250 MICROSCOPIC AND CULTURAL STUDY OF BACTERIA 
of utilizable carbohydrate, diphtheria and tetanus bacilH produce 
their very potent toxins ;i proteolytic bacteria elaborate soluble 
enzymes;- B. coli, B. proteus and other bacteria form indol and 
phenolic bodies from tryptophan and tyrosine respectively; the cholera 
vibrios form nitroso indol, ^ 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 F'olin microscopic method for ammonia; the 
increase in ammonia indicates the extent of deamination.^ The 
rate of hydrolysis of protein is conveniently estimated with the van 
Slyke^ amino-acid apparatus, after removal of ammonia from the 
medium*^ together with a determination of the non-protein nitrogen, and 
the peptid nitrogen. '^ A combination of these methods affords an 
approximate analysis of plain broth media before and after bacterial 
growth. Undoubtedly the application of the Emil Fischer esterifica- 
tion method of amino-nitrogen determination will throw much light 
upon the utilization of various amino-acids by specific bacteria during 
their growth in artificial media. Amino-acids containing aromatic 
nuclei— as tryptophan, tyrosin, histidin— give colored compounds 
with various reagents because they contain the chromophoric group, 
C = C. The formation of indol from tryptophan (see page 72 for 
chemistry) has long been used as a diagnostic test for B. coli and other 
bacteria. The test depends upon the removal of alanin from the 
tryptophan molecule by the activity of the organism, and the addi- 
tion of auxochromic group, NO2 to the beta carbon atom of the pyrrol 
ring. In an acid medium the compound nitroso-indol is brownish-red. 
Procedure, Indol Test.— To a three-day plain broth culture of 
B. coli (or other organism) add 1 cc. of concentrated hydrochloric acid.^ 
Mix thoroughly and overlay the acid broth with 1 to 2 cc. of a 0.1 per 
cent solution of sodium nitrite.^ At the junction of the two solutions 
a brownish-red ring of nitroso-indol develops. 
Gnczda Indol Test.^'> 
Solution 1: 
Para dimethyl amino-benzaldehyde" 1 gm. 
* Ethyl alcohol, 95 per cent 95 cc. 
Hydrochloric acid, concentrated 20 cc. 
Solution 2: 
Saturated, aqueous solution of potassium persulfate, KaSsOa- 
» Theobald Smith: Trans. Assn. Am. Phys., 1896; Jour. Exp. Med., 1899, 4, 373. 
2 Kendall, Cheetham and Hamilton: Jour. Infec. Dis., 1922, 30, 251 
' Kendall: Jour. Med. Res., 1911, 25, 117. 
* Kendall and Farmer: Jour. Biol. Chem., 1912, 12, 13, 215, 219, 465; 13, 63. Ken- 
dall, Day and Walker: Jour. Am. Chem. Soc, 1913, 35, 1201; 1914, 36, 1937. Kendall, 
Cheetham and Hamilton: Loc. cit. 
' Jour. Biol. Chem., 1912, 12, 275; 1913, 16, 121. 
« 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. 
■ For methods see Kendall: Jour. Infec. Dis , 1922, 30, 211. 
* Any strong mineral acid will answer the purpose. 
9 Best accomplished by running the nitrite solution carefully down the side of the 
tube held in a slanting position; a stratification of the two liquids should be obtained. 
1° Gnezda: Compt. rend Acad. Sci., 1899, 128, 128. 
'1 Erhlich's aldehyde. Ehrlich: Med. Wschnschr., 1901, No. 15. 
