20 



time however, it becomes paler, and finally gives place to a dirty greenish 

 brown." 



Considerable work has been carried out on the Voges Proskauer test 

 in the last five years particularly with reference to its constancy, reliability, 

 and methods of determination. 



It was customary to allow the potassium hydroxide-culture-mixture to 

 stand 24 hours, and some investigators did not record until after 

 48 hours. This is extremely unfortunate as it results in unnecessary 

 loss of time. Levine, Weldin, and Johnson found that of 140 strains which 

 gave the Voges Proskauer reaction from glucose, 130 (92.9%) were posi- 

 tive after 5 hours. A similar result was observed with sucrose, where of 

 134 positive carbinol tests, 127 (94% ) were obtained in 5 hours. The 

 same was true of other substances from which acetyl-methyl-carbinol 

 was produced as is shown in the accompanying table. They conclude 

 from this that a period of 5 hours after the addition of the alkali is suf- 

 ficient as a presumptive test for the Voges Proskauer reaction. 



TABLE VIII. COMPARISON OF FIVE HOUR AND TWENTY-FOUR HOUR 

 RECORDS OF TESTS FOR ACETYL-METHYL-CARBINOL. 



West suggested that the reaction could be hastened by heating the 

 mixture and blowing air through it. Levine, Weldin and Johnson employed 

 various oxidizing agents (potassium dichromate, potassium perchlorate, 

 bleaching powder, barium peroxide, and hydrogen peroxide) all of which 

 were capable of accelerating the reaction but best results were obtained 

 with hydrogen peroxide. To 3 c. c. of a 48 hour culture was added an 

 equal volume of 10 percent potassium hydroxide; the mixture was heated in 

 a boiling water bath for 3 minutes and then 2 or 3 drops of hydrogen 

 peroxide were added. The pink coloration appeared in one or two min- 

 utes and persisted for several hours. An excess of hydrogen peroxide or 

 any other oxidizing agent is to be avoided as the coloration will disappear 

 in a very few minutes, or even instantly, if the excess is very great. 



Chen and Rettger suggest the following technique for the Voges Pros- 

 kauer test: Five or six c. c. of the culture are added to an equal volume 

 of 10 percent potassium hydroxide in a test tube, well shaken, and incu- 

 bated at 30 degrees C. for one to three hours, after which the tube is again 

 vigorously shaken until the liquid becomes foamy. A decided eosin-like 

 coloration will develop in an hour or two. 



