Method for preparing microbial culture medium The medium is a nutrient substrate artificially prepared suitable for the growth or accumulation of microorganisms or accumulation of metabolites. Any medium needs to contain the energy, carbon source, nitrogen source, mineral element, water and growth factors necessary for microorganisms, but different nutrient types and different types of microorganisms have great differences in nutrient requirements. The various media that have been used so far are the result of repeated design and comparison design by predecessors. Antioxidants are a very important nutritional supplement as they help our bodies defend against free radical damage. Free radicals are unstable molecules that are produced in our bodies and can cause damage to cells and tissues. Long-term exposure to free radicals can lead to aging, disease, and other health problems. resveratrol,glutathione,quercetin,astaxanthin,alpha lipoic acid Xi'an complex bio-tech CO.,LTD. , https://www.complexpowder.com
Basic components of the medium Energy, carbon source: Autotrophic microorganisms use carbon dioxide as a carbon source, light or inorganic materials as energy, and grow in a medium composed of inorganic substances. For example, in the chemical autotrophic thiobacillus culture medium, powdered sulfur is added as an energy source, and CO2 in the air is used as a carbon source. Heterotrophic microorganisms use organic matter as a carbon source and energy source. It is often necessary to add monosaccharides or disaccharides such as glucose, sucrose or maltose, lactose, etc. in the medium, and some may use polysaccharides such as starch or cellulose, or use sugars in animal tissues. For example, the beef paste peptone medium commonly used for cultivating bacteria, wherein beef extract is the main carbon source and energy source. (See the same formula for the medium, the same below).
Nitrogen source: Autotrophic microorganisms use nitrogen-containing inorganic ammonium salts, nitrates, etc. as nitrogen nutrients, for example, Thiobacillus oxidans uses (NH4)2SO4 as nitrogen source; heterotrophic microorganisms use inorganic ammonium salts, nitrates or nitrogen-containing organic compounds. It is a nitrogen source. The autotrophic nitrogen-fixing bacteria utilize nitrogen in the air for nitrogen nutrition, and there is no need to add a nitrogen source in the medium, which is called a nitrogen-free medium.
Mineral elements, growth factors: Microbial growth and reproduction requires major mineral elements such as P, K, Na, S, Mg, Ca, and trace elements such as Fe and Cu. Therefore, K2HPO4, KH2PO4, MgSO4, NaCl, KCl, FeSO4, etc. are often added to the medium. Inorganic salts; growth factors are mainly B Vitamins that regulate the metabolic activities of microorganisms, and are often provided by yeast extracts, liver extracts, and the like. Many natural ingredients such as beef extract, wort, corn flour, bean sprouts, etc. contain various inorganic elements and growth factors without additional addition.
The type of the medium may be divided into a synthetic medium, a semi-synthetic medium, and a natural medium depending on the source of the raw material.
Synthetic medium: formulated from organic and inorganic substances known in the chemical composition. The composition is precise and repeatable. However, nutrition is limited and microbial growth is slow. Suitable for strain isolation, breeding, genetic analysis and bioassay. For example, a culture medium for culturing actinomycetes, a Clarion medium for cultivating molds, and various chemical autotrophic culture media.
Semi-synthetic media: Formulated from certain natural substances with a small amount of known chemical substances. It is comprehensive in nutrition and can effectively meet the nutritional needs of microorganisms. Widely used in the cultivation of microorganisms. For example, a beef extract peptone medium for cultivating bacteria, a potato glucose culture medium for cultivating mold, various natural substances such as corn flour commonly used in industrial production, and various fermentation media prepared by using inorganic salts.
Natural medium: formulated from natural organic matter with unclear or unregulated chemical composition. The ingredients are complex but nutritious and comprehensive. Often used in experimental research and production. Such as wort medium, corn flour medium, and bran, sawdust and the like used in production.
According to the physical properties of the medium, it can be divided into a liquid medium, a solid medium, and a semi-solid medium.
Liquid medium: It is made by adding various nutrients with water, or by using a natural substance dipping sauce (wort, bean sprouts, etc.). The composition is uniform and suitable for the vegetative growth of various microorganisms. Widely used in experimental research and large-scale industrial production, it is beneficial to obtain a large number of bacteria or metabolites.
Solid medium: a coagulant is added to the liquid medium, or it is prepared with a solid raw material such as bran. The commonly used coagulant is agar (also known as agar, agar), which is prepared by extracting seaweed such as broccoli. Commercially available agar is in the form of strips, flakes or powders. The main component is the galactose of polygalactose, which most microorganisms cannot decompose and only support in the medium. The melting point is about 98 ° C, the freezing point is 42 ° C, and the aqueous solution of 1.5 to 2% is in a gel state at a general culture temperature. Agar solid medium is widely used in the isolation and culture of microorganisms, strain identification and preservation.
Semi-solid medium: 0.2 to 0.5% agar was added to the liquid medium. It is used to observe the movement of bacteria, the identification of strains and the determination of the titer of phage.
According to the use of the medium, it can be divided into a selection medium, a identification medium, a rich medium, a basic medium, and the like.
Selection medium: A medium designed according to the specific nutritional requirements of a certain type or microorganism to increase the separation efficiency of the desired microorganism. For example, a nitrogen-free medium for separating nitrogen-fixing microorganisms, a filter paper strip or cellulose powder is added as a medium for separating the fiber-decomposing bacteria from the carbon source. Adding a compound to the culture medium can effectively separate microorganisms resistant to this compound. For example, adding a few drops of 10% phenol to the actinomycete medium can inhibit the growth of bacteria and mold; The amount of penicillin and streptomycin can inhibit bacterial growth and the like.
Identification medium: According to the metabolic characteristics of the microorganism, an indicator is added to the medium to identify different microorganisms by a color reaction. For example, check the eosin-methylene blue medium used for intestinal bacterial contamination in dairy products and drinking water. When intestinal bacteria such as Escherichia coli grow, the lactose in the fermentation medium discolors the added eosin-methylene blue, deposits purple-black on the colonies, and exhibits metallic luster.
The preparation method of the culture medium is as follows. The conventional method, such as special regulations or requirements in the formulation, is based on the formula.
1. Calculate the amount of various nutrients based on the formula. The general medicine can be weighed by a common drug balance, and the medicine with a small amount can be proportioned into a high-concentration solution, and then sucked by a pipette according to the required amount. The good medicine is placed in a glass beaker or an enamel cup.
2. In a separate container, heat the required amount of water (usually tap water, distilled water if necessary), pour about 1/3 of the total amount into a container for placing the drug, stir with a glass rod, wait for the drug After total dissolution, pour all the remaining hot water.
3. If a solid medium is prepared, weigh 1.5 to 2% of the agar into the dissolved nutrient solution and continue heating until the agar is completely dissolved. Stir at any time during heating to prevent spillage or batter. The burnt-out medium is destroyed by nutrients and produces toxic substances, which should not be reused.
4. After the medium to be dissolved is slightly cooled, adjust the pH according to the formulation requirements. First, take 10 ml of the medium into a test tube, measure the natural pH with a pH test paper, and adjust to the desired pH with 1% NaOH (or 1% HCl). According to the dosage, switch to 10% NaOH (or 10% HCl). Adjust the pH of the entire medium. When adding a base (or acid) solution, it should be stirred while stirring, until the amount should be nearly used, test again, and finally adjust to the required pH.
5. Dispense the medium and dispense it into a test tube or conical flask as needed. A funnel is required for dispensing to prevent the agar from sticking to the nozzle or the mouth of the bottle. The bottling amount is generally 1/3 to 1/2 of the bottle capacity; the test tube is generally 1/5 to 1/4 of the height of the test tube, so as to prevent the medium from overflowing during sterilization and sticking the tampon.
6. Plug the nozzle or bottle mouth with a pre-made tampon. The tampon is beneficial to both ventilation and bacteria filtration, so it should be tight and the size should be appropriate to avoid affecting the operation.
Finally, wrap the tampon with kraft paper or newspaper and fasten it over the bottle neck or test tube to prevent the steam from getting wet or detached during sterilization.
7. The solid medium taken out after sterilization, the tube can be immediately placed obliquely as needed, and then condensed to form a slant medium for expansion and preservation of the strain; the medium in the conical flask is poured into the sterile culture. In the dish, after condensing, it is made into a plate medium, which can be used for separation and identification of strains. After the liquid medium is cooled, the strain can be directly connected as needed.
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Method for preparing microbial culture medium