15 



2 hours IIS'-O growth. 



2 " 15 minutes ... 116 " " 



2 " 30 " .. 115" no growth. 



2 " 45 " .. 115 " " 



3. Capillary tubes with spores in distilled water. 



The temperatures were taken in a large vessel containing 10 pounds of 

 boiling honey. The experiment was repeated, using buckwheat honey instead 

 of clover and with like results. 



Relation to Light. A few experiments were made to ascertain the 

 behaviour of spores toward light. Coverglasses spread with spores and dried, 

 were exposed to bright sunlight during the month of February. The expo- 

 sure was in the opon air and the glasses were on black tile. The temperature 

 varied from — 12" C. to — 22" After exposure, the glasses were placed film 

 side downwards on agar plates, and then incubated at 37° C. 

 Time. Result. 



Results — 3 hours sunlight. Abundant growth in 16 hours 



R t( l« fl It it tt 



g 11 c( (I i< X i( 



These experiments were repeated in September, when the outside tem- 

 perature varied from 24" to 30° 0., with the result, that there was growth 

 after 4, 6, and 7 hours' exposure. 



Agar plates exposed after inoculation showed great differences. Por 

 instances, spores 21 days old was killed by 5 hours' exposure, whilst plates 

 made the day after with spores .2 months and 21 days old, required 7 hours' 

 exposure! Spores 10 days old showed no growth after 5 hours' exposure ; 

 and spores 5 days old, no growth after 6 hours' exposure. From a large num- 

 ber ot determinations, the average length of exposure necessary to kill spores 

 within the above range of temperature was found to be 5 hours. 



Vitality on various media. The cultures seem to live longer on agar 

 than in liquid media. The vitality of old gelatine and bouillon cultures 

 seems to be lessened by the products of the bacilli growing in these media. 

 The epores taken from these sources have also decreasi'd resisting power. 



Effect of growth on reaction of media. Ordinary bouillon becomes slightly 

 more alkaline as growth proceeds, the presence of ammonia being detected 

 by Nessler's reagent ; but control bouillon does not give the reaction. In 

 bouillon, with the addition of glycerine and various sugars, the acidity 

 of the media is increased, but more in the case of glucose broth than in any 

 other. In these experiments accurate titration was made with phenolphtalein 

 as indicator. Cheyne tried the reaction, " making the infusions faintly 

 alkaline, and after the growth of this organism in it, it is faintly alkaline." 



Sensitiveness to Antiseptics and Germicides. This subject is taken up 

 in connection with the chemical remedies used for the disease. 



Pathogenesis. Besides being pathogenic to the larvae of bees, Cheyne 

 has inoculated two mice and one rabbit with spore-bearing cultivations with- 



