218 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 



and freed from sugar with Bacillus coli, 1 contains, on the average, 

 about 300 milligrams of nitrogen per 100 c.c. A small but variable 

 amount of the nitrogen exists as free ammonia. 2 Less than 10 per 

 cent, of the total nitrogen, as a rule, exists as aminonitrogen (deter- 

 mined by the method of Van^Slyke) . 3 



The visible changes in the appearance of broth cultures incidental 

 to the development of bacteria are not of great importance; they 

 consist essentially of turbidity, sediment, and occasionally a ring 

 or pellicle. The development of a pellicle is of importance in the pro- 

 duction of toxin by the diphtheria bacillus, however, because it indi- 

 cates the maximum oxygenation of the bacteria. The character of 

 the turbidity and sediment the viscidity and color may afford 

 some information of the character of the organism. Products of 

 importance are frequently detected by chemical or physiological 

 examination in plain broth cultures of bacteria. Thus, in the absence 

 of utilizable carbohydrate, diphtheria and tetanus bacilli produce 

 their very potent toxins; 4 proteolytic bacteria elaborate soluble 

 enzymes; 5 Bacillus coli, Bacillus proteus and other bacteria form indol 

 and phenolic bodies from tryptophan and tyrosine respectively; the 

 cholera vibrios form nitroso indol, 6 and in sugar-free broth containing 

 freshly drawn, sterile, defibrinated blood, various bacteria produce 

 hemolysis. 



The rate of decomposition of the protein constituents of the broth 

 may be measured by the Folin microscopic method for ammonia; the 

 increase in ammonia indicates the extent of deaminization. 7 The 

 rate of hydrolysis of protein is conveniently estimated with the Van 

 Slyke 8 amino-acid apparatus, after removal of ammonia from the 

 medium. 9 A combination of these methods affords an approximate 

 analysis of plain broth media before and after bacterial growth. Un- 

 doubtedly the application of the Emil Fischer esterification method 

 of aminonitrogen determination will throw much light upon the utili- 

 zation of various amino acids by specific bacteria during their growth 

 in artificial media. Amino acids containing aromatic nuclei as tryp- 



1 Theobald Smith, Cent. f. Bakt., 1897, xxii, 45. 



2 Determined by the Folin Method, Jour. Biol. Chem., 1912, xi, 523. 



3 Jour. Biol. Chem., 1912, xii, 275; 1913, xvi, 121. 



4 Theobald Smith, Tr. Assn. Am. Phys., 1896; Jour. Exp. Med., 1899, iv, 373. 



5 Kendall and Walker, Loc. cit. 



c Kendall, Jour. Med. Res., 1911, xxv, 117. 



7 Kendall and Farmer, Jour. Biol. Chem., 1912, xii, 13, 215, 219, 465; xiii, 63; Kendall, 

 Day and Walker, Jour. Am. Chem. Soc., 1913, xxxv, 1201; 1914, xxxvi, 1937. 



8 Van Slyke, Loc. cit. 



9 The rate of hydrolysis may also be estimated by Sorenson's formol titration method, 

 but this is less accurate for small amounts than Van Slyke's method. 



