DETERMINING BIOLOGICAL ACTIVITIES OF BACTERIA 199 



then determining whether or not acid is formed from these substances 

 by the microorganisms. These tests have been of special importance 

 in the differentiation of the typhoid-colon groups of bacilli. 



Quantitative estimation of the degree of acidity or alkalinity 

 produced by bacteria may be made by careful titration of definite 

 volumes of the medium before and after bacterial growth has taken 

 place. 



The variety of acid formed by bacteria depends largely upon the 

 nature of the nutrient medium. The acids most commonly resulting 

 from bacterial growth are: lactic, acetic, oxalic, formic, and hippuric 

 acids. Qualitative and quantitative estimation of these acids may 

 be made by any of the methods employed by analytical chemists. 



Indol Production by Bacteria. Many bacteria possess the power 

 of producing indol. Though formerly regarded as a regular accom- 

 paniment of protein decomposition, later researches have shown that 

 indol production is not always coexistent with putrefaction processes 

 and occurs only when pepton is present in the pabulum. 



Indol formation by bacteria is determined by the so-called nitroso- 

 indol reaction. Organisms are grown in sugar-free pepton broth 

 or in the pepton-salt bouillon of Dunham. (See page 151.) Media 

 containing fermentable substances are not favorable for indol 

 production since acids interfere with its formation. The cultures 

 are usually incubated for three or four days at 37 C. At the end 

 of this time, ten drops of concentrated sulphuric acid are run into 

 each tube. If a pink color appears, indol is present, and we gather 

 the additional information that the microorganism in question has 

 been able to form nitrites by reduction (e.g., cholera spirillum). If 

 the pink color does not appear after the addition of the sulphuric 

 acid alone, nitrites must be supplied. This is done by adding to 

 the fluid about 1 c.c. of a 0.01 per cent aqueous solution of sodium 

 nitrite. The sodium nitrite solution does not keep for any length 

 of time and should be freshly made up at short intervals. 



VANILLIN TEST. 2 An excellent test for indol is the so-called 

 vanillin test. To 5 c.c. of the culture add 5 drops of 5 per cent 

 vanillin solution in 95 per cent alcohol, and 2 c.c. of concentrated 

 hydrochloric acid or sulphuric acid. If indol is present, an orange 

 color develops within 2 or 3 minutes. Tryptophane gives a reddish 



- Stcensma, Zeit. . Physiol, Chem., 47, 1906; Nelson, Jour, of Biol. Chem., 

 24, 1916. 



