SUBSTANCES PRODUCED BY BACTERIA 185 



Polyfepides and Proteins 



From the tyrothricin complex group of antibiotics produced by B. 

 brevisy two crystalline compounds have been isolated. They are poly- 

 peptides resistant to the action of proteolytic enzymes (201, 208, 450, 

 909). The organism is grown in shallow layers of a suitable medium, 

 such as one containing i per cent casein digest or tryptone and 0.5 per 

 cent NaCl in tap water, adjusted to /)H 7.0. After inoculation, the 

 medium is heated for 20 minutes at 70° C, in order to kill the vegeta- 

 tive cells of the bacteria, leaving only the spores to develop. The cul- 

 ture 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 dissolved and is separated from the residue by filtration j 

 when the alcoholic solution is diluted with 10 volumes of i per cent 

 NaCl, the substance is precipitated out. It carries all the activity and can 

 be desiccated in vacuo, over P2O5, giving a yield of about 100 mg. of 

 final dry substance per liter of culture medium. The protein-free, alco- 

 hol-soluble active material is tyrothricin. When an attempt was made to 

 produce tyrothricin in aerated submerged cultures, none was obtained 

 in complex nitrogenous media j however, simple amino compounds, 

 like asparagine, gave good growth and yielded the antibiotic substance. 

 The presence of cystine in the mixture of amino acids appeared to in- 

 hibit growth (884). 



Gramicidin is obtained by treating tyrothricin with a mixture of 

 equal volumes of acetone and ether, evaporating, and dissolving 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 hydro- 

 carbons. When a solution containing 20 to 50 mg. per milliliter alcohol 

 is diluted to i mg. per milliliter, with distilled water or with glucose 

 solution, an opalescent solution is produced without flocculation. On 

 dilution with electrolyte solutions, an immediate flocculation occurs. 



