First, the basic principle Microorganisms have the characteristics of being easily mutated. Therefore, in the process of preservation, the metabolism of microorganisms must be in an inactive or relatively static state, so that they can not be mutated and maintain their living ability within a certain period of time.
Low temperature, dryness and air isolation are important factors in reducing the metabolic capacity of microorganisms. Therefore, although there are many methods for preservation of strains, they are all designed according to these three factors.
The preservation methods can be roughly divided into the following types:
1. The subculture method also includes slant culture, puncture culture, blister culture, etc. (the latter is used for preservation of anaerobic bacteria), and is stored in a refrigerator at 4-6 ° C after cultivation.
2. The liquid paraffin cover preservation method is a phase change method of subculture, which can prolong the preservation time. It is covered with sterilized liquid paraffin on the slant culture and the puncture culture to prevent the death of the bacteria caused by the evaporation of the culture medium. On the other hand, it prevents oxygen from entering to weaken metabolism.
3. The carrier preservation method is a preservation method in which microorganisms are adsorbed on a suitable carrier such as soil, sand, silica gel, filter paper, and then dried, for example, sand preservation method and filter paper preservation method are widely used.
4. The host preservation method is used for microorganisms that are not currently grown on artificial medium, such as viruses, rickettsia, spirochetes, etc., which must be infected and passaged in living animals, insects, and chicken embryos. Subculture and preservation of microorganisms. Microorganisms such as viruses can also be preserved by other methods such as liquid nitrogen storage and freeze-drying preservation.
5. The cryopreservation method can be divided into a low-temperature refrigerator (-20-30 ° C, -50--80 ° C), dry ice alcohol rapid freezing (about -70 ° C) and liquid nitrogen (-196 ° C) and other preservation methods.
6. The freeze-drying preservation method first rapidly freezes the microorganisms at an extremely low temperature (about -70 ° C), and then removes the water by using a sublimation phenomenon under reduced pressure (vacuum drying).
Some methods, such as filter paper preservation method, liquid nitrogen storage method, and freeze-drying preservation method, require the use of a protective agent to prepare a cell suspension to prevent damage to cells due to freezing or moisture sublimation. Protective solutes stabilize the configuration of cellular components by the affinity of hydrogen and ionic bonds to water and cells. The protective agent includes cow's milk, serum, sugar, glycerin, dimethyl sulfoxide and the like.
Second, equipment bacteria, yeast, actinomycetes and mold;
Meat paste peptone slant medium, sterilized skim milk, sterilized water, chemically pure liquid paraffin, glycerin, phosphorus pentoxide, river sand, thin loess or laterite, ice cubes, salt, dry ice, 95% alcohol, 10% Hydrochloric acid, anhydrous calcium chloride;
Sterilized straw, sterile dropper, sterile culture dish, tubular ampoule, 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 ° C), liquid nitrogen frozen storage.
Third, the operation steps, the 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. The slant surface cryopreservation method inoculates the strain on a suitable solid slant medium. After the bacterium is fully grown, the tampon portion is wrapped with oil paper and transferred to a refrigerator at 2-8 ° C for storage.
The preservation time varies depending on the type of microorganism, and mold, actinomycetes and spore-bearing bacteria are stored for 2-4 months, and transplanted once. Yeast for two months, the bacteria zui is good to transplant once a month.
This method is a commonly used preservation method in laboratory and factory strain chambers. The advantage is that it is easy to operate, easy to use, and requires no special equipment. It can check whether the deposited strains are dead, mutated and contaminated with bacteria at any time. The disadvantage is that it is easy to mutate because the physical and chemical properties of the medium are not strictly constant. Repeated passages will change the metabolism of microorganisms and affect the traits of microorganisms; there are more opportunities to contaminate the bacteria.
2. Liquid paraffin preservation method (1) Dispensing liquid paraffin in an Erlenmeyer flask, stuffing a cotton plug, wrapping it with kraft paper, sterilizing at 1.05kg/cm2>, 121.3°C for 30 minutes, and then placing it in a 40°C incubator So that the water vapor evaporates and is ready for use.
(2) The strain to be preserved is cultured in a suitable slant medium of Zui to make a strong cell or spore.
(3) Aspirate the sterilized liquid paraffin with a sterile pipette and inject it onto the inclined surface of the long-lasting fungus. The amount of the liquid paraffin is 1 cm above the top of the bevel, so that the strain is isolated from the air.
(4) Hold the test tube upright and store it at low temperature or room temperature (some microorganisms are longer than the time stored in the refrigerator at room temperature).
This method is practical and effective. Mold, actinomycetes, spore bacteria can be preserved for more than 2 years, yeast can be preserved for 1-2 years, generally no spore bacteria can be preserved for about 1 year, even meningococcal bacteria that are difficult to preserve by general methods, at 37 ° C It can also be stored for 3 months in the incubator. The advantage of this method is that it is simple to make, does not require special equipment, and does not need to be transplanted frequently. The disadvantage is that it must be placed upright when stored, occupying a large position and not being carried at the same time. After the culture is removed from the liquid paraffin, the culture is easily splashed with the residual liquid paraffin when the inoculating ring is cauterized on the flame, and special care should be taken.
3. Filter paper preservation method (1) Cut the filter paper into 0.5×1.2cm strips, put into 0.6×8cm ampoule tube, 1-2 sheets per tube, plug with cotton plug, 1.05kg/cm2 >, sterilized at 121.3 ° C for 30 minutes.
(2) The strain to be preserved is cultured on a suitable slant medium to allow sufficient growth.
(3) Take 1-2 parts of sterilized skim milk and add it to the sterilized culture dish or test tube, and mix the several ring of bacteria in the milk to make a concentrated suspension.
(4) Using a sterile tweezers, take a filter paper strip from the ampoule tube and immerse it in the bacterial suspension to make it saturated, then put it back into the ampoule tube and plug the cotton plug.
(5) The ampoule tube was placed in a desiccator containing phosphorus pentoxide as a water absorbing agent, and evacuated by a vacuum pump to dryness.
(6) Insert the cotton into the tube, seal it with flame according to Figure VII-13, and store it at low temperature.
(7) It is necessary to use the strain. When the culture is revived, the ampoule can be heated on the flame. A drop of cold water is sprayed on the hot part to break the glass. Then the tweezers are used to knock off the glass at the mouth. The filter paper is taken out, placed in a liquid medium, and cultured in an incubator.
Bacteria, yeasts, and filamentous fungi can be preserved by this method. The former two can be preserved for about 2 years, and some filamentous fungi can be preserved for 14-17 years. This method is simpler than liquid nitrogen and freeze drying, and does not require special equipment.
4. Sand preservation method (1) Add 10% dilute hydrochloric acid to Hesha and heat and boil for 30 minutes to remove organic matter.
(2) Pour off the acid water and rinse with tap water until neutral.
(3) Drying, sifting with a 40 mesh sieve to remove coarse particles and use.
(4) Take the non-cultivated layer of humus-free thin loess or laterite, and wash it with tap water for several times until neutral.
(5) Drying, crushing, and sieving through a 100 mesh sieve to remove coarse particles.
(6) According to the ratio of one loess and three parts of sand (or other proportions as needed, or even all of the sand or all of the soil), mix them evenly into small test tubes or ampoules of 10×100mm, each tube Pack about 1g, stuff the cotton plug, sterilize and dry.
(7) Sampling for sterility inspection, pumping one out of every 10 sand pipes, pour the sand into the broth culture medium, and incubate at 37 °C for 48 hours. If there are still bacteria, all the re-sterilization should be carried out. The bacteria test until the sterility is proved before use.
(8) Selecting mature cultures (generally, the spore layer is full-bodied, and the vegetative cells are not effective in this method). The excellent strains are washed with sterile water to prepare a spore suspension.
(9) Add about 0.5 ml (generally just wet the sand) to each sand tube, and mix well with the inoculation needle.
(10) Put it into a vacuum dryer and drain the water with a vacuum pump. The shorter the drying time, the better, so that it can be drained within 12 hours.
(11) Take one out of every 10 pieces, take a small amount of sand from the inoculating loop, inoculate it on the slant medium, and culture it to observe the growth and the growth of the bacteria. If there are few or no colonies or no colonies If it is long, it means that there is a problem with the sand pipe produced, and further sampling is required.
(12) If there is no problem after inspection, seal the nozzle with flame, and store it in the refrigerator or indoor dry place. Check vitality and bacteria every six months.
(13) It is necessary to use the strain. When the culture is revived, the sand is slightly transferred into the liquid medium and cultured in a thermostat.
This method is mostly used for spore-producing microorganisms such as mold and actinomycetes. Therefore, it is widely used in the production of antibiotics, and the effect is good. It can be stored for about 2 years, but it is not effective for vegetative cells.
5. Liquid nitrogen cryopreservation method (1) Prepare an ampoule for use in liquid nitrogen storage. It is required to withstand sudden changes in temperature without rupture. Therefore, an ampoule tube made of borosilicate glass is required, and the size of the ampoule tube is usually used. 75 × 10mm, or can hold 1.2mm liquid.
(2) When adding a protective agent and sterilizing cells that are easily dispersed, such as bacteria, yeast or mold spores, plug the empty ampoule tube with a tampon, 1.05kg/cm2, and sterilize at 121.3°C for 15 minutes: For the preservation of mold mycelium, a protective agent such as 10% glycerin distilled water solution or 10% dimethyl sulfoxide distilled water solution is added to the ampoule tube, and the amount is limited to the colony block which can be added after immersion, and then used again. 1.05 kg/cm2>, sterilized at 121.3 ° C for 15 minutes.
(3) Accessing the strains The strain is made into a bacterial suspension using a 10% glycerol distilled aqueous solution and filled into a sterilized ampoule; the mold mycelium can be cut from the flat by a sterilized puncher. The block is placed in an ampoule containing a protective agent and then sealed with a flame. Immerse in water to check for leaks.
(4) Freeze and then freeze the sealed ampoule to -30 °C at a slow rate of 1 °C per minute. If the cells are frozen sharply, ice crystals form in the cells, thus reducing the survival rate.
(5) The ampoule tube that has been frozen to -30 °C is immediately placed in a small cylinder of a liquid nitrogen cryopreservation (Fig. VII-14), and then the small cylinder is placed in a liquid nitrogen reservoir. The gas phase in the liquid nitrogen reservoir is -150 ° C, and the liquid nitrogen is -196 ° C.
(6) When it is necessary to restore the cultured cultured species, the ampoule tube is taken out and immediately placed in a water bath at 38-40 ° C for rapid thawing until it is completely melted. The ampoule is then opened and the contents are transferred to a suitable medium for cultivation.
In addition to being suitable for the preservation of general microorganisms, this method can preserve long-term microorganisms such as mycoplasma, chlamydia, hydrogen bacteria, molds, phage and animal cells that are difficult to form by lyophilization, and the traits are not mutated. The downside is that special equipment is required.
6. Freeze-drying preservation method (1) Preparation of ampoule tube The ampoule tube used for the preservation of freeze-dried strains should be made of neutral glass. The shape can be long-necked spherical bottom, also known as tear-drop type ampoule tube (Fig. VII-15). The outer diameter is 6-7.5mm, the length is 105mm, the diameter of the ball is 9-11mm, and the wall thickness is 0.6-1.2mm. A tubular ampoule without a ball can also be used. Plug the tampon, 1.05kg/cm2>, sterilize at 121.3 °C for 30 minutes, and set aside.
(2) Preparation of strains, strains preserved by freeze-drying method, the preservation period of which can be several years to ten years, in order to make no mistakes after many years, the strains used should pay special attention to its purity, that is, there must be no The bacteria are contaminated, and then cultured in a zui suitable medium at a suitable temperature to prepare a good culture. The bacterial age of bacteria and yeast is required to exceed the logarithmic growth phase, and if the strains in the logarithmic growth phase are preserved, the survival rate is reduced. Generally, bacteria require 24-48 hours of culture; yeast should be cultured for 3 days; spore-forming microorganisms should preserve spores; actinomycetes and filamentous fungi are cultured for 7-10 days.
(3) Preparation of bacterial suspension and dispensing Take the bacterial bevel as an example, and add about 2 ml of skim milk to the inclined test tube to prepare a concentrated bacterial solution, and each ampoule is dispensed with 0.2 ml.
(4) The freeze-freezer has a complete set of equipment for sale, which is expensive. The simple method and device are introduced here to achieve the same purpose.
The packaged ampoule tube is frozen in a low-temperature refrigerator. The non-low temperature refrigerator can be used with a refrigerant such as dry ice (solid CO2) alcohol solution or dry ice acetone solution at a temperature of -70 °C. Insert the ampoule into the cryogen and freeze it for 4-5 minutes to freeze the suspension.
(5) Vacuum drying to keep the sample frozen when vacuum drying, prepare a freezing tank, crush the ice cubes and salt in the tank, mix well, and cool to -15 °C. The instrument is shown in Figure VII-16, and the ampoule is placed in a dry bottle in the freezing tank.
If the pumping can reach a vacuum of 93.3Pa (0.7mmHg) in 30 minutes, the dry matter will not melt, and then continue to pump. In a few hours, the dried object can be observed to be dry, generally Pump a vacuum of 26.7Pa (0.2mmHg) and keep the pressure for 6-8 hours.
(6) After vacuum sealing and drying, take out the ampoule tube and connect it to the glass tube for sealing. You can continue pumping with the L-shaped five-way tube (Fig. VII-17), which can reach 26.7Pa in about 10 minutes. 2mmHg). In a vacuum state, a fine flame of a gas burner 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 effective methods for Zui in the method of preservation of bacteria. It is suitable for microorganisms with high viability and their spores and non-spore bacteria, even for some pathogenic bacteria that are difficult to preserve, such as meningococcal and gonorrhea. Can also be saved. It is suitable for long-term preservation of strains. It can be stored for several years to more than ten years, but the equipment and operation are more complicated.
Fourth, the experimental report
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