420 G. S. WILSON 



up within five or ten minutes of preparation of the dilutions, it is 

 immaterial whether tap water, saline, or Ringer's solution be 

 employed. For most purposes in this work tap water, sterilized 

 by autoclaving at 120°C. for forty-five minutes, has been chosen 

 as the diluent, and been found perfectly satisfactory. Just 

 recently however, during the drought of June and Jul}', it was 

 noticed that the three tubes put up for each count during the 

 course of routine work showed a marked disagreement with one 

 another, and, on investigation this was referred to the tap water. 

 Whether any marked change was produced in the saline content 

 as a result of the drought, or whether possibly some metalUc 

 contamination had gained entrance to the water, was not ascer- 

 tained, but on replacing the tap water by Ringer's solution, the 

 tubes again showed close agreement with one another. 



Nature of the medium 



The nutrient agar used was prepared from a three weeks' casein 

 digest at 37°C. (Cole and Jordan Lloyd, 1917). After filtration 

 it was diluted till it had an amino-nitrogen content — as determined 

 by Sorensen's method of formol titration — of O.OS per cent, this 

 being found to be the most suitable proportion for the purpose. 

 An addition of 2.5 per cent agar was then made — liigher amounts 

 being found to exert a deleterious action on the development of 

 the organisms. As no advantage of washed over commercial 

 agar could be substantiated for the growth of Bad. suipestifer — a 

 result which failed to confirm the findings of Ayers, Mudge and 

 Rupp (1920) in the case of mUk bacteria — the latter was uni- 

 formly employed. The hydrogen-ion concentration of the 

 finished medium was allowed to vary from pH 7.4 to pH 7.8. 



Length of incubation and effect of moisture during incubation 



With regard to the length of time that the tubes should be 

 incubated, experiments were made in which the number of 

 colonies was counted on successive days up to a week. The 

 result was to show that though the majority of the bacilli 

 grow within the first twenty-four hours at 37°C., a few colonies 



