原生動物也可用培養基培養,有的原生動物需要較多的營養物質,例如梨型四膜蟲(Tetrahymena pyriformis)的培養基含有10種氨基酸、7種維生素、鳥嘌呤、尿嘧啶及一些無機鹽等,而有些變形蟲可在較簡單的蛋白胨肉湯(peptone broth)中生長。大多數藻類可以利用光能,只需要CO2、水和一些無機鹽就可生長,而某些藻類如眼蟲藻(Euglena)中的一些種可在黑暗條件下利用有機物質生長。有些藻類需要在培養基中補加土壤浸液,培養海洋藻類時可直接利用海水,但如果在特殊情況下需要用合成培養基培養海洋藻類時,則必需在培養基中加入海水中含有的各種鹽。
培養基的pH必須控制在一定的范圍內,以滿足不同類型微生物的生長繁殖或產生代謝產物。各類微生物生長繁殖或產生代謝產物的最適pH條件各不相同,一般來講,細菌與放線菌適于在pH7~7.5范圍內生長,酵母菌和霉菌通常在pH4.5~6范圍內生長。值得注意的是,在微生物生長繁殖和代謝過程中,由于營養物質被分解利用和代謝產物的形成與積累,會導致培養基pH發生變化,若不對培養基pH條件進行控制,往往導致微生物生長速度或(和)代謝產物產量降低。因此,為了維持培養基pH的相對恒定,通常在培養基中加入pH緩沖劑,常用的緩沖劑是一氫和二氫磷酸鹽(如K2HPO4和KH2PO4)組成的混合物。K2HPO4溶液呈堿性, KH2PO4溶液呈酸性,兩種物質的等克分子混合溶液的pH值為6.8。當培養基中酸性物質積累導致H 濃度增加時, H 與弱堿性鹽結合形成弱酸性化合物,培養基pH不會過度降低;如果培養基中OH-濃度增加, OH-則與弱酸性鹽結合形成弱堿性化合物,培養基pH也不會過度升高。
The principle of microbial fermentation preparation culture medium.
Choose the right nutrients.
In general, all microorganisms grow need medium containing carbon source, nitrogen source, inorganic salt, growth factors, water and energy, due to the complex microbial nutrition type, different microorganisms on nutrient requirements is different, so first of all, according to the requirements of different microbial preparation pertinence of medium. Autotrophic microbes can from simple inorganic synthesis of their needs sugar, lipid, protein, nucleic acids, vitamins and other complex organic compounds, thus cultivating autotrophic microbe culture medium can (or should) is composed of simple inorganic matter. For example, sulfur oxide can cultivate the autotrophic thiobacillus, in the process of the culture medium preparation did not specifically to join the other carbon source material, but rely on air and CO2 dissolved in the water for sulfur oxide sulfur bacillus carbon source.
The cultivation of other autotrophic microorganisms is basically similar to that of the above medium, except that the energy material has changed. In addition to all kinds of nutrients, photoautotrophic microorganisms need light to provide energy. The cultivation of heterotrophic microorganisms requires the addition of organic matter in the medium, and the nutritional requirements of different types of heterotrophic microorganisms are very different, so the composition of the culture medium is far away. Culture medium composition of e. coli, for example, is simpler, and some heterotrophic microbial culture medium composition is very complicated, such as intestinal membrane bright beads bacterium need growth factor, if the synthetic medium of preparation training it, need to add as many as 33 kinds of growth factors in the culture medium, so often need to grow and provide it with using natural organic growth factor.
Bacteria, actinomycetes, yeasts, fungi, protozoa, algae and viruses are the main types of microorganisms, and the cultures they need are different. In the laboratory, common beef paste peptone medium (or common broth medium) is used to cultivate bacteria; The culture of actinomycetes was cultured with the no.1 synthetic culture medium. General with malt juice culture yeast culture medium, it is 4 times malt powder and water blending, under the condition of 58 to 65 ℃ heat preservation 3 ~ 4 hours to fully saccharification, adjust the concentration of sugar solution for 10 о bahrain, boil with gauze filter, made from the pH 6.0. The composition of malt powder is complex, which can provide sufficient nutrients for yeast. The cultivation of mildew is generally used in the synthesis culture medium.
Protozoa could also be a medium, some native animals need more nutrients, such as four pear type membrane worm (Tetrahymena pyriformis) culture medium containing 10 kinds of amino acids, 7 kinds of vitamin, guanine, uracil and some inorganic salts, etc., and some of the amoeba can be simpler peptone broth (peptone broth). Most algae use light energy, which requires only CO2, water and some inorganic salts to grow, while some of the algae, such as Euglena, use organic matter to grow in dark conditions. Some algae need adding soil immersion in culture, cultivating Marine algae can be directly used in the water of the sea, but if in special circumstances need to use synthetic medium to develop Marine algae, are required to join in the seawater in culture contains a variety of salt.
2. The nutrient concentration and proportion are suitable.
Medium nutrients concentration of microorganisms can grow well when appropriate, cannot satisfy microorganisms at low nutrient concentration required for normal growth, when the concentration is too high may inhibitory effect on microorganism growth, such as high concentration of sugar matter, inorganic salt, heavy metal ions such as not only unable to maintain and promote the growth of microorganisms, but inhibit or sterilization effect. In addition, the concentration ratio of various nutrients in the medium also directly influences the growth and reproduction of microorganisms and the formation and accumulation of metabolic products, among which the carbon nitrogen ratio (C/N) has a greater influence. Strictly speaking, carbon nitrogen ratio refers to the ratio of the number of moles of carbon to nitrogen in medium, and sometimes the ratio of raw sugar to crude protein in the medium. For example, in the process of using microbial fermentation to produce glutamate, when the medium carbon nitrogen ratio is 4/1, the bacteria multiply and the glutamate accumulation is small. When the carbon nitrogen ratio of the medium was 3/1, the growth of the bacteria was inhibited and the yield of glutamate increased substantially. Again, such as, in the antibiotic fermentation process, can control the medium available nitrogen or carbon source and delayed the ratio between the nitrogen or carbon source to control the bacteria growth and the synthesis of antibiotics.
3. Control pH condition
The pH of the culture medium must be controlled within a certain range to meet the growth and reproduction of different types of microorganisms or to produce metabolites. All kinds of microorganisms grow or produce metabolites optimum pH conditions each are not identical, in general, applicable for bacteria and actinomycetes pH7 ~ 7.5 within the scope of growth, yeast and mold usually grow in pH4.5 ~ 6 range. It is important to note that in the process of microbial growth, reproduction and metabolism, as nutrients are broken down to use and formation and accumulation of metabolites, medium pH changes, if it is not medium to control the pH condition, often leads to microbial growth or (and) metabolites production decrease. Therefore, in order to maintain the relatively constant pH of the medium, the pH buffer is usually added in the medium, and the commonly used buffer is a mixture of hydrogen and dihydrophosphate (such as K2HPO4 and KH2PO4). K2HPO4 solution is alkaline, and KH2PO4 solution is acidic. The pH value of the isogram mixed solution of two substances is 6.8. When the accumulation of acid in the medium resulted in the increase of H concentration, H combined with weak alkaline salt to form a weak acid compound, and the pH of the medium was not excessively reduced; If the concentration of OH- concentration in the medium increased, OH- then combined with weakly acidic salt to form a weakly alkaline compound, the medium pH would not be excessively elevated.
However, the K2HPO4/KH2PO4 buffer system can only be adjusted in a certain pH range (pH6.4~7.2). Some microorganisms, such as lactic acid bacteria can produce more acid, the buffer system, it will be difficult to play a buffer action at this point can be added in the culture medium of refractory carbonate (CaCO3) to adjust, CaCO3 poorly soluble in water, does not make the medium pH rising excessively, but it can counteract acidity of microbes, releases CO2 at the same time, the culture medium pH control within a certain range.
There are also some natural buffer systems in the medium, such as amino acids, peptides and proteins, which are both ampholytes, which can also act as a buffer.