Experiment 29 preservation of bacteria

1、 The basic principle of microorganisms is easy to mutate Therefore, in the process of preservation, the metabolism of microorganisms must be kept in the state of inactive or relatively static Zui, so that they can not mutate in a certain period of time and maintain their living ability Low temperature, drying and air isolation are the important factors to reduce the metabolic capacity of microorganisms Therefore, although there are many ways to preserve bacteria, they are all designed according to these three factors The preservation methods can be roughly divided into the following: 1 Subculture and preservation methods include slant culture, puncture culture, blister culture medium (the latter is used for preservation of anaerobic bacteria), which are stored in refrigerator at 4-6 ℃ 2 The liquid paraffin covering preservation method is a disguised method of subculture, which can appropriately prolong the preservation time It is to cover the sterilized liquid paraffin on the slant culture and puncture culture, on the one hand, it can prevent the death of bacteria due to the evaporation of water in the culture medium, on the other hand, it can prevent the entry of oxygen, so as to weaken the metabolism 3 Carrier preservation method is a kind of preservation method that adsorbs microorganisms on suitable carriers, such as soil, sand, silica gel, filter paper, and then dry them, such as sand preservation method and filter paper preservation method, which are widely used 4 Host preservation method is used for microorganisms that can not grow on artificial culture media at present, such as viruses, Rickettsia, spirochetes, etc they must be infected and passed on in living animals, insects and chicken embryos This method is equivalent to that of general microorganisms Virus and other microorganisms can also be preserved by other methods, such as liquid nitrogen preservation and freeze-drying preservation 5 Cryopreservation can be divided into low-temperature refrigerator (- 20-30 ℃, - 50-80 ℃), quick freezing of dry ice alcohol (about - 70 ℃), and liquid nitrogen (- 196 ℃) 6 The method of freeze-drying is to freeze the microorganism rapidly at very low temperature (- 70 ℃), and then use sublimation phenomenon to remove water (vacuum drying) under reduced pressure Some methods, such as filter paper preservation, liquid nitrogen preservation and freeze-drying preservation, need to use protective agents to prepare cell suspension, in order to prevent cell damage caused by freezing or water sublimation The protective solute can stabilize the configuration of cell components through the affinity of hydrogen and ion bonds to water and cells Protective agents include milk, serum, sugar, glycerin, dimethyl sulfoxide, etc 2、 Equipment bacteria, yeast, actinomycetes and molds; meat extract peptone slant medium, sterilized skimmed milk, sterilized water, chemically pure liquid paraffin, glycerin, phosphorus pentoxide, river sand, thin loess or laterite, ice, salt, dry ice, 95% alcohol, 10% hydrochloric acid, anhydrous calcium chloride; Sterilization straw, sterilization dropper, sterilization culture dish, tubular 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 pipe, refrigerator, low temperature ice box (- 30 ℃), liquid nitrogen cryopreservation device 3、 The following methods can be selected according to the specific conditions and needs of the laboratory 1 The culture was inoculated on a suitable solid slant medium by slant low temperature preservation method After the bacteria were fully grown, the cotton plug was wrapped with oil paper and moved to a refrigerator of 2-8 ℃ for preservation The preservation time varies according to the type of microorganism Mould, actinomycete and spore bacteria are preserved for 2-4 months and transferred once Yeast should be transplanted once a month for two months This method is commonly used in laboratory and factory strain room It has the advantages of simple operation, convenient use, no need of special equipment, and can check whether the preserved strains are dead, mutated and contaminated at any time The disadvantage is that it is easy to mutate, because the physical and chemical characteristics of the medium are not strictly constant Repeated generations will change the metabolism of microorganisms, and affect the characteristics of microorganisms; there are more opportunities to contaminate miscellaneous bacteria 2 Liquid paraffin preservation method (1) divide the liquid paraffin into triangular flasks, plug with cotton plugs, and wrap with kraft paper, 1.05kg/cm2 >, 121.3 ℃ for sterilization for 30 minutes, and then put it in a 40 ℃ temperature box to evaporate the water vapor for standby (2) The strains that need to be preserved will be cultured in the suitable slant medium of Zui to obtain robust bacteria or spores (3) The liquid paraffin which is sterilized is sucked by a sterilization straw and injected into the slant which has grown well The dosage shall be 1cm higher than the top of the slant, so as to isolate the bacteria from the air (4) Keep the test tube upright at low temperature or room temperature (some microorganisms can be stored at room temperature longer than in refrigerator) This method is practical and effective Mould, actinomycete and spore bacteria can be preserved for more than 2 years, yeast can be preserved for 1-2 years, and non spore bacteria can be preserved for about 1 year Even meningococci that are difficult to be preserved by general methods can be preserved for 3 months in 37 ℃ incubator The advantage of this method is that it is easy to make, does not need special equipment, and does not need to transfer seeds frequently The disadvantage is that it must be placed upright, occupying a large position and inconvenient to carry After the culture is taken from the bottom of the liquid paraffin, when the inoculation ring is burned on the flame, the culture is easy to splash with the residual liquid paraffin, so special attention should be paid 3 Filter paper preservation method (1) cut the filter paper into 0.5 × 1.2cm small strips, put them into 0.6 × 8cm ampoule tubes, 1-2 pieces for each tube, plug with cotton plug, 1.05kg/cm2 >, sterilize at 121.3 ℃ for 30 minutes (2) The strains that need to be preserved will be cultured on a suitable slant medium for full growth (3) Take 1-2ml of sterilized skimmed milk and drop it into a sterilized culture dish or test tube, take several rings of bacterial coating and mix them in the milk to make a concentrated suspension (4) Take the filter paper from the ampoule tube with sterilization forceps and immerse it into the bacterial suspension to make it full Then put it back into the ampoule tube and plug it with cotton plug (5) Put the ampoule tube into a desiccator with phosphorus pentoxide as water absorbent, and pump air to dryness with vacuum pump (6) Put the cotton into the tube and seal it with flame according to Fig Ⅶ - 13 Keep it at low temperature (7) It is necessary to use bacteria When resurrecting culture, the ampule nozzle can be heated on the flame, and a drop of cold water can be dropped on the heated part to break the glass Then use tweezers to knock off the glass at the mouth After the ampule tube is opened, take out the filter paper, put it into the liquid culture medium, and put it into the incubator for culture 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 Compared with liquid nitrogen and freeze-drying, this method is simple and does not need special equipment 4 Sand preservation method (1) add 10% diluted hydrochloric acid to river sand, heat and boil for 30 minutes to remove organic matter (2) Pour out the acid water and wash it with tap water until it is neutral (3) Drying, sieving with 40 mesh sieve to remove coarse particles, for standby (4) In addition, the thin loess or laterite without humus in the non cultivated layer shall be soaked and washed several times with tap water until it is neutral （5） Dry, grind and pass through a 100 mesh sieve to remove coarse particles (6) According to the proportion of one part of loess and three parts of sand (or other proportion as required, or even all of them can be sand or all of them can be soil), mix them evenly and put them into 10 × 100mm small test tube or ampoule tube, each tube is about 1g, plugged with cotton plug, sterilized and dried (7) Take samples for sterility test, take one from every 10 sand pipes, pour the sand into the broth culture medium, culture at 37 ℃ for 48 hours, if there are still mixed bacteria, all need to be sterilized again, and then carry out sterility test until it is proved that it is sterile, then it can be used for standby (8) The spore suspension was made by washing the mature strains (generally, the spore layer is full, the effect of this method is not good) with sterile water (9) Add about 0.5ml spore suspension into each sand pipe (generally, it is better to just wet the sand) and mix well with inoculation needle (10) Put it into the vacuum dryer, and use the vacuum pump to dry the water The shorter the time is, the better Make sure to dry it within 12 hours (11) Take one out of every 10, take out a few sand grains with the inoculation ring, inoculate them on the inclined culture medium, culture them, observe the growth condition and whether there is any growth of mixed bacteria If there are few or no growth of mixed bacteria or colonies, it indicates that there is a problem in the sand pipe, and further sampling inspection is needed (12) If there is no problem after inspection, seal the nozzle with flame and store it in refrigerator or dry place indoors Check the activity and bacteria every half a year (13) It is necessary to use bacteria When resurrecting culture, take a little sand and transfer it into the liquid culture medium, and put it in the incubator for culture This method is mainly used for spore producing microorganisms such as mould and actinomycete Therefore, Zui is widely used in antibiotic industry with good effect It can be preserved for about 2 years, but it is not effective in nutritional cells 5 Liquid nitrogen cryopreservation method (1) prepare the ampoule tube for liquid nitrogen preservation It is required to be able to withstand sudden changes in temperature without rupture Therefore, it is necessary to use the ampoule tube made of borosilicate glass The size of the ampoule tube is usually 75 × 10 mm, or 1.2 mm liquid (2) When protective agent and sterilization are added to preserve the easily dispersed cells such as bacteria, yeast or mould spores, the empty ampoule tube shall be plugged with cotton stopper and sterilized at 1.05kg/cm2121.3 ℃ for 15 minutes: if it is used to preserve the mycelium of mould, protective agent such as 10% glycerin distilled water solution or 10% dimethylsulfoxide distilled water solution shall be added in advance in the ampoule tube, and the added amount shall be able to be immersed and then added And then sterilized at 121.3 ℃ for 15 minutes (3) Inoculate the bacteria, make the bacteria suspension with 10% glycerine distilled water solution, and put it into the sterilized ampoule tube; the mycelium of the mold can be sterilized with a hole punch, cut the colony round block from the plate, put it into the ampoule tube containing the protective agent, and then fuse it with a flame Immerse in water to check for leaks (4) Freeze and then freeze the sealed ampoule tube to - 30 ℃ at a slow rate of 1 ℃ per minute If the cells are frozen rapidly, ice crystals will form in the cells, thus reducing the survival rate (5) The ampule tube frozen to - 30 ℃ is immediately put into the small cylinder of liquid nitrogen cryopreservation device (Figure vii-14), and then the small cylinder is put into the liquid nitrogen cryopreservation device The gas phase in the liquid nitrogen reservoir is - 150 ℃, and that in the liquid nitrogen reservoir is - 196 ℃ (6) When it is necessary to recover the cultured and preserved strains, take out the ampoule tube and immediately put it into a 38-40 ℃ water bath for rapid thawing until all of them melt Then open the ampoule tube and transfer the contents to a suitable medium for culture In addition to being suitable for the preservation of general microorganisms, this method can be used for the long-term preservation of some microorganisms that are difficult to preserve by freeze-drying, such as Mycoplasma, chlamydia, hydrogen bacteria, molds, phages and animal cells that are difficult to form spores, and the properties are not variable The disadvantage is the need for special equipment 6 Freeze drying preservation method (1) prepare the ampoule tube for freeze-drying bacteria preservation The ampoule tube should be made of neutral glass The shape can be made of long neck and spherical bottom, also known as teardrop type ampoule tube (Figure vii-15) The size requires an outer diameter of 6-7.5mm, a length of 105mm, a spherical diameter of 9-11mm, and a wall thickness of 0.6-1.2mm A tubular ampoule without a ball can also be used Plug the cotton, 1.05