266 CHEN CHONG CHEN AND LEO F. RETTGER 



Clark and Lubs found that B. coli produced definite changes 

 in these media, with sufficient acid production to give a brilliant 

 red coloration when a few drops of the methyl red indicator are 

 added, whereas a similar test on cultures of the B. aerogenes 

 type resulted in a yellow coloration. More than this, they 

 showed that these reactions correlate in a perfect manner with 

 the source of the strains, and with their gas ratio. These obser- 

 vations were soon followed by fruitful researches of other investi- 

 gators, and were in a large measure corroborated by Levine, 

 Winslow and Kligler, Greenfield, Fulton, Johnson, Burton and 

 Rettger, and Winslow and Cohen. 



There are, however points still undetermined in the methyl 

 red test which require further elucidation. Clark and Lubs 

 emphatically state that the use of peptone other than Witte's 

 would lead to an erroneous reaction. With this brand of peptone 

 there are doubtful reactions which have been termed by Clark 

 and Lubs as the "neutral tints." The typing of bacteria by 

 means of the methyl red test depends upon gradations in color 

 varying from a yellow to a bright red color, and covering a 

 range of 4.2 to 6.0 on the pH scale. Determining the hydrogen 

 ion concentration is comparatively easy when pronounced shades 

 of red are obtained on the addition of the indicator; but when 

 the hydrogen ion concentration is so low that only a yellow color 

 ("neutral tints") is produced by the methyl red, it is imperative 

 that some sort of a scale or limit within the "neutral tints" be 

 established, or the types may be incorrectly placed and the 

 correlations disturbed. Levine (1916) in his first work on the 

 methyl red test encountered this difficulty; and Burton and 

 Rettger (1917) found that the variation in the high ratio cultures 

 was such as to lead them to the conclusion that the biometric 

 method was of little value in the classification. Winslow and 

 Cohen (1918) encountered this difficulty in the Witte peptone- 

 phosphate-glucose medium, as well as in the synthetic medium 

 later devised by Clark and Lubs (1916). 



