1. Basic principles
Microorganisms have the characteristics of easy mutation. Therefore, in the preservation process, the metabolism of microorganisms must be kept in the most inactive or relatively static state in order to maintain their ability to live without mutation in a certain period of time.
Low temperature, dryness, and air isolation are important factors that reduce the metabolic capacity of microorganisms. Therefore, although there are many methods for preserving strains, they are designed based on these three factors.
Second, the preservation methods can be roughly divided into the following types:
1. Subculture and preservation method
There are also slant culture, puncture culture, blister medium culture, etc. (the latter is used for the preservation of anaerobic bacteria), and after culture it is stored in a refrigerator at 4-6 ° C.
2. Liquid paraffin overlay preservation method
It is a phase-change method of subculture, which can prolong the storage time properly. It is covered with sterilized liquid paraffin on the slant culture and puncture culture. Prevent oxygen from entering to weaken the metabolism.
3. Carrier deposit method
Microorganisms are adsorbed on appropriate carriers, such as soil, sand, silica gel, filter paper, and then dried for preservation methods, such as sand preservation method and filter paper preservation method is widely used.
4. Host Deposit Law
Used for microorganisms that cannot be grown on artificial mediums at present, such as viruses, rickettsia, spirochetes, etc., they must be infected and passaged in living animals, insects, chicken embryos, this method is equivalent to the passage of general microorganisms Cultivate preservation methods. Microorganisms such as viruses can also be preserved by other methods such as liquid nitrogen preservation and freeze-dry preservation.
5. Frozen preservation method
It can be divided into preservation methods such as low temperature refrigerator (-20-30 ℃, -50-80 ℃), quick freezing of dry ice alcohol (about -70 ℃) and liquid nitrogen (-196 ℃).
6. Freeze drying preservation method
First, the microorganisms are quickly frozen at a very low temperature (about -70 ° C), and then sublimation is used to remove water under reduced pressure (vacuum drying).
Some methods such as filter paper preservation, liquid nitrogen preservation and freeze-drying preservation require the use of a protective agent to prepare the cell suspension to prevent damage to the cells due to freezing or constant water sublimation. Protective solutes can stabilize the configuration of cellular components through the affinity of hydrogen and ionic bonds to water and cells. Protective agents include milk, serum, sugar, glycerin, dimethyl sulfoxide, etc.
3. Equipment
Bacteria, yeasts, actinomycetes and molds;
Meat peptone slant medium, sterilized skim milk, sterilized water, chemically pure liquid paraffin, glycerin, phosphorus pentoxide, river sand, lean loess or red clay, ice cubes, table salt, dry ice, 95% alcohol, 10% Hydrochloric acid, anhydrous calcium chloride; sterilized pipette, sterilized dropper, sterilized petri dish, tube-shaped ampoule tube, teardrop-shaped ampoule tube (long neck spherical bottom), 40 mesh and 100 mesh sieve, oil paper, filter paper Strip (0.5 × 1.2cm), dryer, vacuum pump, vacuum pressure gauge, blowtorch, L-shaped five-way tube, refrigerator, low temperature refrigerator (-30 ℃), liquid nitrogen freezer.
4. Operation steps, application scope and advantages and disadvantages of each preservation method
The following methods can be selected according to the specific conditions and needs of the laboratory.
1. Slope cryopreservation method
Inoculate the strains on a suitable solid inclined medium. After the bacteria have fully grown, the cotton plug part is wrapped with oil paper and moved to a refrigerator at 2-8 ° C for preservation.
The storage time varies according to the type of microorganisms. Molds, actinomycetes and spore-forming bacteria are stored for 2-4 months, and transplanted once. Yeast for two months, and the bacteria are best transplanted once a month.
This method is a commonly used preservation method in laboratory and factory strain room. It has the advantages of simple operation, convenient use, no special equipment, and can check whether the deposited strain is dead, mutated and contaminated. The disadvantage is that it is easy to mutate, because the physical and chemical characteristics of the culture medium are not strictly constant. Repeated passages will change the metabolism of the microorganisms and affect the characteristics of the microorganisms; there are also more opportunities to contaminate the bacteria.
2. Liquid paraffin preservation method
(1) Pack the liquid paraffin in a triangular flask, put a cotton stopper on it, and wrap it with kraft paper, 1.05 kg / cm2>, sterilize at 121.3 ℃ for 30 minutes, and then put it in a 40 ℃ incubator to evaporate the water vapor. .
(2) The strains to be preserved are cultivated in the most suitable inclined culture medium, so that strong bacteria or spores are obtained.
(3) Aspirate the sterilized liquid paraffin with a sterilizing pipette and inject it into the inclined surface of the grown bacteria. The dosage is 1 cm higher than the top of the inclined surface (Figure â…¦-12) to isolate the bacteria from the air.
(4) Keep the test tube upright and store it at low temperature or room temperature (some microorganisms are stored at room temperature longer than the refrigerator).
This method is practical and effective. Molds, actinomycetes, and spore bacteria can be preserved for more than 2 years without death, yeasts can be preserved for 1-2 years, generally non-spore-free bacteria can also be preserved for about 1 year, and even meningococci that are difficult to preserve by general methods, at 37 ℃ It can also be kept in the incubator for 3 months. The advantage of this method is that it is simple to manufacture, does not require special equipment, and does not require frequent seeding. The disadvantage is that it must be placed upright during storage, occupying a large position, and it is also not portable. After taking the culture under the liquid paraffin and transplanting, when the inoculation ring is burnt on the flame, the culture is easy to splash together with the residual liquid paraffin, so special care should be taken.
3. Filter paper preservation method
(1) Cut the filter paper into 0.5 × 1.2 cm strips, put them into 0.6 × 8 cm ampule tubes, 1-2 sheets per tube, plug with cotton plug, 1.05 kg / cm2>, sterilize at 121.3 ℃ for 30 minutes .
(2) The strains that need to be preserved are cultivated on a suitable slant medium to fully grow.
(3) Take 1-2ml of sterilized skim milk and drop it in a sterilized Petri dish or test tube. Take a few rings of moss and mix it in the milk to make a concentrated suspension.
(4) Using sterile forceps, take the filter paper strip from the ampoule tube and immerse it in the bacterial suspension to make it full, then put it back into the ampoule tube and put a cotton plug on it.
(5) Place the ampoule tube in a desiccator with phosphorus pentoxide as water absorbent, and pump it to dryness with a vacuum pump.
(6) Insert cotton into the tube, seal with flame according to Figure â…¦-13, and store at low temperature.
(7) It is necessary to use strains. When resurrecting the culture, you can heat the ampoule tube mouth on the flame, drop a drop of cold water on the hot part to break the glass, and then use tweezers to knock off the glass at the mouth end. , Take out the filter paper, put it into the liquid medium, and put it in the incubator for cultivation.
Bacteria, yeast and filamentous fungi can be preserved by this method. The first two can be preserved for about 2 years, and some filamentous fungi can even be preserved for 14-17 years. This method is simpler than liquid nitrogen and freeze-drying methods and does not require special equipment.
4. Sand preservation method
(1) Take river sand and add 10% dilute hydrochloric acid, heat and boil for 30 minutes to remove organic matter.
(2) Pour off the acidic water and rinse it to neutrality with tap water.
(3) Drying and sieving with a 40-mesh sieve to remove coarse particles and set aside.
(4) Take another non-cultivated layer of thin yellow soil or red soil without humus, add tap water to wash for several times until neutral.
(5) Dry, crush and sieve through a 100-mesh sieve to remove coarse particles.
(6) According to the ratio of one part of loess and three parts of sand (or other proportions according to needs, or even all or even all of the soil), mix evenly and put into a small test tube or ampoule tube of 10 × 100 mm, each Pack about 1 g of tube, put a cotton plug on it, sterilize and dry.
(7) Sampling for sterility inspection, drawing one for every 10 sand and soil tubes, pouring the sand and soil into the broth culture medium, and cultivating at 37 ℃ for 48 hours. Bacterium test can not be used until it proves to be sterile.
(8) Select the cultured mature (generally refers to the spore layer is full-grown, vegetative cells are not effective with this method), good bacteria, washed with sterile water to make a spore suspension.
(9) Add about 0.5 ml of spore suspension (usually just wet the sand) to each sand tube, mix well with the inoculation needle.
(10) Put it in a vacuum dryer and use a vacuum pump to dry the water. The shorter the drying time, the better. Make sure that it is dried within 12 hours.
(11) Take one out of every ten, take out a few sand grains with the inoculation ring, inoculate it on the slant medium, cultivate it, observe the growth situation and the growth of miscellaneous bacteria, such as the occurrence of miscellaneous bacteria or few or no colonies If it is long, it indicates that there is a problem with the sand pipe made, and further sample inspection is required.
(12) If there is no problem after inspection, seal the tube mouth with flame and store in the refrigerator or indoor dry place. Check the vitality and miscellaneous bacteria every six months.
(13) It is necessary to use strains. When resurrecting the culture, take a little sand and soil into the liquid medium, and place it in a thermostat for cultivation.
This method is mostly used for microorganisms that can produce spores such as molds and actinomycetes. Therefore, it is the most widely used in the industrial production of antibiotics. The effect is also good. It can be stored for about 2 years, but it is not good for vegetative cells.
5. Liquid nitrogen freezing preservation method
(1) Prepare an ampoule tube for liquid nitrogen storage. It must be able to withstand sudden changes in temperature without breaking. Therefore, an ampoule tube made of borosilicate glass is required. The size of the ampoule tube is usually 75 × 10 mm. , Or capable of holding 1.2 mm liquid.
(2) When adding a protective agent and sterilizing and storing bacteria, yeast or mold spores and other cells that are easily dispersed, the empty ampoule tube is plugged with a cotton plug, 1.05 kg / cm2, and sterilized at 121.3 ° C for 15 minutes: For mycelium, a protective agent such as 10% distilled glycerol aqueous solution or 10% dimethyl sulfoxide distilled aqueous solution needs to be added in advance to the ampoule tube. The amount of addition is limited to the colonies that can be added after immersion. cm2>, sterilized at 121.3 ° C for 15 minutes.
(3) Connect the bacteria to make the bacteria suspension with 10% glycerol distilled aqueous solution, and put it into a sterilized ampoule tube; mold mycelium can use a sterilized punch to cut the colony circle from the plate Block, put it in an ampoule tube containing a protective agent, and then seal it with a flame. Immerse in water to check for leaks.
(4) Freeze and then freeze the sealed ampoule tube at a slow rate of 1 ° C per minute to -30 ° C. If the cells are suddenly frozen, ice crystals will form inside the cells, thereby reducing the survival rate.
(5) Store the ampoule tube frozen to -30 ℃ immediately into the small cylinder of the liquid nitrogen freezer (Figure Ⅶ-14), and then put the small cylinder into the liquid nitrogen container. The gas phase in the liquid nitrogen storage is -150 ° C, and the liquid nitrogen is -196 ° C.
(6) When it is necessary to restore the cultured and preserved strains, take out the ampoule tube and immediately put it in a 38-40 ° C water bath for rapid thawing until all are melted. Then open the ampoule tube and transfer the contents to a suitable medium for cultivation.
In addition to being suitable for the preservation of general microorganisms, this method can preserve long-term preservation of some microorganisms that are difficult to preserve by freeze-drying methods, such as mycoplasma, chlamydia, hydrogen bacteria, molds that are difficult to form spores, bacteriophages, and animal cells. The disadvantage is that special equipment is required.
6. Freeze drying preservation method
(1) Prepare the ampoule tube. The ampoule tube used for the preservation of freeze-dried strains should be made of neutral glass. The shape can be a long-necked spherical bottom, also known as a teardrop ampoule tube (Figure Ⅶ-15). The size requires an outer diameter of 6- 7.5 mm, length 105 mm, ball diameter 9-11 mm, wall thickness 0.6-1.2 mm. A tubular ampoule tube without a ball can also be used. Stuff the cotton plug, 1.05 kg / cm2>, sterilize at 121.3 ° C for 30 minutes, and set aside.
(2) Prepare strains and preserve them by freeze-drying method. The preservation period can be several years to ten years. In order to make no mistakes after many years, the strains used must pay special attention to their purity, that is, there can be no Contaminated by bacteria, and then cultivated in the most suitable medium with the most suitable temperature to make a good culture. The bacterial age of bacteria and yeast is required to exceed the logarithmic growth period. If the bacterial growth of the logarithmic growth period is used for preservation, the survival rate is reduced. Generally, bacteria require 24-48 hours of culture; yeast needs to be cultured for 3 days; spore-forming microorganisms should be kept for spores; actinomycetes and filamentous fungi are cultured for 7-10 days.
(3) Preparation of bacterial suspension and sub-packaging Take the bacterial slope as an example, add about 2 ml of defatted cow's milk to the inclined plane test tube to make a concentrated bacterial solution, and each ampoule tube is divided into 0.2 ml.
(4) The freeze freeze dryer has a complete set of equipment for sale, which is expensive. The simple method and equipment described here can achieve the same purpose.
Place the packed ampules in a low-temperature refrigerator for freezing. No low-temperature refrigerator can use refrigerants such as dry ice (solid CO 2>) alcohol liquid or dry ice acetone liquid. The temperature can reach -70 ℃. Insert the ampoule tube into the refrigerant and freeze it for only 4-5 minutes.
(5) Vacuum drying In order to keep the sample frozen during vacuum drying, a freezing tank needs to be prepared, and crushed ice cubes and table salt are placed in the tank, mixed evenly, and can be cooled to -15 ° C. The equipment is shown in Figure Ⅶ-16. The ampoule tube is placed in the drying bottle in the freezing tank.
Generally, if the vacuum can reach 93.3 Pa (0.7 mmHg) vacuum within 30 minutes, the dry matter will not melt, and then continue to evacuate. Within a few hours, it can be observed by the naked eye that the dried object has become dry. The vacuum degree is 26.7 Pa (0.2 mmHg), and the pressure can be maintained for 6-8 hours.
(6) After the sealing is evacuated and dried, take out the ampoule tube and connect it to the glass tube for sealing. You can use the L-shaped five-way tube (Figure â…¦-17) to continue to evacuate, reaching 26.7 Pa (0.2 mmHg) in about 10 minutes . In the vacuum state, a fine flame of a gas blowtorch is used to seal the center of the ampoule neck. After sealing, store in the refrigerator or in a dark place at room temperature.
This method is one of the most effective methods for the preservation of strains. It is suitable for general viable microorganisms and their spores and non-spores, even for some pathogenic bacteria that are difficult to preserve, such as meningococcus and gonorrhea Can also be saved. It is suitable for long-term preservation of bacteria, which can be stored for several years to more than ten years, but the equipment and operation are relatively complicated.
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