Preservation method and operation process of strains

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.

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, store it in the 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. Cryopreservation 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-dry preservation

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.

1. Bevel cryopreservation

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) Separately fill the liquid paraffin in a triangular flask, plug a cotton plug, and wrap it with kraft paper, 1.05kg / cm2>, sterilize at 121.3 ° C for 30 minutes, and then put it in a 40 ° C incubator to evaporate water vapor Off, spare.

(2) The strains to be preserved are cultivated in the most suitable inclined culture medium, so that the 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 based on 1 cm above 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 a 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, yeast 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 small strips of 0.5 × 1.2cm, and put them into 0.6 × 8cm ampule tubes, each tube has 1-2 sheets, plug with cotton plug, 1.05kg / cm2>, 121 ． Sterilized at 3 ℃ for 30 minutes.

(2) The strains to be preserved are cultivated on a suitable slant medium to allow full growth.

(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) Use sterile forceps to 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, use flame to seal 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 need, or even all or all of the soil), mix evenly and put into a small test tube or ampoule tube of 10 × 100mm, each tube Pack about 1g, put on a cotton plug, sterilize and dry.

(7) Sampling for sterility inspection, one for every 10 sand and soil tubes, pour the sand and soil into the broth culture medium, culture at 37 ℃ for 48 hours, if there are still bacteria, you need to re-sterilize all 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 of growth, the vegetative cells are not effective with this method), good bacteria, washed with sterile water to make a spore suspension.

(9) Add about 0.5ml 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 to dry 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 culture medium, cultivate it, observe the growth situation and the growth of miscellaneous bacteria, such as the occurrence of microbes 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 it 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 culture medium and place it in the incubator 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 cryopreservation

(1) Prepare ampoule tubes for preservation of liquid nitrogen. It is required to be able to withstand sudden changes in temperature without breaking. Therefore, ampoule tubes made of borosilicate glass are required. The size of ampoule tubes is usually 75 × 10mm. Or can accommodate 1.2mm liquid.

(2) When adding protective agents and sterilizing to preserve cells that are easily dispersed such as bacteria, yeast or mold spores, plug the empty ampoule tube with a cotton plug, 1.05 kg / cm2, and sterilize at 121.3 ° C for 15 minutes: if For the preservation of mold mycelium, a protective agent such as 10% glycerol distilled aqueous solution or 10% dimethyl sulfoxide distilled aqueous solution needs to be added to the ampoule tube in advance, and the amount added is limited to the colony round block that can be added after immersion, and then reused 1.05kg / cm2>, sterilized at 121.3 ° C for 15 minutes.

(3) Connect the bacteria to make bacteria suspension with 10% glycerol distilled water solution, and put it into a sterilized ampoule tube; mold mycelium can use a sterilizing 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 to -30 ° C at a slow rate of 1 ° C per minute. 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 ° C 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 water bath at 38-40 ° C for rapid thawing until all of it is 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, and their traits do not change. The disadvantage is that special equipment is required.

6. Freeze-dry preservation

(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). 7.5mm, length 105mm, ball diameter 9-11mm, wall thickness 0.6-1.2mm. A tubular ampoule tube without a ball can also be used. Plug a 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. 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 must 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 2ml of skim milk to the inclined test tube to make a concentrated bacterial solution, and each ampoule tube is divided into 0.2ml.

(4) There are complete sets of equipment sold in freeze freeze dryers, which are expensive. The simple methods and devices 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 CO2>) alcohol or dry ice acetone. 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 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 93.3Pa (0.7mmHg) vacuum within 30 minutes, the dry matter will not melt, and then continue to pump down. Within a few hours, the naked eye can be observed that the dried object has become dry, generally Pump to a vacuum of 26.7 Pa (0.2 mmHg) and keep the pressure for 6-8 hours.

(6) After the sealing is vacuum-dried, remove the ampoule tube, connect it to the glass tube for sealing, and use the L-shaped five-way tube (Figure â…¦-17) to continue pumping, which can reach 26.7 Pa (0. 2mmHg). 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.