494 G. C. BUNKER, E. J. TUCKER AND H. W. GREEN 



of a large quantity of dilute solution, the amount of fluid in the^tube- 

 is not increased, and the colour very seldom fails to appear in sufficient 

 quantities to be easily recognizable in two hours. 



In the 1917 edition of "Standard Methods of Water Analysis," 

 a medium consisting of 5 grams of Witte's peptone, 5 grams of c.p. 

 glucose and 5 grams of dipotassium phosphate (K2HPO4) in 

 100 cc. of water, is recommended for the methyl red and Voges 

 and Proskauer tests with the following procedure, after incuba- 

 tion at 30°C. for five days and after the former test has been 

 made: "To the remaining 5 cc. of medium add 5 cc. of a 10 per 

 cent solution of potassium hydroxide. Allow to stand over 

 night. A positive test is indicated by an eosin pink color." 



In the case of both media, after the addition of the sodium 

 hydroxide, the tubes removed from the incubator in the after- 

 noon were returned to it or allowed to stand in the laboratory 

 until the morning of the following day. After a few parallel 

 tests had been made it was observed that, with the same cultures, 

 positive Voges and Proskauer reactions did not always develop 

 in the two media. As a result of this disagreement a study was 

 made of the factors which were thought to exert the greatest 

 influence on the reaction, namely, the concentration of the 

 sodium hydroxide solution and the optimum interval of time 

 necessary for the development of the pink color. 



Without giving the details of all the experiments it was found 

 that the addition of 0.5 cc. of a 45 per cent solution (1 pound 

 per liter of water) of sodium hydroxide to about 5 cc. of the media 

 gave sharper and more distinct colors than that of 5 cc. of a 10 

 per cent solution. At the same time it was observed, in many 

 of the tubes in which positive reactions developed at the end of 

 five hours, that the pink color disappeared on standing over 

 night; that the intensity of the pink color varied with different 

 cultures; that the permanency of the color was usually propor- 

 tional to the intensity; and that the color in the majority of cases 

 developed in an interval of two hours after the addition of sodium 

 hydroxide. The following table furnishes a comparison of the 

 positive Voges and Proskauer reactions resulting from the use 



