METHODS OF GROWING ANTAGONISTIC ORGANISMS 65 



organisms capable of producing antibiotic substances are aerobic, either 

 shallow layers of medium ( 1.5 to 2 cm. in depth) are placed in station- 

 ary vessels (flasks or trays), or shaken cultures are used. In the case of 

 deep vessels or tanks, the medium is properly stirred and aerated by 

 forced draft with sterilized and filtered air. 



The optimum temperature required for the growth of the antagonis- 

 tic organisms and the production of 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 

 on agar slants in order to obtain abundant spore material for the inocu- 

 lation of stationary cultures. For submerged cultures, special spore sus- 

 pensions are produced by growing the organisms in shaken cultures. 



The cultures must be tested carefully in order to establish the opti- 

 mum activity when the culture filtrate is cooled and extraction of active 

 substance is started. 



Tyrothrkin 



For the production of tyrothricin, shallow layers of medium are used 

 most frequently. The media contain complex sources of nitrogen, such 

 as tryptone, casein hydrolysate, soybean meal digest, and pressed juice 

 of waste asparagus. Simple substances, such as glutamic acid, aspara- 

 gine, ammonium salt, plucid citric or malic acid, are also effective in 

 presence of 0.2 per cent tryptone. Glucose, mannitol, or glycerol (3 

 to 5 per cent) can be used as the source of carbon, and calcium, magne- 

 sium, and manganese as required mineral. Maximum yields of more 

 than 2 gm. per liter are obtained in 10 to 16 days' incubation at 35° C. 

 (564). 



Penicillin 



^ For the production of penicillin, the composition of the medium is 

 highly important. At first a simple glucose-nitrate solution known as 

 Czapek-Dox medium was used. It was later found that when yeast ex- 



