生長(zhǎng)現(xiàn)象；細(xì)菌在液體培養(yǎng)基中的生長(zhǎng)現(xiàn)象；細(xì)菌在半固體培養(yǎng)基中的生長(zhǎng)現(xiàn)象；細(xì)菌在固體培養(yǎng)基上的生長(zhǎng)現(xiàn)象。
1.觀(guān)察菌落；2.血瓊脂上的溶血；3.氣味；4.色素
菌落特征包括大小、形狀、突起、邊緣、顏色、表面、透明度和粘度等。
根據(jù)細(xì)菌菌落表面特征不同，可將菌落分為三型：
光滑型（S型）菌落；粗糙型（R型）菌落；粘液型（M型）菌落
2.血瓊脂上的溶血：
α溶血：又叫草綠色溶血，菌落周?chē)囵B(yǎng)基變?yōu)榫G色環(huán)狀。
β溶血：又稱(chēng)完全溶血，菌落周?chē)纬梢粋€(gè)完全清晰透明的環(huán)。
γ溶血：即不溶血，菌落周?chē)呐囵B(yǎng)基沒(méi)有變化；紅細(xì)胞沒(méi)有溶解或無(wú)缺損。
雙環(huán)：上層溶血環(huán)，內(nèi)層為β溶血，外層為α溶血。如產(chǎn)氣莢膜梭菌。
3.氣味：通過(guò)某些細(xì)菌在平皿培養(yǎng)基上代謝活動(dòng)產(chǎn)生的氣味，有助于細(xì)菌的鑒定。
如銅綠假單胞菌（生姜?dú)馕叮?、變形桿菌（巧克力燒焦氣味）、厭氧梭菌（腐敗的惡臭味）、放線(xiàn)菌（泥土味）等。
（二）細(xì)菌在液體培養(yǎng)基中的生長(zhǎng)現(xiàn)象
渾濁生長(zhǎng)：大多數(shù)細(xì)菌。
沉淀生長(zhǎng)：少數(shù)鏈狀排列的細(xì)菌如鏈球菌、炭疽芽胞桿菌。
菌膜生長(zhǎng)（表面生長(zhǎng)）：專(zhuān)性需氧菌如枯草芽胞桿菌、結(jié)核分枝桿菌和銅綠假單胞菌。
（三）細(xì)菌在半固體培養(yǎng)基中的生長(zhǎng)現(xiàn)象
半固體培養(yǎng)基用于觀(guān)察細(xì)菌的動(dòng)力。
有鞭毛的細(xì)菌除了在穿刺接種的穿刺線(xiàn)上生長(zhǎng)外，在穿刺線(xiàn)的兩側(cè)均可見(jiàn)羽毛狀或云霧狀渾濁生長(zhǎng)，為動(dòng)力陽(yáng)性。
無(wú)鞭毛的細(xì)菌只沿穿刺線(xiàn)呈明顯的線(xiàn)狀生長(zhǎng)，為動(dòng)力試驗(yàn)陰性。
五、細(xì)菌L型的檢查：表現(xiàn)為形態(tài)多形性、染色不確定性、可濾過(guò)性、滲透壓敏感性，生化反應(yīng)減弱特性以及對(duì)β-內(nèi)酰胺類(lèi)和其他作用細(xì)胞壁抗生素的抵抗性。
1.標(biāo)本采集
應(yīng)盡量采集無(wú)雜菌污染的組織或體液標(biāo)本。
胸水、腹水及尿液標(biāo)本，應(yīng)加20%蔗糖無(wú)菌溶液，以保持高滲；血液標(biāo)本應(yīng)接種高滲肉湯增菌培養(yǎng)。
2.培養(yǎng)方法——
（1）L型檢查程序：
將標(biāo)本接種到高滲肉湯增菌培養(yǎng)1～7天，然后轉(zhuǎn)種L型平板和血平板37℃培養(yǎng)2～7天。
典型菌落為“荷包蛋”樣，但從患者標(biāo)本中新分離的L型菌落常不典型，多呈顆粒型菌落，涂片染色為多形性。
（2）檢驗(yàn)報(bào)告：
①血平板無(wú)菌生長(zhǎng)，L型平板有菌落生長(zhǎng)，可報(bào)告檢出細(xì)菌L型；
②血平板中菌落細(xì)小，不易刮下。涂片檢查細(xì)菌呈多形性，細(xì)胞壁缺損，L型平板中有L型菌落，報(bào)告檢出L型；
③血平板及L型平板均有菌落生長(zhǎng)。涂片有原菌及L型兩種形態(tài)特征，可報(bào)告細(xì)菌型及L型同時(shí)存在，并分別作藥敏試驗(yàn)以供臨床用藥參考。
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.