In the preparation of media, it is important that the time and temperature of storage of stock solutions and finished media be standardized and adhered to carefully. Many minor variations can be tolerated by cells growing in a mixture of synthetic medium and serum or other biological or proteinaceous supplements. When cells are grown entirely in synthetic media, very minor variants can be critical.
It cannot be overemphasized that all procedures, sources of reagents, and others should be standardized, empirically if need be, and adhered to rigorously.
The media for invertebrate cell culture are based partly on the composition of the hemolymphs of the animal from which tissues are taken for culture and partly on information gained from the experience with other animal cells in culture. Such tissues may be inspected under a biology microscope. The hemolymphs of invertebrates differ from the bloods of vertebrates mainly in the composition of inorganic ions, amino acids, and organic acids, as viewed under a biology microscope.
Invertebrate cell culture media are normally prepared by dissolving similar ingredients together and then combining these solutions to prepare the final medium. The customary solutions are the inorganic salts, the organic acids, the amino acids, the sugars, and the vitamins. Among the amino acids, cystine and tyrosine are dissolved separately in a small amount of 0.1 N HVL and then added in turn to the amino acid solution. The organic acids are neutralized with 5% KOH or other base after they have been dissolved. After all the solutions are prepared, they are combined usually in the following order: inorganic salts, sugars, amino acids, and vitamins. The volume then is adjusted to 90% of the final volume and the CaCL2 is added slowly with continuous stirring. This is very critical because the level of calcium is very near the limit of solubility for the sulfate and phosphate salts, and care must be taken to avoid irreversible precipitation of these calcium salts.
A great diversity exists in the composition of plant culture media, as viewed under a biology microscope. This diversity is to large extent a necessary and desirable feature of working with the multitude of species, organs, and research objectives. It is unrealistic to expect widespread success by simply applying an existing formula. Existing media should serve only as reference. In contrast to the inorganic salts, extensive variability has been the case of the organic constituents. This is expected, inasmuch as many of them appear to serve a growth-regulating role. The only ingredients common to many media are sucrose and thiamine. Additional vitamins especially myo-inositol, nicotinic acid, and pyridoxine have been used in many instances. In the development of nutrient formulations, possible antagonisms and synergisms should be recognized and chemicals should be tested accordingly.
Sterilization of media consists of autoclaving for 15 minutes at 15r pounds per square inch.
