METHODS OF GROWING ANTAGONISTIC ORGANISMS 65 



The media used for the production of antibiotic substances can be 

 classified into two groups: synthetic media and complex organic media. 

 The first contain a source of carbon, usually glucose (2 to 6 per cent) j 

 a source of nitrogen, usually nitrate (0.2 to 0.6 per cent), as well as sev- 

 eral salts, namely, K0HPO4 or KH2PO4 (o.i to 0.2 per cent), 

 MgSO^.yH.O (0.05 per cent), KCl (0.05 per cent), and FeS04.7H.O 

 (0.00 1 per cent) 5 certain supplementary materials, such as yeast ex- 

 tract, meat extract, or corn steep, and other salts, such as NaCl (0.05 

 to 0.5 per cent), ZnS04, MnS04, or CUSO4 ( i to 2 ppm.) may also be 

 added. The organic media contain a complex form of nitrogen, such as 

 tryptone, peptone, casein digest j either no other source of carbon is used 

 or a carbohydrate is added in the form of glucose, starch, brown sugar, 

 molasses, or similar products as well as several salts similar to those 

 listed above. Some media are supplemented with CaCO.j, and others 

 are not, depending upon the extent of acidity produced by the organism. 



The medium may be solid or liquid, but the latter type is more com- 

 mon. Agar and bran are used as solid media. Several types of culture 

 vessels are used, depending on the condition of aeration. Since so far as 

 is known all the microorganisms capable of producing antibiotic sub- 

 stances are aerobic, either shallow layers of medium (1.5 to 2 cm. in 

 depth) are placed in stationary vessels (flasks or trays), or deep vessels 

 (tanks) are filled with the medium and properly aerated by forced draft 

 with sterilized and filtered air. 



For the production of penicillin, a constant-flow apparatus similar to 

 the quick-vinegar process has been suggested (134), the medium trick- 

 ling over a column of wood shavings. The establishment and operation 

 of large-scale production of penicillin under submerged conditions have 

 been described in detail by Callaham (103). 



The optimum temperature required for the growth of the antagonis- 

 tic organisms and production of the antibiotic substances ranges be- 

 tween 20° and 30° C. The length of incubation varies from 2 to 6 days 

 for submerged cultures and from 3 to 20 days for stationary cultures. 



A knowledge of the preliminary treatment of the inoculum or spore 

 material is essential. For the growth of spore-forming bacteria, the use 

 of a pasteurized spore suspension is advisable in order to avoid the vari- 

 able factor due to vegetative cells. Actinomycetes and fungi are grown 



