IDENTITY OF THE LACTIC-ACID STREPTOCOCCUS 167 



tococci. Rogers and Davis (1912) considered the reduction of 

 this compound of differential value in their study of the lactic- 

 acid bacteria of milk. As was noted earlier, Hastings (1911) 

 has used the action on litmus in milk to distinguish the lactic- 

 acid organism from other streptococci. 



A preliminary survey of the various stains indicated that 

 methylene blue, neutral red, Htmus and indigo carmine might 

 be of service in the present work. According to Fred (1912), 

 bacteria, at least those types common in milk, reduce stains 

 more actively in a milk medium than in broth, and a few tests 

 made at the beginning of this study verified this conclusion. 

 Tests were made by adding the dye to sterilized whole milk, 

 the advantage of unskimmed milk being that the fat forms a 

 layer over the surface which excludes the air quite effectively 

 and thus reduction is not hindered. The litmus milk was pre- 

 pared in the ordinary way by adding sufficient litmus solution 

 to the milk to give a rather dark lavender color and then ster- 

 ilizing. The other dyes were made as follows: 



Methylene blue 



Medicinal meth} lene blue 0.5 gram 



Distilled water jqqq cc. 



Indigo carjnine 



Indigo carmine (Kahlbaum's) 1.0 gram 



Distilled water jqqo cc. 



Neutral red 



Neutral red (Griibler's) 0.1 gram 



Distilled water 2000 cc. 



The stain solutions and milk were sterilized separately and then 

 mixed in the proportion of 1 cc. of stain to 10 cc. of milk. 



In making the tests twenty-four hours old cultures of the 

 organisms in milk were used to inoculate from, and the stain 

 culture so prepared was incubated at 37°C. Observations were 

 then made on three points, (1) reduction of stain, (2) time 

 required to reduce, and (3) whether reduction was before or after 

 curdling of milk. When no reduction was evident the cultures 

 were allowed to remain six days before final examination was 

 made. 



THE JOURNAL OF BACTERIOLOGY, VOL. Ill, NO. 2 



