Methods of preserving microbial strains in incubators and points to note

1, the transmission preservation method: some microorganisms when encountering freezing or drying and other treatments, will soon die, so in this case, can only resort to the transmission culture preservation method. Succession culture is to regularly transfer strains, culture and then save, it is the most basic microbial preservation method, such as yogurt and other common production of bacteria preservation.

When preserving by passaging, the concentration of the culture medium should not be too high, and the nutrients should not be too rich, especially the concentration of carbohydrates should be minimized within the possible range. The culture temperature is usually slightly below the optimal growth temperature. If it is an acid-producing strain, a small amount of calcium carbonate should be added to the medium.

Generally speaking, the preservation temperature of most strains is 5℃, and microbial strains such as anaerobic bacteria, Vibrio cholerae and some pathogenic fungi can be preserved at 37℃, while strains of large edible mushrooms can be preserved directly at room temperature.

Succession culture preservation method is simple, but its shortcomings are also obvious, such as: ① strain tube cotton plug is often easy to mold; ② strains of genetic traits are prone to mutation; ③ repeated substitution, the pathogenicity of strains, the formation of physiologically active substances, as well as the formation of spores, etc., have been reduced; ④ the need for regular transfer of seeds, the workload is large; ⑤ the chance of contamination of more stray bacteria.

2、Liquid paraffin covering preservation method: this method is longer than the previous method of preserving strains of bacteria, applicable to the preservation of molds, yeasts, actinomycetes and aerobic bacteria. This method can prevent drying, and by limiting the supply of oxygen to achieve the purpose of weakening the metabolism of microorganisms. It has the advantage of simple method, but also suitable for the preservation of microorganisms that are not suitable for freeze-drying (e.g., filamentous bacteria with low spore-producing ability), while some bacteria such as nitrogen-fixing bacteria, lactobacillus, Streptomyces, Mycobacterium, Rhizobium and Salmonella, etc. and some fungi such as curly molds, small grams of silverhammer molds, Trichoderma, Rhizoctonia and so on should not be used in this method for preservation.

3, carrier preservation method: that is, the microorganisms adsorbed on the appropriate carrier for dry preservation method. Commonly used methods include the following, such as ① soil preservation method: mainly used for the formation of spores or sporocysts of microbial strains of preservation. The method is to add the bacterial liquid in the sterilized soil, immediately dry at room temperature or make the bacterial propagation and then dry, and then refrigerated or sealed at room temperature preservation. The soil used for preservation is in principle fertile cultivated soil, and the soil needs to be air-dried, crushed, sieved and sterilized.

Make microorganisms in the soil reproduction and then dry preservation method is: take the appropriate amount of soil (5 grams) in a test tube stuffed with cotton plugs, add water or add fully diluted liquid medium (to the water content of the soil for the maximum water holding capacity of 60% is appropriate), and then autoclaved. Then the microorganisms to be preserved for a large number of inoculation, culture to the extent that the mycelium can be confirmed with the naked eye until the extent of the move into the desiccator after a short period of drying or air-drying after sealing, refrigeration or room temperature preservation.

② sand preservation method: take clean sand, 60 mesh sieve to remove large sand particles, and use a magnet to absorb the sand in the iron debris, and then NaOH solution, 10% HCl solution and water alternately cleaned several times, after drying, placed in a test tube or ampoule tube to maintain a 2-3cm deep, and then sterilized by dry heat, add 1ml of strain culture solution, after sufficient mixing, put into a vacuum desiccator, completely dry and then melt-sealed preservation. Can also be used two washed sand (pre-treated with HCl) and a barren, sifted loess mixed and sterilized, and then strain preservation.

③ silica gel preservation method: 6 to 16 purposes of colorless silica gel instead of sand, dry heat sterilization, add bacteria liquid. When adding bacterial liquid, the adsorption heat of silica gel often raises the temperature, and thus needs to be cooled.

④ Magnetic bead preservation method: the bacterial solution is immersed into the vegetarian burned magnetic beads (or porous glass beads) and then dry preservation of a method. In the spiral mouth test tube filled with 1/2 tube high silica gel (or anhydrous CaSO4), on the top of the glass wool, and then put on the 10-20 magnetic beads, after dry heat sterilization, access to the bacterial suspension, and finally refrigeration, room temperature preservation, or decompression drying and sealing preservation. This method is very effective for yeast, especially for rhizobium, can be preserved for up to two and a half years.

⑤ Bran preservation method: add 60% water in bran, inoculate the culture after sterilization, and finally dry and preserve.

⑥ paper (filter paper) preservation method: sterilized paper is immersed in culture solution or bacterial suspension, dried at atmospheric pressure or reduced pressure, and then placed in containers with desiccant for preservation.

4, suspension preservation method: even if the microorganisms are suspended in the appropriate solution for preservation methods. Commonly used, ① distilled water preservation method: for molds, yeasts and most of the actinomycetes, the bacteria will be suspended in distilled water can be preserved at room temperature for several years. This method should be careful to avoid the evaporation of water.

② Sugar solution preservation method: applicable to yeast, such as its body suspended in 10% sucrose solution, and then stored in a cold dark place, can be up to 10 years. In addition to this, buffer solution or saline can also be used for preservation.

5, host preservation method: that is, to make microorganisms invade their hosts to be preserved.

6. Cryopreservation method: it is suitable for microorganisms with strong resistance to freezing. These microorganisms can suffer freezing outside the cells of their organisms without damage, while for most other microorganisms, whether frozen outside the cell or frozen inside the cell, will cause damage to the organism, so when using this method of preservation, should pay attention to the following points, ① to select the age of the cells suitable for freeze-drying bacteria; ② to select the appropriate medium, because some microorganisms on the resistance of the freezing of Because the resistance of some microorganisms to freezing often shows great differences with the changes in the composition of the medium; ③ To choose the appropriate concentration of the bacterial solution, usually the higher the concentration of the bacterial solution, the higher the survival rate, the longer the preservation period; ④ It is best not to add electrolytes within the bacterial solution (such as salt, etc.); ⑤ Glycerol can be added to the bacterial solution in the bacterial solution and other protective agents, in order to prevent a large number of bacterial deaths occurring in the freezing process. Similarly, solvents with good protective effects such as various sugars, defibrillated blood and skimmed buttermilk can be added, but for some microorganisms they are more effective when no protective agent is added.

(vi) In principle, cryoprocessing should be carried out as soon as possible, but when a protective agent is added, it can be left to stand for some time before processing.

(7) in terms of animal cells, should be in the range of -20 ℃ to 1 ℃ / min about the rate of slow cooling, and thereafter must be as soon as possible to the storage temperature; for the vast majority of microorganisms, it is not necessary to do so, such as the structure of the more complex protozoa can be within the range of -35 ℃ for slow cooling, and phage must be used in the two-stage method for frozen; (8) the above-mentioned Stage method for freezing; ⑧ if for long-term preservation, the lower the storage temperature the better; ⑨ access to cryopreservation of strains, should be taken to take measures for rapid melting, that is, in the 35 ~ 40 ℃ warm water gently oscillate to make it quickly thawed. In the case of anaerobic bacteria, it should choose the measures of static thawing. When the frozen bacteria thawed, should try to avoid re-freezing, otherwise the survival rate of the bacteria will be significantly reduced.

Commonly used cryopreservation methods include:

① low-temperature refrigerator preservation method (-20 ℃, -50 ℃ or -85 ℃): low-temperature cryopreservation is more convenient to use the spiral mouth test tube, can also be wrapped in plastic film outside the cotton plug test tube. Preservation of bacterial liquid should not be added too much, some can add protective agent. In addition, can also be used φ5mm glass beads to adsorb the bacterial fluid, and then put the glass beads in plastic containers, and then into the low-temperature refrigerator for preservation.

② dry ice preservation method (-70 ℃ or so): the strain tube will be inserted into the dry ice, and then placed in the refrigerator for freezing preservation.

③ Liquid nitrogen preservation method (-196℃): it is the most widely applicable microorganism preservation method. The operating steps are as follows, a, ampoule tube: use the thickest possible bacteria suspended in a sterilized solution containing appropriate antifreeze (preservation of mold without antifreeze), 0.2 ~ 1ml of this solution is divided into ampoules, or in the ampoules containing dispersant directly inoculated, or mycelium agar block directly suspended in the dispersant.

b, melt-seal the ampoule tube: if it is stored directly in gas-phase liquid nitrogen (-150 ℃ ~ -170 ℃), there is no need to melt-seal.

c, check whether the ampoule tube is well fused: that is, at 4 ℃, the fused ampoule tube will be soaked in the appropriate pigment solution for 2 ~ 30 minutes, to observe whether there is no pigment into the ampoule.

d, slow cooling: the fusion-sealed ampoule tube is placed in a small tank, and then cooled down to -25 ℃ with liquid nitrogen gas at a speed of about 1 ℃ / min, or can be slowly cooled down for 30 to 60 min in the refrigerator at -20 ℃ to -25 ℃.

e. Quick cooling: finally immersed in liquid nitrogen for rapid cooling to -196℃.

7, freeze-drying preservation method: its principle is to first freeze the microorganisms, and then use the sublimation phenomenon to remove water under reduced pressure. In fact, after removing most of the water from the bacterium, the physiological activity of the cell will stop, so it can achieve the purpose of maintaining the life state for a long time. This method is suitable for the preservation of most microbial strains (including phage and rickettsiae, etc.).

When freeze-drying, the following problems should be noted:

a. Cultivation conditions before freeze-drying: firstly, test the purity of strains. Generally speaking, the microorganisms to be preserved in the nutrient-rich and easy to increase the bacterial medium for culture is appropriate; the age of the bacteria to reach the logarithmic growth period is good; if there are spores or spores of microorganisms, it is good to be preserved after the formation of spores and spores; the concentration of bacterial fluid is good to be high, such as bacteria should be 109 ~ 1010/ml.

b. Labeling of strain number and so on: it can be labeled on the outside of the ampoule or sealed into the label inside the ampoule.

c, preparation of ampoules: soak the ampoules in 2% HCl overnight, rinse with tap water for 3 times, brush with distilled water, dry, plug cotton plugs, label, dry heat sterilization or warm sterilization, dry at a constant temperature of 60 ℃.

d, add protective agent: commonly used protective agents are skim milk, 12% sucrose, add 7.5% glucose common broth and add 7.5% glucose serum and so on.

8, Bordelli's method: take a clean small test tube (8 × 60mm) stuffed with cotton plugs, sterilized. Use sterilized skimmed buttermilk to elute the bacterial moss on the slant of the test tube to make a thick bacterial suspension. The bacterial suspension was then dropped into the bottom of the small test tubes using a sterile pipette. One drop per tube and then rotate the small test tube to disperse the bacterial solution on the wall at the bottom of the test tube. Mark the name of the bacteria. The small test tubes were packed in 15×150 mm test tubes with 1.5 cm high of (P2O5 or KOH) at the bottom of the large test tubes beforehand, and the mouths of the tubes were plugged tightly with rubber stoppers with glass tubes and wax-sealed. Connect the large test tube with the vacuum pump, vacuum to 0.1 ~ 0.2 mm Hg, the glass tube fused seal, placed in a dark place at room temperature or refrigerator storage.

Instruments and materials 1, slant strains: filamentous fungi, yeast, bacillus and bacillus-free each one.

2、One set of inoculation utensils.

3, Sterilization items: 1ml, 5ml pipette, long dropper, ampoule tube, 9ml sterile water test tube, sand soil tube, liquid paraffin, skimmed buttermilk. Among them, the liquid paraffin was treated with 1.5kg/cm2 pressure sterilization for 1 hour or 1kg/cm2 pressure sterilization for 3 times, 30 minutes each time. Next, it was checked for complete sterility, i.e., it was accessed into the broth to check for the growth of stray bacteria. Finally it was placed in 60-80°C blast roast to remove moisture. Degreased buttermilk is processed by centrifugation at 2000 rpm for 10 minutes to degrease, then sterilized at 0.5kg/cm2 pressure for 20 minutes, or intermittently sterilized for 3 times, and then prepared for use after checking for sterility.

4、P2O5, anhydrous CaCl2 each bottle.

5、Vacuum dryer, vacuum pump.

Method 1, liquid paraffin covering method: take the strain slant to be preserved, add sterilized paraffin into it with 5ml sterile pipette, the liquid paraffin is required to be higher than the bacterial slant about 1cm, if not enough for this height, it will often cause the slant to dry out phenomenon.

2、Sand soil tube method: use 1ml sterile pipette to suck 0.2～0.5ml bacterial suspension, drop into the sand soil tube, mix well, put the sand soil tube into the vacuum desiccator, dry, vacuum desiccator need to be placed in anhydrous CaCl2. finally, take out the dry sand soil tube, quickly in the flame on the wax sealing tube mouth, and preserved at room temperature or in the refrigerator.

3、Freeze drying preservation method:

① Take out 2 drops (about 0.1ml) of sterile skimmed buttermilk with a sterilized long burette, put it in a sterile ampoule tube, then take out an equal volume of concentrated bacterial solution with another sterile burette and put it in this ampoule tube as well, mix it well.

② Concentrate the ampoule tube, put it in 100ml beaker, put the beaker into the bell jar of vacuum freeze dryer, turn on the machine and turn on the cooling water, so that the bacterial liquid freezes rapidly into solid at the temperature of -20℃, and then pump the vacuum to make the moisture sublimation, after 2 hours, you can stop freezing and dry it at a slightly higher temperature until it is completely dry.

③ Finally take out the ampoule tube, and connect it with the vacuum pump, and then vacuum, quickly melt the ampoule tube, and finally refrigerator storage.

4, glycerol tube preservation method: in 5ml strain preservation tube, equal volume of bacterial suspension and 40% of glycerol fully mixed, in -20 ℃ refrigerator storage.