簡述：培養(yǎng)基定義與分類
發(fā)布時間：
2022-11-16
作者：
買賣培養(yǎng)基的公司
簡述：培養(yǎng)基定義與分類
培養(yǎng)基是做什么用的？培養(yǎng)基(medium)培養(yǎng)基是人工配制的適合微生物生長繁殖或積累代謝產(chǎn)物的營養(yǎng)基質(zhì)。任何培養(yǎng)基中均需含有微生物所必須的能源、碳源、氮源、礦質(zhì)元素、水和生長因素，但不同營養(yǎng)類型、不同種類的微生物對營養(yǎng)元素的要求又有很大差異。目前已使用的各種培養(yǎng)基都是前人經(jīng)過反復(fù)實踐，比較設(shè)計的成果。
培養(yǎng)基的基本成分
能源、碳源：自養(yǎng)微生物以二氧化碳為碳源，光或無機物為能源，在無機物組成的培養(yǎng)基中生長。例如化能自養(yǎng)型的氧化硫桿菌培養(yǎng)基中，加進粉末狀硫為能源，以空氣中的CO2為碳源。異養(yǎng)微生物以有機物為碳源和能源，培養(yǎng)基中常需加進葡萄糖、蔗糖或麥芽糖、乳糖等單糖或雙糖，有的可利用淀粉、纖維素等多糖，或利用動物組織中的糖類。例如培養(yǎng)細菌常用的牛肉膏蛋白胨培養(yǎng)基，其中牛肉膏即為主要碳源和能源。(培養(yǎng)基配方見后，下同)。
氮源：自養(yǎng)微生物以含氮無機物銨鹽、硝酸鹽等為氮素營養(yǎng)，例如氧化硫桿菌以(NH4)2SO4為氮源；異養(yǎng)微生物以無機物銨鹽、硝酸鹽或含氮有機物為氮源。自生固氮菌利用空氣中的N2為氮素營養(yǎng)，其培養(yǎng)基中無須加進氮源，稱為無氮培養(yǎng)基。
礦質(zhì)元素、生長因子：微生物生長繁殖需要P、K、Na、S、Mg、Ca等主要礦質(zhì)元素以及Fe、Cu等微量元素，因此培養(yǎng)基中常加進K2HPO4、KH2PO4、MgSO4、NaCl、KCl、FeSO4等無機鹽類；生長因子主要是調(diào)節(jié)微生物代謝活動的B族維生素，常由酵母膏、肝浸出液等提供。很多自然成分的原料如牛肉膏、麥芽汁、玉米粉、芽菜汁等，含有各種無機元素和生長因子，不需另外添加。
培養(yǎng)基的類型
根據(jù)原料來源不同，可將培養(yǎng)基分為合成培養(yǎng)基、半合成培養(yǎng)基與自然培養(yǎng)基。
合成培養(yǎng)基：由化學(xué)成分已知的有機物和無機物配制而成。成分精確，重復(fù)性強。但營養(yǎng)局限，微生物生長緩慢。適用于菌種分離、選育、遺傳分析及生物測定等。如培養(yǎng)放線菌的高氏培養(yǎng)基、培養(yǎng)霉菌的察氏培養(yǎng)基以及各種化能自養(yǎng)菌培養(yǎng)基等。
半合成培養(yǎng)基：由某些自然物質(zhì)與少量已知成分的化學(xué)物質(zhì)配制而成。營養(yǎng)全面，能有效地滿足微生物對營養(yǎng)的需求。廣泛應(yīng)用于微生物的培養(yǎng)。如培養(yǎng)細菌用的牛肉膏蛋白胨培養(yǎng)基，培養(yǎng)霉菌的土豆葡萄糖培養(yǎng)基，產(chǎn)業(yè)生產(chǎn)中常用的玉米粉等自然物質(zhì)加無機鹽配制的各種發(fā)酵培養(yǎng)基等。
自然培養(yǎng)基：由化學(xué)成分不清楚或不衡定的自然有機物配制而成。成分復(fù)雜，但營養(yǎng)豐富全面。常用于實驗研究和生產(chǎn)。如麥芽汁培養(yǎng)基、玉米粉培養(yǎng)基，以及生產(chǎn)中使用的麩皮、鋸末等。
根據(jù)培養(yǎng)基的物理性質(zhì)，可分為液體培養(yǎng)基、固體培養(yǎng)基和半固體培養(yǎng)基。
液體培養(yǎng)基：用各種營養(yǎng)成分加水配成，或用自然物質(zhì)的浸汁(麥芽汁、芽菜汁等)制成。組分均一，適宜各類微生物的營養(yǎng)生長。廣泛應(yīng)用于實驗研究及大規(guī)模產(chǎn)業(yè)生產(chǎn)中，有利于廣泛獲得大量菌體或代謝產(chǎn)物。
固體培養(yǎng)基：在液體培養(yǎng)基中加進凝固劑，或用麩皮等固體原料配制。常用的凝固劑是瓊脂(又稱瓊膠、洋菜)，由石花菜等海藻中提取加工制成。市售瓊脂為條狀、片狀或粉末狀，主要成分為多聚半乳糖的硫酸酯，盡大多數(shù)微生物不能將其分解，在培養(yǎng)基中僅起支撐作用。其熔點約98℃，凝固點42℃，1.5～2％的水溶液在一般培養(yǎng)溫度下呈凝膠狀態(tài)。瓊脂固體培養(yǎng)基廣泛應(yīng)用于微生物的分離培養(yǎng)、菌種鑒定和收躲。
半固體培養(yǎng)基：液體培養(yǎng)基中加進0.2～0.5％瓊脂制成。用于觀察細菌的運動、菌種鑒定及測定噬菌體的效價等。
根據(jù)培養(yǎng)基的用途，可分為選擇培養(yǎng)基、鑒別培養(yǎng)基、加富培養(yǎng)基、基本培養(yǎng)基等。
選擇培養(yǎng)基：根據(jù)某一類或某種微生物的特殊營養(yǎng)要求而設(shè)計的培養(yǎng)基，用于進步所需微生物的分離效率。如分離固氮微生物的無氮培養(yǎng)基、加進濾紙條或纖維素粉為碳源分離纖維分解菌的培養(yǎng)基。在培養(yǎng)基中加進某種化合物，可有效地分離出對這種化合物有抗性的微生物，例如在放線菌培養(yǎng)基中加進數(shù)滴10％的酚，可抑制細菌和霉菌的生長；加進一定量的青霉素、鏈霉素，可抑制細菌生長等。
鑒別培養(yǎng)基：根據(jù)微生物的代謝特點，在培養(yǎng)基中加進某種指示劑，通過顯色反應(yīng)以鑒別不同的微生物。例如檢查乳制品和飲用水中是否有腸道細菌污染所用的伊紅-美藍培養(yǎng)基。當(dāng)大腸桿菌等腸道細菌生長時，發(fā)酵培養(yǎng)基中的乳糖，使加進的伊紅-美藍變色，在菌落上沉積為紫玄色，并呈現(xiàn)金屬光澤。
以下為常規(guī)方法，如配方中有特殊規(guī)定或要求，以配方為依據(jù)。
1.根據(jù)配方，計算各種營養(yǎng)成分用量。一般藥品可用普通藥物天平稱量，用量少的藥品，可按比例配成高濃度溶液，再按所需量用移液管吸取。稱好的藥品放進玻璃燒杯或搪瓷杯中。
2.在另一容器中將所需量的水(一般可用自來水，有特殊要求時需用蒸餾水)加熱，取全量的1/3左右倒進放藥品的容器中，用玻璃棒攪拌，待藥品全溶后，再將其余熱水全部倒進。
3.若配制固體培養(yǎng)基，則稱取1.5～2％的瓊脂放進已溶化的營養(yǎng)液中，繼續(xù)加熱至瓊脂全部溶解。加熱中隨時攪拌，防止溢出或糊底。燒糊的培養(yǎng)基營養(yǎng)物質(zhì)破壞，并產(chǎn)生有毒物質(zhì)，不宜再用。
4.待溶化的培養(yǎng)基稍冷卻后，按配方要求調(diào)整pH值。先取10毫升培養(yǎng)基裝進試管中，用pH試紙測其自然pH，再用1％NaOH(或1％HCl)調(diào)至所需pH，根據(jù)用量計算，換用10％NaOH(或10％HCl)調(diào)整所配全量培養(yǎng)基的pH。加堿(或酸)溶液時，應(yīng)邊滴加邊攪拌，至應(yīng)加量將近用完時，再次測試，最后調(diào)至要求的pH值。
5.配好的培養(yǎng)基，根據(jù)需要趁熱分裝至試管或錐形瓶中。分裝需用漏斗，以免瓊脂粘在管口或瓶口上。裝瓶量一般為瓶容量的1/3～1/2；裝試管一般為試管高度的1/5～1/4，以免滅菌時培養(yǎng)基上溢，粘濕棉塞。
6.用預(yù)先制好的棉塞塞住管口或瓶口。棉塞既有利于通氣，又有濾菌作用，故松緊、大小應(yīng)適當(dāng)，以免使用時影響操縱(如圖)。
最后用牛皮紙或報紙包住棉塞，扎緊在瓶頸或試管上方，以免滅菌時水蒸汽沾濕棉塞或脫落。
7.滅菌后取出的固體培養(yǎng)基，根據(jù)需要可將試管立即斜放，冷凝后即成斜面培養(yǎng)基，用于菌種擴大培養(yǎng)及收躲；錐形瓶中的培養(yǎng)基，倒進無菌培養(yǎng)皿中，冷凝后即制成平板培養(yǎng)基，可用于菌種的分離、鑒定等。液體培養(yǎng)基冷卻后可直接根據(jù)需要接進菌種。
培養(yǎng)基配方舉例
氧化硫桿菌培養(yǎng)基
粉狀硫10克MgSO40.5克(NH4)2SO40.4克FeSO40.01克KH2PO44克CaCl20.25克
水1000毫升自然pH
牛肉膏蛋白胨培養(yǎng)基(常用的細菌培養(yǎng)基)
牛肉膏5克蛋白胨10克NaCl5克水1000毫升pH7.2～7.4(瓊脂15～20克)
高氏一號培養(yǎng)基(常用的放線菌培養(yǎng)基)
可溶性淀粉20克KNO31克K2HPO40.5克 MgSO40.5克NaCl 0.5克FeSO40.01克水1000毫升瓊脂15～20克pH7.4～7.6
察氏培養(yǎng)基(培養(yǎng)霉菌用)
蔗糖 20克 NaNO33克K2HPO41克KCl1克MgSO40.5克FeSO40.01克瓊脂20克水1000毫升自然pH
無氮培養(yǎng)基(培養(yǎng)自生固氮菌用)
葡萄糖 10克 NaCl0.2克KH2PO40.2克CaSO4·2H2O0.1克MgSO40.2克CaCO35克
瓊脂 20克水1000毫升自然pH
伊紅-美藍培養(yǎng)基(檢查腸道細菌用)
蛋白胨10克 K2HPO42克乳糖10克瓊脂25克，水1000毫升，pH7.6
2％伊紅水溶液2毫升0.5％美藍水溶液2毫升將乳糖、伊紅、美藍分別滅菌后，加進滅菌的培養(yǎng)基中搖勻，倒平板。
Description: definition and classification of culture medium
Medium is a kind of artificial nutrient substrate suitable for microbial growth and reproduction or accumulation of metabolic products. Any medium are both necessary to include microbial energy, carbon source, nitrogen source, mineral elements, water, and growth factors, but different kinds of nutrition and different kinds of microorganisms to the requirement of nutrient elements and have very big difference. All kinds of media used at present are the result of repeated practice and comparative design.
The basic ingredients of the culture medium
Energy source and carbon source: autotrophic microbes grow in a culture medium composed of inorganic substances with carbon dioxide as the carbon source and light or inorganic materials as the energy source. For example, in the culture medium of self-cultured thiobacillus oxygenation, powdered sulfur is added as the energy source, and CO2 in the air is the carbon source. Heterotrophic microorganisms to organic carbon and energy source, medium often need to be added into glucose, sucrose and maltose, lactose and other monosaccharides and disaccharides, some available polysaccharides such as starch, cellulose, or use sugar in animal tissues. For example, beef paste peptone culture medium commonly used for cultivating bacteria, in which beef paste is the main carbon source and energy source. (see the formula of culture medium, same as below).
Nitrogen source: autotrophic microorganisms take ammonium salt and nitrate containing nitrogen inorganic substances as nitrogen nutrients, for example, thiobacillus oxides take (NH4)2SO4 as nitrogen source. Heterotrophic microorganisms are nitrogen sources of inorganic ammonium salt, nitrate or nitrogen-containing organic matter. N2 in the air is used as nitrogen nutrient by self - nitrogen-fixing bacteria.
Mineral elements, growth factors, microbial growth need to P, K, Na, S, Mg, Ca and so on main mineral elements and trace elements such as Fe, Cu, therefore media often include K2HPO4, KH2PO4, MgSO4, NaCl, KCl and FeSO4 inorganic salts; Growth factors are mainly B vitamins that regulate microbial metabolism and are often provided by yeast extract and liver extract. Many natural ingredients, such as beef paste, malt juice, corn meal and sprout juice, contain various inorganic elements and growth factors, which need not be added.
The type of culture medium
According to different sources of raw materials, medium can be divided into synthetic medium, semi-synthetic medium and natural medium.
Synthetic media: prepared from organic and inorganic substances with known chemical composition. Accurate composition, strong repeatability. But nutrients are limited and microbes grow slowly. It is suitable for bacteria separation, breeding, genetic analysis and biological determination. For example, the culture of actinomycetes, such as high culture, culture of mold, and various culture of chemoautotrophic bacteria.
Semi-synthetic medium: a mixture of some natural substances and a small amount of chemicals of known constituents. Comprehensive nutrition can effectively meet the needs of microorganisms for nutrition. It is widely used in microbial culture. Such as cultivating bacteria in beef extract peptone medium, develop mold potato dextrose medium, commonly used in industrial production of corn flour and other natural material and various inorganic salt preparation fermentation medium, etc.
Natural culture medium: made from natural organic matter with unclear or unbalanced chemical composition. The ingredients are complex, but the nutrition is rich and comprehensive. It is often used in experimental research and production. Such as malt juice culture medium, corn meal culture medium, and the bran used in production, sawdust, etc.
According to the physical properties of the medium, it can be divided into liquid medium, solid medium and semi-solid medium.
Liquid culture medium: it is made by adding various nutrients and water, or by using the immersion juice of natural substances (malt juice, sprout juice, etc.). The composition is uniform, suitable for the nutrition growth of all kinds of microorganisms. Widely used in experimental research and large-scale industrial production, it is beneficial to obtain a large number of bacteria or metabolic products.
Solid medium: coagulant is added into liquid medium or prepared with solid materials such as bran. Commonly used coagulants are agar-agar (also known as qiongjiao, cabbage), made from seaweed and other seaweed extraction and processing. Market agar-agar is a strip, sheet or powder, the main component is polygalactose sulfuric acid ester, most of the microorganism can not decompose it, only plays a supporting role in the medium. Its melting point 98 ℃, freezing point 42 ℃ and 1.5 ~ 2% aqueous solution under the general temperature in gel state. AGAR solid medium is widely used in isolation culture, identification and collection of microorganisms.
Semi-solid medium: the liquid medium was prepared by adding 0.2-0.5% AGAR. Used to observe the movement of bacteria, identify strains and determine the titer of bacteriophages.
According to the use of the medium, it can be divided into selective medium, differential medium, enriched medium, basic medium and so on.
Selective medium: a medium designed for the special nutritional requirements of a particular type or microbe to improve the separation efficiency of the desired microbe. For example, the nitrogen-free culture medium for the separation of nitrogen-fixing microorganisms, and the culture medium for the carbon source separation of fibrous decomposition bacteria is added with filter paper or cellulose powder. Inserting some compounds in the culture medium, can be separated effectively from microbes resistant to this kind of compounds are, for example in actinomycetes medium add few drops of 10% phenol, can inhibit the growth of bacteria and moulds; Adding a certain amount of penicillin, streptomycin, can inhibit bacterial growth.
Differentiation medium: according to the metabolic characteristics of microorganisms, a certain indicator is added to the medium to identify different microorganisms by coloration reaction. For example, the iran-methylene blue culture medium used to check for intestinal bacterial contamination in dairy products and drinking water. When intestinal bacteria such as escherichia coli grow, lactose in the fermentation medium changes color, and the added iran-methylene blue is deposited on the colonies in a dark purple color, which presents a metallic luster.
Preparation of culture medium
The following is a general method, if there are special requirements or requirements in the formula, based on the formula.
1. Calculate the dosage of various nutrients according to the formula. General drugs can be weighed on the scale of ordinary drugs, small dosage of drugs, can be proportioned to a high concentration of solution by proportion, and then according to the required amount with the pipette. Weigh the medicine into a glass beaker or enamel cup.
2. Will the required amount of water in another container (generally available water, have special requirements with distilled water heating, 1/3 of take all put into drug container, stirring with a glass rod, after being soluble drugs, then all the rest of the hot water to pour into.
3. If the solid medium is prepared, the AGAR between 1.5 and 2% is added into the dissolved nutrient solution and then heated until all the AGAR is dissolved. Stir at any time during heating to prevent overflow or paste. The nutritive substance of burning medium damages, and produces poisonous substance, should not be used again.
4. After the medium to be dissolved cools slightly, pH value is adjusted according to the requirements of the formula. First take 10 ml of culture medium in the test tube, with its natural pH pH test paper test, with 1% NaOH (or 1% HCl) to the required pH, according to the amount of calculation, switch to 10% NaOH (or 10% HCl) with total quantity of medium pH adjustment. When adding a base (or acid) solution, stir it in drops until it is nearly used up. Test it again and adjust to the required pH.
5. The prepared culture medium is divided into test tube or conical bottle as needed. A funnel is used to separate the containers so that AGAR does not stick to the mouth of the tube or bottle. The quantity of bottle filling is generally 1/3 ~ 1/2 of the bottle capacity. The test tube is usually 1/5 ~ 1/4 of the height of the test tube, so as to avoid the overflow of the medium and the sticky cotton plug during sterilization.
6. Plug the pipe or bottle with a premade cotton stopper. Cotton plug is not only good for ventilation, but also has the function of filtering bacteria. Therefore, it should be loose and of appropriate size, so as not to affect the operation when using (as shown in the figure).
Finally, cover the cotton stopper with kraft paper or newspaper and fasten it to the top of the bottleneck or test tube, so as not to wet the cotton stopper or fall off when sterilizing.
7. The solid culture medium taken out after sterilization can be immediately tilted in the test tube as required, and then formed inclined culture medium after condensation, which can be used for the expansion of culture and collection of bacteria; The culture medium in the conical bottle is poured into the sterile culture dish, which is then made into flat culture medium after condensation, which can be used for the separation and identification of bacteria species. After the liquid medium is cooled, it can be directly connected to the strains as required.
Examples of culture medium formulations
Thiobacterium oxide culture medium
Powder 10 g MgSO40.5 g (NH4)2SO40.4 g feso4001 g KH2PO44 g CaCl20.25 g
Water has a natural pH of 1000 ml
Beef paste peptone culture medium (commonly used bacterial culture medium)
Beef paste 5 g peptone 10 g NaCl5 g water 1000 ml pH7.2 ~ 7.4(AGAR 15 ~ 20 g)
Cochrane no. 1 medium (commonly used actinomycetes medium)
Soluble starch 20 g KNO31 g K2HPO40.5 g MgSO40.5 g NaCl 0.5 g FeSO40.01 g water 1000 ml AGAR 15 ~ 20 g pH7.4 ~ 7.6
Chap's culture medium (for mold culture)
20 g sucrose NaNO33 g K2HPO41 g KCl1 g MgSO40.5 g FeSO40.01 g AGAR 20 g water 1000 ml natural pH
Nitrogen free medium (for cultivation of nitrogen-fixing bacteria)
Glucose 10 grams NaCl0.2 grams kh2po40. 2 grams CaSO4· 2h2o0.1 gram MgSO40.2 grams CaCO35 grams
AGAR 20 grams of water 1000 ml of natural pH
Iran-methylene blue culture medium (for checking intestinal bacteria)
Peptone 10 g K2HPO42 g lactose 10 g agarose 25 g water 1000 ml pH7.6
After sterilizing lactose, irus and methylene blue respectively in 2ml 0.5% methylene blue solution of 2ml solution of 2% irus red solution, add them to sterilized medium and shake well. Pour over the plate.