SUBSTANCES PRODUCED BY BACTERIA 167 



tionship was found to exist between the activity of the enzyme in vitro 

 and its protective power in the animal body. 



Polypeptides. The credit for first isolating, in crystalline form from 

 spore-forming aerobic soil bacteria, specific chemical compounds of the 

 polypeptide type is due Dubos (190, 193, 203, 436, 530). The antago- 

 nistic organism {B. brevis) is grown in shallow layers of a medium 

 containing i per cent casein digest or tryptone and 0.5 per cent NaCl 

 in tap water, adjusted to /jH 7.0. After inoculation, the medium is 

 heated for 20 minutes at 70° C, in order to kill the vegetative cells of 

 the bacteria, leaving only the spores to develop. The culture is allowed 

 to grow for 72 hours. The reaction of the culture is then adjusted to /)H 

 4,5 by the use of about 3 or 4 cc. concentrated HCl per liter of culture. 

 A precipitate is formed which is removed by filtration through paper j 

 it is then suspended in 95 per cent alcohol (20 cc. of alcohol per liter of 

 culture) and allowed to stand 24 hours. The active substance is dis- 

 solved and is separated from the residue by filtration j when the alco- 

 holic solution is diluted with 10 volumes of i per cent NaCl, the sub- 

 stance is precipitated out. It carries all the activity and can be desiccated 

 in vacuo, over PoO-,, giving a yield of about 100 mg. of final dry sub- 

 stance per liter of culture medium. The protein-free, alcohol-soluble ac- 

 tive material was designated as tyrothricin. When an attempt was made 

 to produce tyrothricin in aerated submerged cultures, none was ob- 

 tained in complex nitrogenous media 5 however, simple amino com- 

 pounds, like asparagine, gave good growth and yielded the antibiotic 

 substance. The presence of cystine in the mixture of amino acids ap- 

 peared to inhibit growth ( 856) . 



Tyrothricin can be separated into two crystalline preparations, grami- 

 cidin and tyrocidine. Gramicidin is obtained by treating tyrothricin with 

 a mixture of equal volumes of acetone and ether, evaporating, and dis- 

 solving in boiling acetone. On cooling, it crystallizes out as spear- 

 shaped colorless platelets, melting at 228° to 230° C, with a yield of 

 about 10 to 15 grams from 100 grams of the crude material. Gramicidin 

 is soluble in lower alcohols, acetic acid, and pyridine, and moderately 

 soluble in dry acetone and dioxanej it is almost insoluble in water, 

 ether, and hydrocarbons. When a solution containing 20 to 50 mg. per 



