280 CHEN CHONG CHEN AND LEO F. RETTGER 



The technique finally adopted in this work was as follows: 

 At the end of one, three and five days' incubation periods 5 to 

 6 cc. of the culture fluid were well shaken in the test tube with an 

 equal amount of 10 per cent potassium hydroxide solution. The 

 tubes were placed in an incubator having a constant temperature 

 of 30°C. At the end of one to three hours the tubes were again 

 vigorously shaken until the liquid became foamy. As a rule, the 

 eosin-like coloration made its appearance quickly, and the color 

 deepened throughout the tube, instead of becoming visible as 

 a ring or a surface layer. Without an exception, a decidedly 

 positive Voges and Proskauer reaction (maximum color produc- 

 tion) could be observed within a few hours (two to three) by 

 this method. West used a more or less similar shake method 

 ten years ago. He said: "After heating the tube, shake well, 

 or blow through it to bring the red color out." 



By the use of the method just described a total of 3725 individ- 

 ual Voges and Proskauer tests were made in the three different 

 glucose-phosphate media, following three different incubation 

 periods. The results are shown in the following chart (VI). 



The chart shows clearly that there was no difference in the 

 color reaction as the result of differences in the length of the 

 incubation periods, one, three and five days, or of the different 

 brands of peptone employed. Although the coloration in the 

 synthetic medium was not so uniformly strong as in the peptone 

 media, the reaction was always sufficiently pronounced to be 

 called distinctly positive 



Different strains manifested a striking difference in the degree 

 of coloration which they gave. Not a single aerogenes culture, 

 however, failed to give the reaction. On the other hand, not 

 one of the colon strains, either from soil or feces, responded to 

 the test. 



An attempt was made further to determine the minimum 

 incubation period in which color production can be obtained. 

 A small collection of typical and representative strains of B. 

 aerogenes was tested in the same three media after four, ten and 

 fourteen hours of incubation of the cultures at 30°C. The results 

 showed that a positive Voges and Proskauer reaction may be ob- 



