"Zhongguang" bacteria vacuum freeze-drying preservation method.

Vacuum freeze-drying preservation method, which has a long shelf life (about 5 years or more), small variation and wide scope of application, is the most ideal method of preservation at present, and is the main method widely used by various preservation institutions.
example, fewer than 100 of the more than 6,500 strains reported to have been held by ATCC could not be protected in this way.
principle is to create a dry and low temperature preservation environment.
The basic method is to freeze microbial cells or spores quickly at a lower temperature (-15 degrees C), keep the cells intact, and then subliminalize moisture under decompression, so that life activities such as cell growth and metabolism are stopped and preserved for a long time.
its operating procedures are as follows: (1) the handling of ampoules is generally 8 to 10mm, the length is not less than 100m.
First with a capillary tube added washing liquid soaked overnight, and then washed with distilled water after drying, the mouth of the bottle with cotton stuffed, and with a paper bag, 121 degrees C sterilization 30min, take out in a 60 degrees C oven dry spare.
(2) The protective agent's effect is to keep the bacteria's cells alive and minimize the freezing damage caused to microorganisms during freeze drying.
protective agents can also act as a supportive force, allowing microorganisms to be loosely fixed on top of them.
protectors can be used in low-molecular and polymer compounds, such as amino acids, organic acids, sugars, proteins, polysaccharides and so on.
commonly used are skimmed milk and serum.
such as horse serum or horse serum to add 7.5% glucose.
the protective agent before use with appropriate methods of sterilization.
(3) Bacteriom suspension preparation should be used in the most appropriate culture conditions (e.g. medium, temperature, culture time, etc.) culture species, in order to obtain good culture for long-term preservation.
culture time should be kept in the later stages of growth, as cells that are several years old are less resistant to freeze-drying, and spore-producing microorganisms should extend the culture time appropriately in order to obtain mature spores.
general bacteria culture 24 to 48h, yeast 72h, line bacteria and silky fungi 7 to 10d or more.
operation, in sterile conditions, first add a small amount of protective agent to the slope, gently scrape off the moss or spores, to make a bacteriop suspension.
with capillary droppers to take 0.1 to 0.2 ml of bacteria suspension added to the sterilized a bottle tube, stuffed cotton plug.
(4) freeze-drying freezing temperature of -15 degrees C to -30 degrees C can be.
the ampoule tube filled with the bacterial suspension should be frozen as soon as possible to prevent the bacteria from precipitate into uneven bacterial suspension and the re-growth or germination of microorganisms spores.
temperature is reached, the vacuum pump is activated immediately, bringing the vacuum to 66.661Pa within 15 min.
temperature rises above 13.3322 Pa, the temperature can be raised to 25 to 30 degrees Celsius.
the sample will not melt at this time as sublime continues.
is finished drying, turn off the vacuum pump, exhaust, remove the ampoule tube on the porous pipe and vacuum and seal.
(5) The preservation of the ampoule tube can be maintained for 5 to 10 years at a temperature of 4 to 5 degrees C, and the preservation effect at room temperature is not good.
Factors affecting the preservation effect in addition to bacteria species, bacteria age, but also directly related to the water content in the sample, usually water content in 1% to 3% when the preservation effect is better, 5% to 6% when the preservation effect is reduced accordingly, water content of more than 10%, the sample is difficult to preserve.
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