第三章 培养基.docx

1、第三章 培养基第三章培养基Chapter 3 Culture Media Beginning of Microbiology Almost exactly 300 years ago Anton van Leeuwenhoek described the first bacteria seen through the microscope, thus providing the technical basis for studying the morphology of micro-organisms Almost exactly 300 years ago Anton van Leeuwen

2、hoek described the first bacteria seen through the microscope, thus providing the technical basis for studying the morphology of micro-organisms伟大的科赫总结的“科赫三原则”: 细菌要恒定的与该病的病理症状有关；能在病人中找到并分离，培养，纯化；放到健康的动物上也能引出相同的症状和病理特点。Agar - Agar Frau HessescontributionAgar Agar Solid medium is made by adding Agar A

3、gar is obtained from Sea weeds (New Zealand agar is more) Agar contain long chain polysaccharides, Inorganic salts and protein like substance Melts at 980c and sets at 420c Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not degradated by bacteria Liquefies a

4、t 98C Solidifies 42C Agar is melted by boiling Liquid medium can be converted into solid by the addition of Agar (1%-2%) or semisolid medium (0.6%) Used as solidifying agent for culture media in Petri plates, slants, and deeps Generally not degradated by bacteria Liquefies at 98C Solidifies 42C Agar

5、 is melted by boiling Liquid medium can be converted into solid by the addition of Agar (1%-2%) or semisolid medium (0.6%) Plate: provide large surface for isolation and observation of colonies Using a sterile loop or a sterile swab streak your sample on the petri plate Important let your sterilized

6、 loop cool before you pick up your sampleMedia and Culture Media: Nutrients (agar, pH indicators, proteins and carbohydrates) used to grow organisms outside of their natural habitats Culture: The propagation of microorganisms using various mediaCulture media A cultural medium is the substance in whi

7、ch a specific micro-organism lives and grows. It must contain the essential nutrients needed for the microbe to grow. 培养基是指维持微生物生长繁殖和产物形成的营养物质。 工业发酵培养基是指提供微生物生长繁殖和生物合成各种代谢产物所需要的，按一定比例配制的多种营养物质的混合物。 Culture medium: Nutrients prepared for microbial growth Sterile: No living microbes Inoculum: Introduc

8、tion of microbes into medium Culture: Microbes growing in/on culture medium A good medium is very important to the success of an industrial fermentation. The medium supplies nutrients for growth, energy, building of cell substance, and biosynthesis of fermentation products. 3.1 培养基的类型 Classification

9、 of Culture media 按纯度分类： -合成培养基 chemically defined media: compound identity and concentration of all components are known. More expensive, Synthetic media -天然培养基 chemically undefined media: ingredients include one or more chemically complex substances such as protein hydrolysates and extracts. Cheap

10、er, Crude media Synthetic Media Defined media are media composed of pure ingredients in carefully measured concentrations dissolved in double distilled water i.e., the exact chemical composition of the medium is known. Typically, they contain a simple sugar as the carbon and energy source, an inorga

11、nic nitrogen source, various mineral salts and if necessary growth factors (purified amino acids, vitamins, purines and pyrimidines. The choice of defined or undefined medium is dependent upon its application. - Chemically defined media are useful in biochemical or metabolic studies of organisms. -

12、General laboratory growth media and industrial media are often chemically undefined media. Such as 花生饼(peanut meal)、蛋白胨(peptone)等。一般不需外加微量元素 (trace elements)、维生素等。 按状态分类： -Solid media：suitable to culture and preserve species and spores; cultivating fungi. -Liquid media: 80%-90%water, industrial ferm

13、entation media. -Semi-solid media: liquid medium + agar 主要用于鉴定细菌、观察细菌运动特征等。Classification of Culture mediaBased on the consistency: Liquid - Peptone water, Nutrient broth Semisolid - Nutrient agar Solid - Blood agar, Serum agar 按用途分类 斜面培养基、种子培养基和发酵培养基1.斜面培养基(slant media) 供微生物细胞生长繁殖或保藏菌种用的固体培养基。细菌、酵母

14、等斜面培养基，霉菌、放线菌生产孢子的孢子培养基。 作用：供给细胞生长繁殖所需的各类营养物质。富含有机氮源。 斜面培养基宜加少量无机盐，必要的生长因子 孢子培养基 (spore media) : 供菌种繁殖孢子的一种常用固体培养基。 要求：使菌体迅速生长，产生较多的优质孢子，不易引起菌种发生变异。 基本配制要求： not rich in nutrition, especially organic nitrogen source. optimal mineral salt concentration optimal pH and humidity 生产上常用斜面培养基： 由葡萄糖、蛋白胨、牛肉膏和

15、食盐等配制的琼脂斜面培养基。 麸皮(bran)、大米(rice)、小米(millet)孢子培养基； 大米和小米常用作霉菌孢子培养基，因它们含氮少、疏松、表面积大，是较好的孢子培养基。水分控制在2125。2、种子培养基 inoculum media 种子扩大培养的目的是短时间内获得数量多、质量高的大量菌种，以满足发酵生产需要 要求：营养比较丰富和完全，氮源和维生素的含量要高些。 一般种子培养基中各种营养物质浓度不需太高 可添加易被吸收利用的碳源和氮源，如葡萄糖、酵母粉 (yeast)、蛋白胨 通常不要求积累产物 一般种子培养基常包括有机氮源和无机氮源 最后一级的种子培养基的成分最好接近发酵培养基

16、。 3、发酵培养基 production media 供菌体生长、繁殖和合成产物之用。 要求：既要使种子接种后能迅速生长，达到一定的菌丝浓度，又要使长好的菌体迅速合成所需产物。 配制：其组成除有菌体生长所必需的元素和化合物外，还需有合成产物所需的特定元素、前体和促进剂等。3.2 发酵培养基的成分及来源Composition of media and their sources 微生物需从外界吸收营养物质，经过一系列生物化学反应，获得能量并形成新的细胞物质，同时排出废物。 微生物种类繁多，营养物质需求和利用不同。 从细胞物质成分分析可知，水占80%左右。 20%干物质中，炭素约占50%、氮素约占

17、5%-13%、矿物质约占3%-10%。 配制培养基时需有足够的碳源、氮源、水和无机盐。还可添加生长因子。Basic requirements of culture media Nutrients - Energy source - Carbon source - Nitrogen source Mineral salts Sulphate, phosphates, chlorides & carbonates of K, Mg & Ca. A suitable pH 7.2 7.4Accessory growth factors - Tryptophan for Salmonella typh

18、i3.2.1 碳源 carbon sources Carbon source is one of main composition of medium. The functions are: providing carbons and energy; the components for synthesis of products. These can include simple sugars, complex carbohydrates, alcohols, amino and other organic acids, and short-chain lipids. 常用的碳源有糖类、油脂

19、、有机酸和低碳醇。蛋白质、氨基酸也可用作碳源。 3.2.1.1 糖类 sugars Sugars used mainly: glucose, molasses (糖蜜), dextrin (糊精). Glucose: most microbes can utilize it. 速效碳源、 high concentrationspeed respiration upDO no enoughintermediates (中间产物）accumulation inhibiting growth and synthesis molasses (糖蜜) Beet(甜菜) and cane molasses

20、 are by-products of the sugar industry. containing 50% -75% fermentable sugars, mainly sucrose. Also containing nitrogenous compounds, vitamins and minerals. Cheaper Starch and dextrin (糊精) Starch: -widely used in fermentation industry- starch hydrolyze using microbial amylase (淀粉酶) dextrin glucose-

21、Most common used starches: corn, wheat, potato starches. Also using corn flour. -advantages: 缓效碳源、cheaper than glucose3.2.1.2 油和脂肪 oils and fats Oil and fats are also used as carbon sources because some microbes contain lipase (脂肪酶) Fat glycerol(甘油) and fatty acids oxidize CO2+ water, releasing much

22、 energy Providing enough O2 is important. 常用的油：豆油、菜子油、猪油、鱼油、棉子油等。 3.2.1.3 有机酸 organic acids Some microbes can use organic acids as carbon sources, such as lactic acid, citric acid, acetic acid. pH of cultural medium will increase. CH3COONa + O2 2CO2 + H2O + NaOH 3.2.1.4 烃和醇类 hydrocarbon and ethanol

23、石油及裂解产物如正烷烃也可用作 carbon sources. 自然界中能同化乙醇的微生物和能同化糖质的微生物一样普遍。 3.2.2 氮源 nitrogen sources 氮源主要用于构成菌体细胞物质（氨基酸、蛋白质、核酸等）和合成含氮代谢物。 常用的氮源可分为：有机、无机氮源两类。 有机氮源 organic nitrogen sources 常用的: 花生饼、棉子饼、玉米浆、玉米蛋白粉、蛋白胨、酵母粉、鱼粉、蚕蛹粉、尿素、废菌丝体和酒糟等。 organic nitrogen sources microbial proteinase amino acids metabolized by mi

24、crobes. organic nitrogen sources contain rich proteins, peptides, amino acids, vitamins and growth factors. 在含有机氮源的培养基中，微生物常表现出生长旺盛、菌丝或细胞浓度增长迅速的特点。 有些微生物对氨基酸有特殊的需求。如缬氨酸可提高红霉素的发酵单位。 玉米浆 Cornsteep Liquor Cornsteep liquor is the water extract by-product resulting from the steeping (浸泡) of corn during t

25、he commercial production of corn starch and other corn products. 玉米浆是一种优良的氮源，因为它含有丰富的氨基酸（丙氨酸、赖氨酸、缬氨酸、苯丙氨酸等）、还原糖、磷微量元素和生长素。 Of the 50% solids of cornsteep liquor, nearly half is lactic acid. The rest includes amino acids, glucose and other reducing sugars, salts, vitamins, and precursors (前体)such as

26、those for the penicillin molecule. The high lactic acid content of cornsteep liquor results from the growth of lactic acid bacteria during its manufacture. This variation in composition, at times, can lead to poor reproducibility of an industrial fermentation. 尿素 urea Urea is also a commonly used ni

27、trogen source. Its feature: -single component, no has nutritional feature of complex nitrogen sources; -cheaper, often used in production of penicillin and glutamic acid (谷氨酸). yeast extract (酵母抽提物) Peptones (蛋白胨) 有机氮源除了作为菌体生长繁殖的营养外，有的还是产物的前体。 缬氨酸、半胱氨酸和-是合成青霉素和头孢菌素的主要前体；甘氨酸可作为L-丝氨酸的前体等。无机氮源 inorgani

28、c nitrogen source 常用的：氨水(ammonia)、铵盐(ammonium)和硝酸盐(nitrate) Microbes can utilize it faster. 速效氮源 After it is utilized, the pH of medium will be changed. (NH4)2SO4 2NH3 + H2SO4 NaNO3 + 4H2 NH3 + 2H2O +NaOH 生理酸性物质：NH4 酸性物，硫酸铵 生理碱性物质： NO3 碱性物，硝酸钠 正确使用生理酸碱性物质 Except using as a nitrogen source, ammonia i

29、s often used to adjust pH of media. Stirring is necessary. 无机氮源的影响：硫酸铵硝酸铵硝酸钠尿素 3.2.3 无机盐及微量元素 mineral salts and trace elements Mineral salts and trace elements are needed when microbial cells grow and reproduce. Most common mineral salts and trace elements: 磷(phosphorus)、镁 (magnesium)、硫 (sulphur)、钾(

30、potassium)、钠(sodium)、铁(iron)、氯(chlorine)、锰(manganese)、锌(zinc)、钴(cobalt)、 钙(calcium)等。 作为微生物细胞生理活性物质组成或生理活性作用的调节物。 When their concentration is low, they show a stimulation function for cell grow and product synthesis. When their concentration is high, they will inhibit the cell growth. Their optimal

31、concentrations depend on the species and strains of microorganisms. 无机盐成分一般所用的浓度范围 (见书上表) These elements are added into cultural media in the form of their salts. Except synthetic media, cobalt钴, copper, iron, manganese锰, zinc, molybdenum钼 are not added into crude media. Phosphorus element is one component of nucleic acids and proteins; one component of ATP, energy transfer. Phosphorus can stimulat