ARAO ITANOAND JAMES NEILL 423 



a drop of medium of each pH value was put upon a sterile cover-slip 

 which was then carefully sealed upon the ring of the moist chamber. 

 The moist chamber preparations were then kept for. 12 hours at the 

 temperature to be used in the subsequent incubation. 



Preparation of Dilute Emulsion of Spores. — ^Two drops of the medium 

 of each pH value were put in the cells of sterile concave slides in sterile 

 Petri dishes. With a straight needle a small inoculum of spores was 

 put into the first drop of broth of each pH value. From this drop the 

 second drop of the same pH value was then inoculated by use of the 

 straight needle. The second drop was used as the source of the final 

 inoculum. 



Procedure in this manner with the medium of each pH value gave a 

 very dilute emulsion of free spores in broth of each pH value. By this 

 procedure we obtained for the .inoculation of the hanging drops a 

 satisfactory source for a small number of spores, well washed of all 

 metabolic products; moreover, any of the medium carried over with 

 the final inoculum was of the same pH value and of the same composi- 

 tion as the drop inoculated. 



Inoculation of Drops. — The hanging drops of the moist chamber 

 preparations were then inoculated; those of each pH value were in- 

 oculated from the dilute emulsion of spores in broth of the same pH 

 value. By using a straight needle for transferring and by stirring the 

 inoculated drop, a drop was obtained containing a satisfactory num- 

 ber of spores well distributed throughout the drop. 



Examination of Preparations and Conditions of Observation. — Im- 

 mediately after inoculation the preparations were placed under a mi- 

 croscope and examined for absence of vegetative forms and for even 

 distribution of the spores. After examination the preparations were 

 placed at the temperature of incubation. Observations were made at 

 intervals of 30 minutes for the first 5 hours and then at intervals of 

 1 hour. 



RESULTS. 



Series I {5°C.). — This series (pH 1 to 13) was incubated at 5°C. 

 for 20 days.^ No apparent change took place except a slight swelling 

 of the spores in all hydrogen ion concentrations. This temperature 



^ The cold storage room of the Dairy Department of this institution was used. 



