To make sporulation medium.
Spores are formed when the culture medium by sodium nitrate, potassium hydrogen phosphate, sulfate, potassium chloride and ferrous sulfate and all or part of the preparation of sugar, PH of 4 ~ 7, optimization for 5 ~ 6.
5.
In the preparation add AGAR culture medium, the heating sterilization after cooling solidification, its product as the sporulation medium.
As long as it can make the mycelia proliferation and sporulation, no special requirements for sporulation medium, is characterized by the PH adjustment to fit the spore formed within 4 ~ 7.
specific examples, such as in Czapek AGAR medium (
2 g/L sodium nitrate, potassium hydrogen phosphate 1 g/L, sulfate beauty & middot;
7 crystallization water is 0.
5 g/L, potassium chloride, 0.
5 g/L, ferrous sulfate & middot;
7 crystallization water is 0.
01 g/L, sugar 30 g/L, AGAR 13 g/L)
Adding hydrochloric acid or sulfuric acid to PH to 6.
0 medium.
Another example, such as in Czapek -
Dox AGAR medium (
2 g/L sodium nitrate, potassium hydrogen phosphate 1 g/L, sulfate beauty & middot;
7 crystallization water is 0.
5 g/L, potassium chloride, 0.
5 g/L, ferrous sulfate & middot;
7 crystallization water is 0.
01 g/L, 30 g/L glucose AGAR 13 g/L)
Adding hydrochloric acid or sulfuric acid to PH to 6.
0 medium.
cant made with this method in vitro culture medium (
Slant culture medium)
Or in a petri dish plate culture medium (
Plate culture medium)
, on the culture medium inoculated hypha and spore, solid cultivation under aerobic conditions.
Temperature keep in 0 ~ 40 ℃, optimizing 10 ~ 35 ℃, preferred in 15 ~ 30 ℃ the proliferation and sporulation.
Temperature can be on the way to change, for example, can make its proliferation, under the condition of 25 ℃ under the condition of 5 ℃.
after confirm the sporulation, sterilization, water is added in the solid culture hyphae stir with common way to get the spore suspension.
Add in sterilizing the additives in the water without special requirement, for pure water, can also be adding surfactants, inorganic salts, etc. , can also use modulation of physiological saline in advance.
Mixing method can from the outside to solid culture vessel strength, can also use sterilization brush, directly mixing hyphae in advance.
will get the spore suspension of or the spore and mycelium suspension as a preservation dope.
The preservation dope sterilizing water or surfactant or available again after dilute aqueous solution containing inorganic salts as a preservation dope.
After the preservation dope add cryoprotectants.
The use of cryoprotectants generally no special requirements, from the AGAR powder, bovine serum, DMSO, glycerin, inositol, polyvinyl alcohol, skim milk, etc. Select one or more to add.
Specific examples, such as for the preservation of glycerin concentration of 10%, can add glycerol as cryoprotectants.
add cryoprotectants, will preserve fluid packing in preservation container.
Container sterilization plastic cryopreserved tubes, etc. , should be used to illustrate, such as the preservation of liquid in the sterile operation capacity under the condition of packing in 1.
2 ml of cryopreserved tube medium.
After the storage container at cryogenic refrigerator preservation.
The temperature of the cryogenic refrigerator need to control in -
100℃～-
20 ℃ range, the optimization
90℃～-
30 ℃, the preferred
85℃～-
50℃。
as a result, within the cryogenic refrigerator preservation of strain can be a long time, steady preservation.
