Microbial strain preservation method

Microorganisms are prone to contamination, variability, and even death during use and passage, which often causes the decline of strains and may result in the loss of good strains. The important significance of strain preservation is to maintain the stability of its original traits and vitality as much as possible, to ensure that the strains do not die, do not mutate, and are not polluted, so as to meet the needs of research, exchange and use. What are the methods for preservation of strains?

Passage preservation method

Some microorganisms die quickly when they are subjected to freezing or drying, so in this case, they can only resort to subculture preservation. 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.

Liquid paraffin cover preservation method

This method is longer than the former method for preserving strains and is suitable for the preservation of molds, yeasts, 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.

Suspension preservation method

A method in which microorganisms are suspended in a suitable solution for preservation. Commonly used are:

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: It is suitable for yeast. If it is suspended in 10% sucrose solution, it can be stored in cold and dark place for up to 10 years. In addition to this, it can also be stored using a buffer solution or saline solution.

Vector preservation method

A method in which microorganisms are adsorbed on a suitable carrier for drying and storage. There are several commonly used methods including the following:

1 soil preservation method

It is 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 the clean sand, remove the large sand particles through the 60 mesh sieve, and use the magnet to remove the iron scraps from the sand, then wash it with NaOH solution, 10% HCl solution and water alternately several times. After drying, put it in the test tube or ampule tube to keep 2 ~3cm deep, after dry heat sterilization, add 1ml strain culture solution, mix well, put it into a 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

The sand is replaced by 6 to 16 mesh colorless silica gel, and after dry heat sterilization, the bacterial liquid is added. 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 solution is immersed in the magnetic beads (or porous glass beads) and then stored in a dry state. 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 was added to the bran, inoculated and cultured after sterilization, and finally dried and preserved.

6 paper (filter paper) preservation 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.

Host preservation method

A method by which a microorganism invades its host and preserves it.

Cryopreservation

Suitable for microorganisms with strong freezing resistance. 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 preserving methods, you should pay attention to the following points:

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 and 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 bacterial deaths 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 to stand for a while before processing.

7 For animal cells, the temperature should be slowly lowered at a rate of about 1 °C/min in the range of -20 °C, and then it should be lowered to the storage temperature as soon as possible; for most microorganisms, this is not necessary, if the structure is complicated 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 is carried out, the lower the storage temperature, the better;

9 When using cryopreserved strains, rapid-melting measures should be taken, that is, gently vortex in warm water at 35-40 °C to melt 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.

Common cryopreservation

1 Low-temperature refrigerator storage method (-20 °C, -50 °C or -85 °C): It is convenient to use the spiral tube in cryopreservation, and the plastic film can also be wrapped outside the tampon 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): Insert the strain tube into dry ice, and then store it in the refrigerator for cryopreservation.

3 Liquid nitrogen storage method (-196 ° C): It is the most widely used microbial preservation method. The steps are as follows:

a. Install the ampoule tube: use as much thick bacteria as possible 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 Directly inoculate the ampoule with the dispersing agent, or suspend the mycelial agar block directly in the dispersing agent.

b. Melting the ampoules: If stored directly in the vaporous liquid nitrogen (-150 ° C ~ -170 ° C), no need to seal.

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 or not 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 a refrigerator at -20 ° C ~ -25 ° C. Cool for 30 to 60 minutes.

e. Quick cooling: Finally immersed in liquid nitrogen and rapidly cooled to -196 °C.

Freeze-drying preservation

Its principle is to first freeze the microorganisms and then use the sublimation phenomenon to remove water under reduced pressure. 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 check the purity of the strain. 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, serum with 7.5% glucose, and the like.

Bordelli's method

Take a clean small tube (8 × 60mm) and put a tampon to sterilize. The bacterial moss on the inclined surface of the test tube was eluted with sterilized skim milk to prepare a thick bacterial suspension. The suspension is then dropped into the bottom of the vial with a sterile pipette. One drop per tube, then turn the small tube to disperse the bacteria solution on the wall at the bottom of the tube. Mark the name of the fungus. The small test tube was placed in a 15 x 150 mm test tube, and 1.5 cm high (P2O5 or KOH) was preliminarily attached to the bottom of the large test tube. The tube was stoppered with a rubber stopper with a glass tube and sealed with a wax. The large test tube is connected to the vacuum pump, and when vacuum is applied to 0.1 to 0.2 mm of mercury, the glass tube is sealed and stored in a dark room or in a refrigerator.