Microbial strain preservation method
1. Passage preservation method: Some microorganisms will die quickly when they are treated with freezing or drying. Therefore, in this case, they can only resort to the subculture preservation method. Subculture is to regularly carry out the transfer of strains, and then preserve them after cultivation. It is the most basic method of microbial preservation, such as the preservation of commonly used strains such as yogurt. When subcultured, the concentration of the medium should not be too high, and the nutrient content should not be too rich, especially the concentration of carbohydrate should be reduced as much as possible. The culture temperature is usually preferably slightly lower than the optimum growth temperature. If it is an acid-producing strain, a small amount of calcium carbonate should be added to the medium.
In general, the preservation temperature of most strains is preferably 5 ° C. Microbial strains such as anaerobic bacteria, Vibrio cholerae and some pathogenic fungi can be stored at 37 ° C, while strains of large edible fungi such as mites It can be stored directly at room temperature. Subculture and preservation method is simple, but its shortcomings are also obvious, such as: 1 strain of tube tampon is often prone to mold; 2 strains of genetic traits are prone to variability; 3 repeated passage, the pathogenicity of the strain, the formation of physiologically active substances The ability and the ability to form spores are reduced; 4 need to be transferred regularly, the workload is large; 5 bacteria have more pollution opportunities.
2, liquid paraffin cover preservation method: This method saves the strain for a longer period of time than the former method, and is suitable for the preservation of mold, yeast, actinomycetes and aerobic bacteria. This method prevents drying and reduces the metabolism of microorganisms by limiting the supply of oxygen. It has the advantages of simple method, and is also suitable for the preservation of microorganisms that are not suitable for freeze-drying (such as filamentous bacteria with low sporulation ability), and some bacteria such as nitrogen-fixing bacteria, lactobacillus, Leuconostoc, mycobacteria, red Spirulina and Salmonella, and some fungi such as Rolling Fungus, H. oxysporum, Mucor, Rhizopus, etc. should not be preserved by this method.
3. Carrier preservation method: a method in which microorganisms are adsorbed on a suitable carrier for drying and preservation. Commonly used methods include the following, such as 1 soil preservation method: mainly used for the preservation of microbial strains capable of forming spores or cysts. The method is to add the bacterial liquid to the sterilized soil, immediately dry at room temperature or make the cells propagate after drying, and then refrigerate or seal at room temperature for storage. The soil used for preservation is in principle suitable for fertile tillage, and the soil needs to be air-dried, pulverized, sieved and sterilized. The method of drying and preserving the microorganisms in the soil is as follows: take an appropriate amount of soil (5 g) in a test tube with a tampon, add water or add a fully diluted liquid medium (the water content is the maximum water holding capacity of the soil). 60% is suitable) and then autoclaved. The microorganism to be preserved is inoculated in a large amount, and cultured until the hyphae can be visually confirmed, and then transferred to a desiccator for drying for a short time or air-dried, sealed, refrigerated or stored at room temperature.
2 sand preservation method: take clean sand, remove large sand particles from the sieve, and use the magnet to suck off the iron filings in the sand, then wash it with NaOH solution, 10% HCl solution and water alternately several times, after drying, put it in test tube or ampoules Keep 2~3cm deep, and then dry heat sterilization, add 1ml strain culture solution, mix well, put it into vacuum dryer, completely dry and then seal and store. It is also possible to use two portions of washed sand (pretreated with HCl) and a barren, sieved loess and post-sterilized, and then preserved.
3 Silica gel preservation method: replace the sand with 6~16 mesh colorless silica gel, and add the bacterial liquid after dry heat sterilization. When the bacteria solution is added, since the heat of adsorption of the silica gel often raises the temperature, it is necessary to try to cool it.
4 magnetic bead preservation method: a method in which the bacterial liquid is immersed in the magnetic beads (or porous glass beads) and then dried and stored. Insert 1/2 tube high silica gel (or anhydrous CaSO4) into the screw tube, place the glass wool on top, and put 10 to 20 magnetic beads. After dry heat sterilization, connect the bacterial suspension and finally Store in a refrigerated, room-temperature or dry-pressure oven. This method is very effective against yeast, especially for rhizobium, which can be stored for up to two and a half years.
5 bran preservation method: 60% water is added into the bran, inoculated and cultured after sterilization, and finally dried and preserved.
6 Paper (filter paper) storage method: The sterilized paper is immersed in the culture solution or the bacterial suspension, dried under normal pressure or reduced pressure, and then stored in a container containing a desiccant.
4. Suspension preservation method: a method in which microorganisms are suspended in a suitable solution for preservation. Commonly used,
1 Distilled water preservation method: It is suitable for mold, yeast and most actinomycetes. It can be stored in distilled water for several years at room temperature. This method should take care to avoid evaporation of water.
2 sugar liquid preservation method: suitable for yeast, such as the suspension of the bacteria in 10% sucrose solution, and then stored in cold and dark, up to 10 years. In addition to this, it can also be stored using a buffer solution or saline solution.
5. Host preservation method: a method in which microorganisms invade their host and preserve them.
6, cryopreservation method: suitable for microorganisms with strong anti-freezing ability. These microorganisms can be damaged without being damaged by the extracellular cells of the cells, and for most other microorganisms, whether they are frozen outside the cells or frozen inside the cells, the cells are damaged, so when using this When paying attention to the preservation method, the following points should be noted.
1 to select bacterial age cells suitable for freeze drying;
2 to choose the appropriate medium, because some microorganisms resistance to freezing, often show a huge difference with the composition of the medium;
3 to choose the appropriate concentration of bacteria, usually the higher the concentration of bacteria, the higher the survival rate, the longer the shelf life;
4 It is best not to add electrolytes (such as salt) to the bacteria solution;
5 A protective agent such as glycerin may be added to the bacterial liquid to prevent a large number of deaths of the bacteria during the freezing process. Similarly, a solvent having a good protective effect such as various sugars, defibrinated blood, and defatted milk may be added, but for some microorganisms, it is more effective without a protective agent.
6 In principle, the freezing treatment should be carried out as soon as possible, but when the protective agent is added, it can be left for a while before being processed.
7 In terms of animal cells, it should be slowly cooled at a rate of about 1 ° C / min in the range of -20 ° C, and then must be lowered to the storage temperature as soon as possible; for most microorganisms, this is not necessary, such as more complicated structure The protozoa can be slowly cooled in the range of -35 ° C, and the phage must be frozen by the two-stage method described above;
8 If long-term storage, the lower the storage temperature, the better;
9 When using the cryopreserved strain, fast-melting measures should be taken, that is, gently oscillate in warm water of 35-40 °C to melt it quickly. In the case of anaerobic bacteria, the measures of standing and melting should be chosen. When the frozen bacteria are melted, try to avoid freezing again, otherwise the survival rate of the cells will be significantly reduced.
Commonly used cryopreservation methods include:
1 Low-temperature refrigerator storage method (-20 ° C, -50 ° C or -85 ° C): It is convenient to use a spiral tube when cryopreservation, or to wrap a plastic film outside the tube. When the preservation, the amount of the bacterial liquid should not be excessive, and some may be added with a protective agent. In addition, φ5mm glass beads can also be used to adsorb the bacterial liquid, and then the glass beads are placed in a plastic container and stored in a low temperature refrigerator.
2 dry ice preservation method (about -70 ° C): the bacteria tube is inserted into the dry ice, and then placed in the refrigerator for cryopreservation.
3 liquid nitrogen storage method (-196 ° C): is the most widely used microbial preservation method. The operation steps are as follows.
a. Install the ampoule tube: Use as much thick bacteria as possible to suspend in a sterile solution containing appropriate antifreeze (preserving mold without antifreeze), and dispense 0.2~1ml of this solution into the ampoule, or in the package. Directly inoculate the ampoule with the dispersing agent, or suspend the mycelial agar block directly in the dispersing agent.
b. Melting the ampoule tube: If it is directly stored in the vaporous liquid nitrogen (-150 ° C ~ -170 ° C), then no sealing is required.
c. Check whether the ampoules are well sealed: that is, after immersing the sealed ampoules in a suitable pigment solution for 2 to 30 minutes at 4 ° C, observe whether the pigment enters the ampoules.
d, slow cooling: the sealed ampoule tube is placed in a small tank, and then cooled to about -25 ° C with liquid nitrogen at a rate of about 1 ° C / min, or slowly in the refrigerator at -20 ° C ~ -25 ° C Cool for 30 to 60 minutes.
e, rapid cooling: finally immersed in liquid nitrogen and rapidly cooled to -196 °C.
7. Freeze-drying preservation method: The principle is to first freeze the microorganisms, and then use the sublimation phenomenon to remove water under reduced pressure. In fact, after most of the water is removed from the cells, the physiological activities of the cells are stopped, so that the long-term state of life can be achieved. This method is suitable for the preservation of most microbial species, including phage and rickettsia. When performing freeze-drying, pay attention to the following issues:
a. Culture conditions before freeze-drying: First, the purity of the strain is checked. Generally speaking, it is preferred to culture the microorganisms to be preserved on a nutrient-rich and easily enriched medium; the age of the bacteria is preferably in the logarithmic growth phase; if the microorganisms have spores or spores, the spores and spores are used. It should be preserved after formation; the concentration of the bacterial liquid should be high, for example, the bacteria should reach 109-1010/ml.
b. Marking of strain number, etc.: It can be marked on the outside of the ampoule or sealed in the ampoule.
c. Preparation of ampoules: soak the ampoules in 2% HCl overnight, rinse 3 times with tap water, brush with distilled water, dry, plug tampon, label, dry heat or warm sterilize, 60 °C constant temperature dry.
d, adding a protective agent: commonly used protective agents are skim milk, 12% sucrose, ordinary broth with 7.5% glucose and serum with 7.5% glucose and so on.
9. Glycerol tube preservation method: In a 5 ml strain collection tube, an equal volume of the bacterial suspension and 40% glycerin are thoroughly mixed, and then stored in a refrigerator at -20 °C.
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