19 



there is a slowly incrcusing alkalinity, which is first clearly visible on 

 about the third to fifth day. Toward the close of the first week, or 

 during the second week, a very slow separation of the casein (paraca- 

 sein?) takes place. The first visible separation is usually apparent 

 about the fifth to sixth or eighth to tenth day in the form of a 

 millimeter-deep layer of clear whey on top of the milk, which is still 

 entirely fluid. By the end of the third or fourth w^eek the fine white 

 casein has settled so that it occupies onl}' about one-half of the fluid, 

 the supernatant whey being pale yellow and transparent. Above this 

 whey in old cultures there is always a 5 to 7 mm. wide, dense, bright- 

 yellow bacterial rim on the tube. The casein does not set on the start 

 and is never coarse flocculent. It finally becomes packed together in 

 the bottom of the tube, but for a long time it consists of tiny separate 

 particles Avhich roll over each other easily w4ien the tubes are shaken. 

 This precipitated casein finally changes from white to 3'ellowish and 

 is slowl}^ redissolved (peptonized). At no time during this precipita- 

 tion and re-solution of the casein is there an}^ acid reaction or any 

 formation of gas. The whey from such cultures had a slightly bitter 

 taste. 



The reaction of the medium is best observed by adding to the milk 

 enough litmus water to make it a deep lavender color; i. e., 10 c. c. of a 

 saturated watery solution of c. p., blue, dry, lime-free litmus to 200 c. c. 

 of milk. Man}^ cultures were made in this medium with exact results. 

 During the first 8 or 10 days the blue color very slowly deepens and 

 the separation of the casein begins.^ During the next 10 da3^s or so 

 the casein slowly settles and is still blue. Subsequently the litmus 

 becomes more or less reduced, but at no time is there any appearance 

 indicating the formation of an}^ organic acid. When the litmus is not 

 reduced the whey is pale wine red by transmitted light (noraial color of 

 the litmus), but is not red by reflected light. If after several weeks or 

 months of growth such reduced or parth^ reduced cultures are killed 

 by heating for 10 minutes at 56° C, and are then exposed to the air 

 for some weeks, the color of the litmus returns. The undissolved 

 casein is now distinctly blue and the whey is not red by reflected light. 



Numerous tiny, white, centrally constricted, sheaf -like crystals of 

 tyrosin appeared in old milk cultures. Crystalline plates presumed to 

 be leucin also appeared. 



Ps. campestrls and Ps. 2yhaseoli both act upon milk and litmus milk 

 in much the same way. Neither produces any acid or gas. Both cause 

 a slowly increasing alkalinity in the milk w4th the separation of the 

 casein from the whey ])y means of a lab ferment. In both, a portion 

 of this casein is redissolved (peptonized) with the formation of tyrosin 

 and leucin. Ps. stewarti., on the contrary, does not precipitate the 



^ In one instance whey appeared in two tubes of blue litmus milk the fourth day. 

 See table under Reduction. 



