The application of the hottest strain preservation

Application of strain preservation technology (Part I)

microorganisms have a short growth cycle and can be produced in industry, but the defects associated with human continuous passage are easy to cause genetic variation. Therefore, there have been research reports on strain preservation since the 1930s, and many methods have been explored. Many countries have established special strain preservation institutions, such as the American standard strain collection (ATCC), the British national standard strain collection (NCTC), the Osaka fermentation Institute (LFO), and the Global World Federation for microbial conservation (WFCC). These institutions sell and exchange strains and publish strain catalogs. There are more than 300 species preservation institutions in the world. China established the China microbial species preservation Committee (CCCCM) in 1979, formulated organizational and management conditions, and set up six preservation centers. The purpose of strain preservation is clear. In the basic research work, the same strain can obtain repeated experimental results during and after the work. For the production bacteria with economic value, it is necessary to maintain its high-yield performance. The recombinant bacteria obtained by bioengineering technology must maintain the stability of its genetic characteristics. In short, during the preservation period, these strains should be available at any time, and genetic variation should be reduced or even not produced as much as possible. This paper introduces several methods of strain preservation

1. Slope preservation method

this is the most basic method, which can be applied to a wide range of bacteria, fungi and actinomycetes. When the microorganism grows well in the appropriate inclined medium and temperature, it can be preserved for 3~6 months at about 4 ℃. Replant after expiration. Of course, the preservation temperature and time are not absolute. Some strains are even better preserved at 37 ℃, and some need to be subcultured once a week or two. The disadvantage of this method is that more passages are prone to variation, such as decreased spore production ability, weakened fermentation ability, reduced toxicity and even gene loss. More passages are also easy to increase the chance of pollution. At present, many laboratories use spiral mouth test tubes with good sealing performance to replace the traditional cotton plugs and reduce the content of carbohydrates, which is more conducive to the preservation of bacteria

2. puncture preservation method

this method is an improved method of inclined plane preservation, which is commonly used to preserve all kinds of bacteria that need gas. The method is to make the culture medium into soft agar (the fat content in this scheme is 1/2 of the inclined plane, usually 1%), and fill it with 1.2 × In 10cm small test tube or spiral mouth small test tube, the height is 1/3 of the test tube. After high-pressure sterilization at 121 ℃, do not make an inclined plane, and puncture the strain into 1/2 of the culture medium with a needle shaped inoculation needle. The cultured microorganisms can grow at the puncture site and on the agar surface. Then cover with sterile liquid paraffin of 2~3mm. Liquid paraffin must be autoclaved twice. Such tubules can be preserved in the refrigerator to reduce microbial metabolism. Therefore, the preservation effect is better than the slope. If the liquid paraffin is directly added to the well-developed slope without puncture method, a similar effect can be obtained, and the scope of application is wide, which can be applied to fungal actinomycetes. However, if liquid paraffin is found to be reduced, it should be supplemented in time. Puncture method and liquid paraffin covering method are simple, but the preservation period varies greatly due to different microbial species. Some fungi can be preserved for as long as 10 years. For some filamentous fungi with poor spore forming ability, liquid paraffin covering method is effective. However, other strains such as Azotobacter, Mycobacterium, Salmonella and Mucor are not suitable. In addition, when transplanting seeds from the liquid paraffin coating, the bacteria will splash with the liquid paraffin when the inoculation needle is burned on the flame. If the culture is a pathogen, it should be noted. The first generation of culture will have the residue and rejuvenation of liquid wax, and the second generation is suitable for experimental use

3. dry preservation method

this method is applicable to spore forming bacteria, spore forming filamentous fungi and actinomycetes. The principle is to adsorb microorganisms on various carriers and preserve them after drying. Generally, the garden soil or cultivated soil is ground and sieved (24 mesh), and used after drying. It can also be sieved with sand and washed alternately with sodium hydroxide and hydrochloric acid as the carrier. In 1.2 × Put soil or sand into a 10cm small test tube to a height of 1cm, and autoclave at 121 ℃ for 2~3 times. Bacteria can be added with concentrated suspension, fungi and actinomycetes can be directly scraped off spores to mix, and generally, the carrier can be slightly wet by visual inspection. Put the small test tube into a vacuum dryer or add phosphorus pentoxide to the dryer as a water absorbent for drying. The mouth of the test tube can be fused or sealed with paraffin, and then put it in the drying instrument for preservation at 5 ℃. Its preservation period is generally 2 years, and some microorganisms can be as long as 10 years. The preservation effect at room temperature is poor. When in use, only a small amount of soil or sand need to be dumped evenly on the slope, and then transplanted for use after growing well. In addition to using soil or sand as the carrier, silica gel, magnetic beads or porous glass beads with 6~12 mesh sieve can also be used, with 2) the diameter of the round sample ф： 2 ~ 20mm, and Qu Zi and wheat grains. Silica gel must be colorless. Why does the universal test opportunity slip? Do you know that silica gel indicator is toxic to microorganisms. When bacteria are added to silica gel, the temperature will increase correspondingly due to the production of adsorption heat. When inoculating, put the small test tube containing silica gel in water for cooling. Sand and soil are more commonly used carriers

4. suspension preservation method

the basic principle of suspension method is to suspend microorganisms in nutrient free solutions such as distilled water, 0.25m phosphate buffer (pH6.5) or normal saline for preservation. Applicable to filamentous fungi, yeast fungi and bacteria in the intestinal flora. Most of them can be preserved for 1 year or longer. The key of this method is to use spiral mouth test tubes with good sealing performance or general test tubes with rubber stoppers to prevent the evaporation of water. Store at 4 ℃, 10 ℃ or room temperature (18~20 ℃). At present, domestic manufacturers use corn fermenting mash to preserve seeds in the refrigerator. When inoculating, the old fermenting mash is connected to the fresh corn mash, and then heat-treated for heat preservation culture

5. Liquid nitrogen preservation method: at present, liquid nitrogen tanks are easier to purchase in laboratories. Using liquid nitrogen tank to preserve strains has good effect, simple method and the most extensive preservation objects. The method is to add the concentrated suspension to the sterilized dispersant, and add the bacteria with the final concentration of 10% glycerol or 5% dimethyl sulfoxide (DMSO) (added after sterilization as a antifreeze protective agent) ph7.6. This nutritional gelatin can be made into a good bacterial suspension, and many bacteria can be preserved for 4 years. This method is simple to operate but requires high sterility

6. Sordelli [4] vacuum drying preservation method: the basic principle of this method is to directly drain and preserve the bacterial solution in the small test tube without freezing. Generally 0.8 × 6cm small test tube with cotton stopper, add bacterial suspension made of dispersant such as skimmed milk. The concentration of bacteria in the suspension should be high and the number of small test tubes added should be small, generally no more than 5 drops. Oscillate the small test tubes to attach the suspension to the tube wall. After labeling, put it in 12 × In the 150cm large test tube, a small amount of phosphorus pentoxide and several small glass beads are added at the bottom of the large test tube so that the small test tube does not directly contact with the desiccant. The mouth of the large test tube is equipped with a rubber stopper with a glass tube inserted in the middle. Connect the vacuum pump with the rubber tube and vacuum to about 0.05 Torr. The rubber stopper and glass tube can be completely sealed with paraffin, or most of the test tubes can be narrowed at the lower part of the rubber stopper, and then vacuumized and sealed for the second time. When preserved at 5 ℃, NCTC reported that 2724 strains were preserved, and the survival rate after 14 years was 83%. Yeast and filamentous fungi can be preserved for several years, but they are not suitable for some sensitive beads

7. vacuum drying preservation method: this method is to directly dry the liquid sample under high vacuum, which is often called l-drying preservation method (drying from the liquid state for short). For viruses, bacteriophages and some spirochetes, the effect is better than freeze-drying. Bacteria and yeast can also be used. Add 1 ~ 2 drops of microbial suspension into the ampoule tube, cut off the cotton plug at the tube mouth and push the cotton plug part into the ampoule tube so as to narrow the tube at the upper end of the cotton plug. The tightness of cotton stopper is the key point of operation. Such ampoule tube is directly installed on the hole tube and dried by vacuum pump under reduced pressure. Its vacuum degree is about 0.1 Torr. After the same basic drying, it can be melted and sealed by pumping for 1H. The temperature of the sample is maintained in a 20 ℃ water bath, and some devices can reduce the temperature to 10 ℃, but it will not reach the freezing point. The drying time needs 10 ~ 20h or overnight. This method may not be used much, and the preservation period is less reported. The storage period of bacteriophage is about 5 years