Everything You Need to Know about Microorganism Preservation

Microorganism preservation refers to techniques for maintaining the vitality and genetic traits of microbial strains. Microorganisms can easily become contaminated, mutate, or even die during use and passaging, often resulting in strain deterioration. The purpose of strain preservation is to keep the strain alive, avoid loss, prevent contamination by foreign microbes, and minimize or prevent mutation, and to retain the original viability and characteristics of the strain. The basic principle of strain preservation is to put the life activities of the microorganisms into a state of semi-permanent dormancy, in other words, to constrain microbial metabolism to the lowest range.

Common laboratory preservation methods include glycerol, slant, puncture, liquid paraffin overlay, desert soil preservation, and freeze-drying. Based on these preservation methods, microbial organisms have developed a ceramic bead preservation method that is more effective in preserving strains.

1. Glycerol Preservation Method

This method is suitable for medium to long-term storage of strains and can be stored for more than 1 year. Because glycerol itself is very thick and hard to measure, it is usually mixed with physiological saline at a ratio of 1:1 to prepare a final concentration of 50% glycerol solution. When used, it should be mixed with bacterial liquid in a ratio of 50% glycerol: 1:1 bacterial liquid, so that the final concentration of glycerol is 20-30%, neither too high nor too low concentration of glycerol is conducive to strain preservation. Transfer the glycerol strain to the -80°C refrigerator for long-term freezing. When you need to use the strain, put the frozen tube into the 37°C water bath to thaw quickly until the strain is completely melted, and then you can take out the strain for the experiment.

This method has a wide application range, is simple to operate, has a long preservation period, and the strain is not prone to mutation.

2. Slant Preservation Method

The strain is inoculated into a suitable solid slant culture medium. After the bacteria grow fully, they are transferred to a 4°C refrigerator for storage. The storage time depends on the type of microorganism. Generally, molds, actinomycetes and spore-forming bacteria need to be passaged every 2-4 months, yeast every two months, and bacteria are best passaged once a month. When you need to use the strain, you can take a small amount of bacteria from the slant with an inoculation loop to inoculate.

The advantage of this method is that it is easy to operate, convenient to use, does not need special equipment, and can check at any time whether the preserved strains are dead, mutated or contaminated with foreign bacteria. The disadvantage is that the preservation period is short, the strain is prone to mutation, because the physical and chemical properties of the culture medium are not strictly constant, repeated passage will change the metabolism of microorganisms, and the chance of contamination by foreign bacteria in this method is higher.

3. Stab Preservation Method

Prepare a tube of semi-solid culture medium, pick the strain with an inoculation needle, puncture vertically from the center of the semi-solid culture medium, until it is close to the bottom of the test tube, but do not puncture, then pull out the inoculation needle along the original puncture line, and store it in a 4°C refrigerator. This method needs to be passaged regularly like the slant preservation method.

4. Liquid Paraffin Overlay Preservation Method

This method is an auxiliary method of slant preservation. The strain is inoculated into a suitable solid slant culture medium. After the bacteria grow fully, aseptic liquid paraffin is injected to cover the entire slope, and then it is stored upright in a 4 °C refrigerator. Liquid paraffin can prevent the death of strains caused by evaporation of water in the culture medium, and can also isolate the strain from air, reduce the metabolic activity of the strain, and prolong the storage time. When you need to use the strain, you can pick a small amount of strain from the slope with an inoculation loop.

The advantage of this method is that it is easy to operate, does not need special equipment, and does not need to be passaged frequently, and it has a longer storage time. The disadvantage is that it must be placed upright during storage, which takes up more space.

5. Desert Soil Preservation Method

The cultivated strain is mixed with aseptic desert soil, and the bacteria are adsorbed on the desert soil carrier. After drying, they are transferred to a 4°C refrigerator for storage. To use the strain, take out a few grains of sand.

This method is simple to operate and has a long storage time for strains.

6. Freeze-drying Method

The strain is frozen and the water is removed by sublimation under pressure. This reduces the physiological activities of the cells and maintains the survival state of the strain for a long time. When you need to use the strain, use sterile water or culture medium to resuspend the cells, and then transfer the cell suspension to a solid culture medium plate for cultivation.

This method is suitable for long-term storage of the bacterial body, but the related equipment and operations are more complex than the other preservation methods.

7. Ceramic Bead Preservation Method

Based on the above several preservation methods, microbial organisms have developed ceramic bead preservation methods. Compared with traditional preservation methods, this method has better preservation effect, it’s convenient and can reduce the damage caused by repeated freezing and thawing.

Each ceramic bead-freezing tube contains 12 frosted ceramic beads and strain preservation solution. Pick up the logarithmic growth phase strain and transfer it to the freezing tube, turn it upside down to mix, at this time, the strain will be evenly distributed in the preservation solution. Use a pipette to remove the preservation solution, and under the action of the surface tension, each ceramic bead will be covered with a layer of preservation solution, in which the strain is preserved. The freezing tube containing the strain can be transferred to -80°C for long-term freezing (generally can be preserved for about 1 year).

When you need to use the strain, take out the freezing tube from the -80°C refrigerator, gently shake it to disperse the ceramic beads, pick up one ceramic bead and inoculate it on the culture medium plate for streaking, or inoculate it in liquid culture medium for shaking culture. After inoculation, quickly return the freezing tube to the refrigerator.

For anaerobic strains, we can also provide anaerobic preservation fluid containing reducing agents and vacuum pack the strain to avoid death of the strain due to exposure to oxygen.

There are many methods for strain preservation, but the principles are similar.

First of all, we must select excellent pure breeds. Use the spores, bud spores, and vegetative bodies of microorganisms;

Secondly, create conditions such as low temperature, dryness, or anoxia to suppress the metabolic function of microorganisms, reduce their life activities to the lowest level or put them into a dormant state, thereby extending the life of the strains and preserving the original shape of the strains to prevent mutations. Regardless of the preservation method used, it is required that the strain does not die, is not contaminated by foreign bacteria and does not degrade during the preservation process.

Short term: Slant preservation

Slant preservation is currently the most common short-term preservation method, easy to operate, convenient to use, but needs to be inoculated periodically. Therefore, the workload is large, and many passage times are prone to mutation. Therefore, it is only suitable for short-term strains that are often used.

1. 1. Prepare the culture medium, the concentration of nutrients should be slightly lower, especially the sugar as much as possible, if the strain produces acid, then a small amount of calcium carbonate should be added;
2. 2. The amount of slant filling, the slant should not exceed 1/2 of the length of the test tube after tilting is appropriate;
3. 3. Slant culture, temperature is slightly lower than optimal, time is slightly longer;
4. 4. After growth is complete, seal it and store it in a 2-8°C refrigerator. Each passage, molds, actinomycetes, and spore-forming bacteria can be stored for 1-3 months, yeast for 2 months, bacteria for 1 month.

Activation and use:

Take the slant out of the refrigerator, pick the colonies to propagate the bacteria, or cut the mold to propagate and culture, and then it can be used after the culture is done.

Long term: Frozen storage

Frozen storage is one of the most commonly used long-term preservation methods at present, suitable for all types of microorganisms. Compared with slant storage, it reduces the workload and extends the storage time.

1. 1. Prepare a fresh slant or plate culture, 10% sterile glycerol solution;
2. 2. Use glycerol solution to wash off the bacteria or spores to form a bacterial suspension;
3. 3. Divide the bacterial suspension, fill the cryo-tube with 1mL/tube, and then place in the program cooling box;
4. 4. Put the program cooling box in the -80℃ refrigerator for freezing;
5. 5. It can be stored for 1-2 years.

Activation and use:

1. 1. Remove the frozen strain from the cryo-box, prepare to dissolve it in a water bath, and do not melt it at room temperature;
2. 2. Put the strain quickly into the water bath for rapid dissolution, bacteria at 40℃, fungi at 30℃, after about 2 minutes all dissolved, then inoculate it into the appropriate medium;
3. 3. Complete the culture under appropriate conditions.

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