Microbial sample preservation refers to techniques that preserve the viability and genetic characteristics of microbial strains. Microorganisms are susceptible to contamination, mutation, and even death during use and passage, often leading to strain degradation. The purpose of culture preservation is to maintain the vitality of the culture, avoid loss, prevent contamination by foreign microorganisms, minimize or prevent mutation, and maintain the original vitality and characteristics of the culture. The basic principle of culture preservation is to bring the life activities of microorganisms into a semi-permanent dormant state, in other words, to limit the metabolism of microorganisms to a minimum.
Commonly used preservation methods in the laboratory include glycerol preservation, inclined plane preservation, puncture preservation, liquid paraffin cover method, desert soil preservation, and freeze-drying method. On the basis of these preservation methods, microbial organisms have developed a ceramic bead preservation method that can preserve strains more efficiently.
1.Glycerol preservation
This method is suitable for medium and long-term storage of cultures, which can be stored for more than 1 year. Because glycerol itself is thick and difficult to dose, it is usually mixed with normal saline in a 1:1 ratio to make a glycerol solution with a final concentration of 50%. When using, it should be mixed with the bacteria solution in the ratio of 50% glycerol:1:1 to make the final concentration of glycerol 20%-30%, and too high or too low glycerol concentration is not conducive to the preservation of the culture. Move the glycerol culture to a -80 freezer for long-term freezing. When the culture needs to be used, place the cryovial in a 37°C water bath and quickly thaw until the culture is completely thawed, and then remove the culture for experimentation.
This method has a wide range of applications, simple operation, long shelf life, and is not easy to mutate.
2.Inclined plane preservation method
Inoculate the strains into a suitable solid slope medium. Once the bacteria have grown sufficiently, transfer it to a 4°C freezer for storage. The shelf life depends on the type of microorganism. In general, molds, actinomycetes, and sporoformers need to be passaged every 2-4 months, yeasts are passaged every two months, and bacteria are best passaged monthly. When a culture is required, a small amount of bacteria can be inoculated from an inclined plane with an inoculation loop.
The advantages of this method are that it is simple to operate, easy to use, does not require special equipment, and can be checked at any time for the preserved bacteria to die, mutate or be contaminated with foreign bacteria. The disadvantage is that the shelf life is short, and the strain is prone to mutation, because the physical and chemical properties of the medium are not strictly constant, and repeated passage will change the metabolism of microorganisms, and the probability of contamination by foreign bacteria is high in this method.
3.Piercing preservation method
Prepare a tube of semi-solid transport medium, pick the culture with the inoculation needle, puncture vertically from the center of the semi-solid medium until it is close to the bottom of the tube, but do not puncture, and then pull out the inoculation needle along the original puncture line and place it in the 4 freezer for storage. This method, like the inclined plane preservation method, requires regular passaging.
4.Liquid paraffin overlay preservation method
This method is an auxiliary method to the inclined plane preservation method. Inoculate the strains into a suitable solid slope medium. Once the bacteria have grown sufficiently, inject sterile liquid paraffin to cover the entire slope and store it upright in a 4 freezer. Liquid paraffin can prevent the death of the strain due to the evaporation of water in the medium, and can also isolate the strain from the air, reduce the metabolic activity of the strain, and prolong the storage time. When strains are required, a small amount of strains can be picked from the inclined plane with an inoculation loop.
The advantages of this method are that it is simple to operate, does not require special equipment, does not need to be passed frequently, and has a long storage time. The disadvantage is that it must be placed upright when stored, which takes up a lot of space.
5.Desert Soil Conservation Act
The cultured strains are mixed with sterile desert soil to adsorb the bacteria on the desert soil carrier. After drying, transfer to a 4°C freezer for storage. When using the culture, just take out a few grains of sand.
This method is simple to operate and has a long shelf life for the culture.
6.Freeze-drying
The culture is frozen and the water is removed by pressurized sublimation. This can reduce the physiological activity of the cells and keep the bacteria alive for a long time. When the culture needs to be used, resuspend the cells using sterile water or medium, and then transfer the cell suspension to a solid medium plate for culture.
This method is suitable for long-term storage of bacteria, but the related equipment and operation are more complex than other preservation methods.
7.Ceramic bead preservation method
On the basis of the above-mentioned preservation methods, microorganisms have developed a ceramic bead preservation method. Compared with traditional preservation methods, this method has a better preservation effect, which is convenient and reduces the damage caused by repeated freeze-thaw cycles.
Each ceramic bead cryovial contains 12 frosted ceramic beads and culture preservation solution. Take the logarithmic growth strains and transfer them into a cryovial, invert and mix, at which point the strains will be evenly distributed in the preservation solution. Pipette to remove the preservation solution, and under the action of surface tension, each porcelain bead is covered with a layer of preservation solution, and the strain is stored in this layer of preservation solution. Cryovials containing cultures can be transferred to -80°C for long-term freezing (typically around 1 year).
When the culture needs to be used, take the cryovial out of the -80 freezer, gently shake to disperse the pottery beads, take a pottery bead and inoculate it on the medium plate for strip culture, or inoculate it into a liquid medium for ** culture. After inoculation, quickly put the cryovial back in the freezer.
For anaerobic strains, we can also provide anaerobic preservation solutions containing reducing agents and vacuum-pack the strains to avoid death due to exposure to oxygen.
There are many ways to preserve cultures, but the principles are similar.
The first thing to do is to choose a good purebred. use of spores, spores and clones of microorganisms;
Secondly, create conditions such as low temperature, dryness, and hypoxia to inhibit the metabolic function of microorganisms, so that their life activities can be reduced to the lowest level or enter a dormant state, so as to prolong the life of the strain, preserve the original form of the strain, and prevent mutation. No matter which preservation method is used, it is required that the bacteria do not die, are not contaminated by foreign bacteria, and do not degrade during the preservation process.
Short-term storage Bevel saving
Inclined storage is currently the most commonly used short-term storage method, which is simple to operate and easy to use, but requires regular vaccination. Therefore, the workload is large, and the number of passes is easy to mutate. Therefore, it is only suitable for short-term cultures that are used frequently.
1.To prepare the medium, the concentration of nutrients should be slightly lower, especially the sugars should be as much as possible, and a small amount of calcium carbonate should be added if the strain produces acid;
2.The amount of inclined surface filling, the inclined plane should not exceed 1 2 of the length of the test tube after inclination;
3.Slope culture, the temperature is slightly lower than the optimal temperature, and the time is slightly longer;
4.Once growth is complete, store tightly sealed in a 2-8 freezer. For each channel, molds, actinomycetes, sporophiles can be stored for 1-3 months, yeasts can be stored for 2 months, and bacteria can be stored for 1 month.
Activation and Use:
Remove the slope from the refrigerator and pick the colony to propagate bacteria, or cut the mold to propagate and culture, and it is ready to use after the culture is complete.
Long-term storage: Freeze storage.
Cryopreservation is one of the most commonly used long-term storage methods and is suitable for all kinds of microorganisms. Compared with inclined storage, it reduces the workload and prolongs the storage time.
1.Prepare fresh bevel or plate cultures, 10% sterile glycerol solution;
2. 2.Bacteria or spores are washed away with a glycerol solution to form a bacterial suspension;
3. 3.Aliquot the bacterial suspension, 1 milliliter per tube, into cryovials, and place in the program cooling box;
4. 4.Place the program cooling box in the -80 freezer to freeze;
5.Can be stored for 1-2 years.
Activation and Use:
1.Remove the frozen culture from the freezer and prepare to dissolve in a water bath, do not melt at room temperature;
2.The strains were quickly put into a water bath to quickly dissolve, 40 bacteria and 30 fungi, all of which were dissolved after about 2 minutes, and then inoculated into the corresponding medium;
3.Complete the culture under the appropriate conditions.