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      細菌在培養基上的培養與分離技術要領、方法和實驗

      發布時間:

      2022-11-16

      作者:

      國內培養基供應商


      內容講解摘要:細菌培養是將細菌接種到培養基內,并在適當的環境內,使細菌生長和繁殖。
      分離培養是指從標本中培養出細菌或者從混有多種細菌的標本中將各個菌種分別同時培養出來。
      一、基本條件:細菌實驗室;無菌實驗室;基本設備和器具;細菌實驗室。
      標本接種、培養、分離、鑒定及藥敏試驗等工作都要在此完成。
      1.細菌室必須安裝嚴密的門窗,以防室內環境受到外界的污染。且室內禁用風扇,避免細菌的播散。 
      2.細菌室必須安裝供空氣消毒的紫外線燈,置于操作臺上面lm處,每天開始工作前照射20min。對其消毒效果要定期檢查,及時更換失效的燈管。
      3.室內應備有消毒劑,用于試驗中發生菌液灑濺時的及時消毒處理。同時還應備有供工作人員浸手用的盛有消毒劑的水盆、肥皂及自來水源等。
      4.室內操作臺須每日用消毒劑擦洗,地面至少一周用消毒劑擦洗l次。
      5.對接收的標本、無菌器具、用過的物品等應明顯分開并放在指定位置。同時要對用過的物品及時進行滅菌處理。
      6.細菌室根據當地的氣溫特點,安裝空調機,以適合細菌實驗工作。同時室內應設置必要的消防設備。
      無菌實驗室是細菌實驗室內用于無菌操作的小室,其內部裝飾、消毒條件要求更嚴格。
      1.無菌室應完全封閉,人員出入應有兩道門,其間應隔有緩沖區。
      2.用前應以紫外線消毒30min,定期用乳酸或甲醛熏蒸,徹底消毒。
      3.在無菌室中一般僅限于分裝無菌的培養基及傳染性強的細菌的接種,不進行有菌標本的分離及其他操作。
      4.無菌室內應僅限操作人員進入,而且進入無菌室應著隔離衣和專用鞋,操作時戴口罩,隨時保證室內的無菌狀態。
      5.條件有限的實驗室,可用超凈工作臺代替無菌實驗室進行相應的操作。超凈工作臺應選擇垂直氣流通風方式。
      6.無菌室應配備空調設備,保證不因室溫而影響工作。
      (三)基本設備和器具:溫箱、C02培養箱、厭氧培養設備;顯微鏡;高壓蒸氣滅菌器、干烤箱;冰箱和冷藏柜;接種器具,包括接種環和接種針;pH計;火焰燈或酒精燈;平皿、試管、吸管等玻璃器皿,以及離心機、天平等。
      二、細菌的接種與分離技術
      選擇好合適的培養基,根據待檢標本的來源、培養目的及所使用培養基的性狀,采用不同的接種方法。 
      平板劃線分離法:連續劃線分離法,主要用于雜菌不多的標本。分區劃線分離法,適用于雜菌量較多的標本。
      斜面接種法,用于單個菌落的純培養、保存菌種或觀察細菌的某些特性。
      液體接種法(避免氣溶膠的產生),多用于一些液體生化試驗管的接種。
      穿刺接種法,此法主要用于半固體培養基、明膠及雙糖管的接種。
      傾注平板法,測定牛乳、飲水和尿液等標本細菌數時常用此方法。
      涂布接種法,常用于紙片法藥物敏感性測定,也可用于被檢標本中的細菌計數。
       
      三、細菌培養的方法:需氧培養法;二氧化碳培養法;厭氧培養法
      (一)需氧培養法:臨床細菌室最常用的培養方法,適于一般需氧和兼性厭氧菌的培養。
      置于35℃溫箱中孵育18~24h。
      (二)二氧化碳培養法:有些細菌初次分離培養時須置5%~l0%C02環境才能生長良好。
      1.二氧化碳培養箱:是一臺特制的培養箱,既能調節CO2的含量,又能調節所需的溫度。CO2從鋼瓶通過培養箱的CO2運送管進入培養箱內,調節好所需CO2濃度自動控制器后,將接種好的培養基直接放入培養箱中培養即可。適于大型實驗室應用。
      2.燭缸法:將已接種好的培養基置干燥器內,并放入點燃的蠟燭。干燥器蓋的邊緣涂上凡士林,蓋上蓋子,燭光經幾分鐘后自行熄滅,此時干燥器內CO2含量約占5%~10%,然后將干燥器放入35℃溫箱內培養。培養時間一般為18~24h,少數菌種需培養3~7d或更長。
      3.化學法: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。
      (三)厭氧培養法
      適用于專性厭氧菌和兼性厭氧菌的培養。
      常用的厭氧培養法有:
      ①皰肉培養基法;②焦性沒食子酸法;③厭氧罐法;④氣袋法;⑤厭氧手套箱法。
       
      四、細菌的生長現象:細菌在固體培養基上的生長現象;細菌在液體培養基中的生長現象;細菌在半固體培養基中的生長現象;細菌在固體培養基上的生長現象。
      1.觀察菌落;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。
      根據細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,菌落周圍血培養基變為綠色環狀。
      β溶血:又稱完全溶血,菌落周圍形成一個完全清晰透明的環。
      γ溶血:即不溶血,菌落周圍的培養基沒有變化;紅細胞沒有溶解或無缺損。
      雙環:上層溶血環,內層為β溶血,外層為α溶血。如產氣莢膜梭菌。
      細菌的生長現象圖_青島日水生物_平皿培養基圖
      3.氣味:通過某些細菌在平皿培養基上代謝活動產生的氣味,有助于細菌的鑒定。
      如銅綠假單胞菌(生姜氣味)、變形桿菌(巧克力燒焦氣味)、厭氧梭菌(腐敗的惡臭味)、放線菌(泥土味)等。
      (二)細菌在液體培養基中的生長現象
      渾濁生長:大多數細菌。
      沉淀生長:少數鏈狀排列的細菌如鏈球菌、炭疽芽胞桿菌。
      菌膜生長(表面生長):專性需氧菌如枯草芽胞桿菌、結核分枝桿菌和銅綠假單胞菌。
      (三)細菌在半固體培養基中的生長現象
      半固體培養基用于觀察細菌的動力。
      有鞭毛的細菌除了在穿刺接種的穿刺線上生長外,在穿刺線的兩側均可見羽毛狀或云霧狀渾濁生長,為動力陽性。
       無鞭毛的細菌只沿穿刺線呈明顯的線狀生長,為動力試驗陰性。
       
      五、細菌L型的檢查:表現為形態多形性、染色不確定性、可濾過性、滲透壓敏感性,生化反應減弱特性以及對β-內酰胺類和其他作用細胞壁抗生素的抵抗性。
      1.標本采集
      應盡量采集無雜菌污染的組織或體液標本。
      胸水、腹水及尿液標本,應加20%蔗糖無菌溶液,以保持高滲;血液標本應接種高滲肉湯增菌培養。
      2.培養方法——
      (1)L型檢查程序:
      將標本接種到高滲肉湯增菌培養1~7天,然后轉種L型平板和血平板37℃培養2~7天。
      典型菌落為“荷包蛋”樣,但從患者標本中新分離的L型菌落常不典型,多呈顆粒型菌落,涂片染色為多形性。
      (2)檢驗報告:
      ①血平板無菌生長,L型平板有菌落生長,可報告檢出細菌L型;
      ②血平板中菌落細小,不易刮下。涂片檢查細菌呈多形性,細胞壁缺損,L型平板中有L型菌落,報告檢出L型;
      ③血平板及L型平板均有菌落生長。涂片有原菌及L型兩種形態特征,可報告細菌型及L型同時存在,并分別作藥敏試驗以供臨床用藥參考。
      日水培養基公司圖片

      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.
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