Ⅰ.Components of Microbial Culture Media
Microbial culture medium is a nutrient matrix artificially prepared for the growth and reproduction of microorganisms or to accumulate metabolites. According to the properties and functions of nutrients, it can be divided into six nutrients: carbon source, nitrogen source, energy, growth factor, inorganic salt and water.
1.carbon source
Carbon is the most demanded element of microbial cells, accounting for 50% of the dry weight of cells, and the types are extremely wide. Simple inorganic carbon-containing compounds, relatively complex organic matter, complex organic macromolecules, and natural carbon-containing substances are all carbon sources that can be utilized by different microorganisms.
2.Nitrogen sources
The main function of nitrogen source is to provide nitrogen in the synthesis of cellular substances and metabolites, generally not as energy. Most microorganisms can only use ammonium salts, other nitrogen-containing salts, and organic nitrogen-containing compounds as nitrogen sources, while a few nitrogen-fixing microorganisms can use molecular nitrogen as nitrogen sources to synthesize their own amino acids and proteins.
3.Energy
Energy is a substance that can provide the energy needed for the life activities of microorganisms, such as the light energy of sunlight, which can provide energy for microorganisms under photosynthesis. Energy sources vary with different types of microorganisms. For example, the carbon source of heterotrophic microorganisms is energy; autotrophic microorganisms include photoautotrophs that use sunlight as energy, and chemoautotrophs that can oxidize inorganic substances to obtain energy.
4.Growth Factor
Some microorganisms must also supplement trace amounts of organic substances to grow, and these trace amounts of organic substances are called growth factors. The main function is to provide microbial cells with important chemical substances (proteins, nucleic acids and lipids), components of cofactors (coenzymes, prosthetic groups) and participate in metabolism. Yeast extract, peptone, wort juice, corn steep liquor, animal and plant tissue or cell extract, etc., are all natural substances that can provide growth factors.
5.Inorganic salt
Inorganic salt is an essential component of living matter. The life activities of microorganisms require elements including sulfur, phosphorus, sodium, potassium, magnesium, calcium, iron, etc., as well as certain trace metal elements, such as cobalt, zinc, molybdenum, nickel, Tungsten, copper, etc. Most of these elements are provided to microorganisms in the form of salts, so they are also called inorganic salts or mineral nutrients.
6.water
Water is one of the main chemical components of microbial cells. Most of the chemical reactions in microbial life activities are carried out in water, and the exchange of intracellular and extracellular substances is usually carried out in water.
When determining the composition of the medium, it should be formulated according to the nutritional characteristics of the microorganisms. Generally speaking, it should contain all the nutrients that the microorganisms are most suitable for growth, because microorganisms of different nutritional types have different requirements for nutrients.
Ⅱ.Culture medium design
1. Composition and ratio of nutrient concentration in the mediumAfter the composition of the medium is determined, the concentration and ratio of each nutrient must be appropriate, and the microorganisms can grow rapidly only when the optimum conditions for microbial growth are reached. If the concentration of nutrients is too low, it cannot meet the growth needs of microorganisms, and if it is too high, it will inhibit the growth of microorganisms.
The carbon-nitrogen ratio (C/N) is an important factor affecting the growth, reproduction and metabolic accumulation of microorganisms. It is often used as an indicator to measure the combination. The general medium carbon-nitrogen ratio (C/N) is 100: (0.5-2) .
2. Adjust the pH value of the mediumThe pH value is an important environmental indicator for the growth, reproduction and metabolic accumulation of microorganisms. Generally, the suitable pH value is 7.0-7.5 for bacteria, 3.8-6.0 for yeast, 4.0-5.8 for mold, and 7.5-8.5 for actinomycetes. However, during the growth and metabolism of microorganisms, the use of nutrients accumulates in the form of metabolites, which sometimes causes changes in the pH value. Therefore, in order to maintain the relative stability of the pH value of the medium, it is necessary to add a pH buffer to the medium in special cases. agent to adjust the pH value to meet the growth range of microorganisms.
3. Adjust the osmotic pressure of the mediumThe cell membrane of microorganisms is a semipermeable membrane. Under the condition of low osmotic pressure, when the concentration of the external environment solution is lower than the internal environment of the cell, water molecules will enter the cell from the low osmosis solution, causing the cell to expand or even rupture; otherwise, when the external environment solution Concentrations are higher than the internal environment of the cell, and osmosis forces water to diffuse out of the cell interior, causing the cell to shrink and the cell wall to separate from the cell membrane (plasmolysis).
Therefore, microorganisms can only grow under isotonic conditions. Therefore, in the process of preparing the medium, the concentration of each nutrient should be well controlled, and sodium chloride is usually properly added to the medium to increase the osmotic pressure.
Ⅲ.Optimization of culture medium
At present, there are single factor test method, orthogonal experimental design and response surface optimization design method commonly used in the laboratory to optimize the medium to meet the growth needs of microorganisms.
1. Single factor test methodA single factor experiment refers to an experiment in which only one factor is changing in an experiment and the other factors are kept constant under the premise that there is no interaction between the factors. By changing the level of one factor at a time while keeping the other factors constant to determine the effect of the factor in the whole experiment, and then carry out the optimization method of the experiment one by one.
However, when there are many experimental factors, multiple tests and a long test time are required to complete the screening and optimization of each factor, and finally determine the composition and conditions of the medium. The single factor test method is usually used to determine the carbon source and nitrogen source and their ratio, and then carry out the orthogonal test.
2. Orthogonal test methodThe orthogonal test method is to find out the representative multi-factors in the test according to the needs of the test, use a set of tables to select multiple factors and levels, design a reasonable test, find out the interaction between them, and find the best The combination of factor levels greatly reduces the number of experiments and obtains experimental results faster.
The factors and levels of the orthogonal test design are reasonable, the distribution is uniform, no repeated trials are required, and the error is small. Especially when there are many experimental factors and levels, and there are many interactions between the factors, the orthogonal test method has obvious advantages. Single factor Trials are almost impossible to complete. But it cannot find out the relationship between all factors and levels, it can only be an approximate value, and cannot accurately give an optimal value of factors and levels.
3. Response surface optimization methodThe response surface optimization method is to use mathematical statistics to find out the best conditions for the combination of multiple factors and the response surface, and to obtain a mathematical function equation, which is used to model and analyze the response surface affected by variables, and then the response surface Optimization, through rapid modeling of the functional relationship between factors and response surfaces, reducing the number and time of experiments, conducting comprehensive experimental research with the most economical method and the shortest time, and accurately finding the functional relationship between factors and surfaces in the entire area, so as to obtain better experimental results.