Vacuum freeze-drying method of microbial species preservation method.

Biotechnology Channel News: vacuum freeze-drying method will be microorganisms frozen, under decompression using sublimation to remove moisture, so that the physiological activities of cells tend to stop, so as to maintain long-term survival.
operation steps are as follows: aerobic bacteria freeze-drying tube preparation 1, ampoule tube preparation ampoule tube material to neutral glass is appropriate.
When cleaning the ampoule tube, first soaked with 2% hydrochloric acid overnight, after washing clean tap water, soaked with distilled water to pH neutral, dried, labeled, labeled with bacteria number and time, added to the skimmed cotton plug, 121 degrees C autocultension 15-20 minutes, spare.
2, the choice and preparation of protective agents to be selected according to the microbial category.
preparation of protective agents, attention should be paid to their concentration and pH, as well as sterilization methods.
such as serum, can be filtered to sterilise; milk should be skimmed first, with centrifugal method to remove the upper fat, generally boiled 2-3 times at 100 degrees C intermittently, 10-30 minutes at a time, spare.
3. Preparation of freeze-dried samples The cells are cultured to a static or mature stage under the most suitable culture conditions, after purity inspection (see the Office of Joint Management of natural science and technology resources platform of the Ministry of Science and Technology document "Technical Procedures for The Testing of Microbial Species Purity" (trial)), mixed evenly with the protective agent and divided into protective agents. the concentration of
microbial cultures is suitable for cells or spores not less than 108-1010 /ml (E. coli, for example, in order to obtain 1010 live cell fluid per milliliter 2 ml-2.5 ml, only 10 ml agar slope two).
using a longer capillary dropper, directly drip into the bottom of the ampoule tube, pay attention not to spill the upper tube wall, each tube assembly volume of about 0.1-0.2 ml, if it is a spherical ampoule tube, the loading capacity of half the ball.
if it is a liquid cultured microorganism, the medium should be centrifuged to remove the medium, and then the culture is mixed with the protective agent, and then divided into ampoule tubes.
as short as possible, preferably within 1-2 hours and prefrozen.
should be operated under sterile conditions when packing.
4, prefreeze generally prefreeze more than 2 hours, the temperature reached -20 degrees C to -35 degrees C or so.
5, freeze drying using a freeze dryer for freeze drying.
the frozen sample ampoule tube is placed in the drying box of the freezer and begins to freeze and dry for 8-20 hours.
6, vacuum seal and vacuum test the neck of the ampoule tube with a strong flame, and then use a vacuum pump to pump a vacuum, under vacuum conditions to heat the neck of the ampoule tube fuse.
after the furnace can be used high-frequency electric spark vacuum meter to determine the vacuum degree.
7, the preservation of ampoule tube should be low temperature to avoid light preservation.
8, quality inspection after freezing drying to extract a number of ampoule tubes to carry out various indicators of inspection, such as survival rate, production capacity, morphological variation, clutter pollution and so on.
Anaerobic bacteria freeze-drying tube preparation main procedures and aerobic bacteria operation is the same, pay attention to the choice and preparation of protective agents, protective agents should be boiled in boiling water at 100 degrees C for about 15 minutes, degassed and put in cold water to cool, remove the solubility of the protective agent oxygen.
Recovery method First wipe the upper part of the ampoule with 70% alcohol cotton, heat the top of the ampoule tube, wipe a circle on the top with sterile cotton swabs, cracks appear on the top, tap with a knife or tweezer neck, knock down the top of the cracked ampoule tube, dissolve the fungi block with sterile water or culture solution, use a sterile straw into the fresh culture base, and use a sterile straw to transfer to a fresh culture.
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