EXPERIMENT STATION BULLETINS. 483 



It was not possible to adhere to natural oondition.s for the develop- 

 ment of tyi>h(>i«i bacilli introduced, as the lactic organisms groAving 

 in milk, their most favorable nutrient medium, soon outnund)ered the 

 typhoid bacilli, necessitatino- consequently high dilutions for satisfactory 

 ]>lating; the comparatively few typhoid bacilli entering through natural 

 infection would be lost sight of entirely in this way. Because of this 

 condition, the milk was inoculated with a large number of the typhoid 

 organisms and with comparatively few of the lactics. 



After a few hours incubation, plates were made in ordinary agar from 

 the combined culture. The 24 hour colonies on these plates were in- 

 distinguishable from one another unless microscopic examinations were 

 made. 



At this point, it was evident that a medium for differentiating the 

 typhoid and lactic organisms was necessary. To this end, litmus lactose 

 agar containing calcium carbonate in suspension was tried for plating. 



This experiment was carried out at some length to determine the effi- 

 ciency of the special plating medium. 



Method of Procedure. — The agar for this experiment was made accord- 

 ing to the methods advocated in the "Standard Methods of Water Analy- 

 sis, 1904," for the preparation of litmus lactose agar. 



To the clear filtered agar was added 1 per cent of a solution of Merck's 

 purified litmus and washed calcium carbonate in the proportion of 3 

 grams to 100 cc. of agar.* This was stirred until the calcium carbonate 

 was in even suspension throughout the agar, which was then immediately 

 tubed and sterilized. 



The medium for the typhoid-lactic culture was freshly separated milk 

 obtained from the college dairy. Its acidity to phenolphthalein was 

 tested and recorded. Each lot of milk was -(-15°. 



200 cc. of the milk was poured into each of several 875 cc. Erlen- 

 meyer flasks and sterilized. Fresh brotb cultures of B. typhosus and the 

 lactic organism were used in inoculating the milk flasks. 



The broth cultures were transferred to the milk flasks by means of 

 sterile 1 cc. pipettes. Dilutions for plating and counting were made in 

 sterile salt solution. 



Plates were made directly from the broth cultures in order to deter- 

 mine the nundier of typhoid and lactic organisms introduced. 



After inoculation, the milk flasks were placed in the incubator at 

 37°C. Having previously fomid that. i>. typhosus persists in milk after 

 curding, the flasks were allowed to remain in the incubator until the 

 milk had curded, before plating. 



The acidity of the milk at the time of jdating was determined, and 

 recorded with the day and the hour of plating. 



The counts are recorded in the number of organisms per cc. of milk. 



Colonies which could not be differentiated were identified by their 

 characteristic growths on other media and by microscopical examina- 

 tion. 



The culture of B. typhosus used in the following experiments was trans- 

 ferred from the laborator\^ stock culture and Bact. lactis acidl (lab.) 

 was isolated from sour milk bv one of the laboratorv assistants. 



*In preparing a second lot of agar, calcium carbonate in the proportion of 1 gram to 100 

 cc. of agar was found to give sufficient density and enable the subsurface colonics to be 

 seen more easily. 



