Microorganisms are a group of microscopic organisms that are widely distributed in nature and invisible to the naked eye, including bacteria, spirochetes, rickettsias, chlamydia, mycoplasma, actinomycetes, fungi, viruses, etc., with simple structure, and exist in single cell, cell group or non-cell state. Under the conditions of providing nutrients and suitable environment, they can generally live independently and grow rapidly. In order to avoid the death and pollution of microorganisms, keep their original properties basically stable, and facilitate research and use, it is necessary to use the technology of strain preservation. Here are some common methods for preservation of bacteria.
Cryo preservation
This method is suitable for most microorganisms and viruses that need long-term preservation. The principle is that in the ultra-low temperature environment, microbial metabolic activity stops, and generally the lower the temperature, the better the effect.
Saving method
Use a cryo preservation tube to preserve the bacteria, and at the same time need protective agents, such as glycerin, degreased sheep powder or dimethyl sulfoxide. The frozen storage tube containing bacteria is placed in a refrigerator at - 60~- 80 ℃ for freezing and storage.
Liquid paraffin preservation method
Liquid paraffin preservation method is applicable to some bacteria that cannot decompose paraffin, such as yeast, bacillus, penicillium, aspergillus, etc. Some bacteria and fungi are not suitable for preservation by this method. Liquid paraffin method uses liquid paraffin to cover, microbial metabolism is inhibited, and bacterial aging is delayed. Paraffin method can prevent oxygen from entering, making aerobic bacteria unable to continue to grow. At the same time, if the water in the culture medium evaporates, it is easy to cause bacterial death. Paraffin method can prevent the water in the culture medium from evaporating, and prolong the preservation time of bacteria.
Saving method
Under sterile conditions, inject sterilized liquid paraffin into the newly cultivated slant culture, the liquid level is 1cm~2cm higher than the top of the slant, add rubber stopper and seal with solid paraffin to isolate the bacteria from the air. The strain injected with paraffin oil shall be stored upright in a dry place at low temperature (4 ℃~6 ℃) or at room temperature.
Freeze-drying preservation method
This method is suitable for the preservation of most microorganisms, but not for filamentous fungi that do not produce spores. Freeze-drying preservation method is to preserve bacteria under low temperature, dry and anoxic conditions. That is, quickly freeze the strain under low temperature to keep the microorganism intact, and then decompress and extract the air to sublimate the ice until it is dehydrated and dried, and seal and isolate the air in the empty state to stop the microbial metabolic activity, so as to preserve it for a long time.
Saving method
Add protective agent into ampoule tube according to certain formula, and then add bacteria to prepare bacterial suspension. Pre-freeing the ampoule tube to - 15~- 20 ℃, then dry the ampoule tube with a freeze vacuum dryer, and seal the ampoule tube. Store at low temperature and away from light.
Sand tube preservation method
This method is suitable for the preservation of sporogenous actinomycetes, bacillus, etc., but not for the preservation of fungi and pathogenic fungi that mainly focus on hyphal development. The use of fine sand to create dry conditions can slow down the metabolic activities of those microorganisms that can produce spores or spores in a dry environment, inhibit their reproduction speed, and have strong resistance to death. This method can reduce strain mutation and prolong survival time.
Saving method
Sieve river and sea sand with 60 meshes, discard large particles and impurities, and then sieve with 80 meshes to remove fine sand. Absorb the iron with iron absorption stone, put it into the container and soak it with 10% hydrochloric acid. If there are many organic matters in the river sand, soak it with 20% hydrochloric acid. After 24h, remove hydrochloric acid, soak it in tap water for several times until it is neutral, and bake or dry the sand. In addition, ten soils below the uncontaminated surface were screened for 100 days, washed to neutral with tap water, and dried. Mix evenly according to sand: soil=2:1, sub-pack into ampoule tube or small test tube, the height is about 1cm, plug the cotton plug, autoclave at 121 ℃ for 1h~1.5h, or autoclave at normal pressure for 3 times, 16 times a day. Dry it under 50 ℃ (the temperature should not be too high, otherwise the soil organic matter will be decomposed by high temperature to produce harmful substances, which will affect the performance of the strain), and it can be used only if there is no microbial growth after inspection.
Then prepare the bacterial suspension and add the bacterial suspension into the sand pipe. Put the sand pipe containing bacterial suspension into the vacuum dryer for vacuum drying. Use flame to seal and melt the nozzle, store it at low temperature (4 ℃~10 ℃) or dry place at normal temperature, and regularly check the vitality and miscellaneous bacteria.
Porcelain bead preservation method
There are usually finished porcelain bead preservation tubes that are easy to use on the market for low-temperature preservation of bacteria. The porcelain bead preservation tube is usually composed of a frozen storage tube, as well as the internal preservation solution and porcelain beads. Porcelain beads with concave and convex surface are used for adsorption and preservation of bacteria. The strain can be stored for a long time at - 20 ℃ or - 80 ℃. It is easier to operate whether in storage or in resuscitation.
Saving method
Scrape the pure culture of bacteria from the plate and put it into the culture preservation tube, and roughly configure it into 3-4 Michaelis concentrations. Shake to make bacteria emulsified, wash the preservation solution, tighten the preservation tube, and freeze at low temperature.
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