EUROPEAN FOULBROOD. 15 
of the rod with the protoplasm distributed along the opposite side and the two 
ends (fig. 5; Pl. VII, H). The rod, together with the spore within it, measures 
about 2.44 in length and 1.24 in width. This relation of spore and rod persists 
in cultures on a solid medium for a long period, especially at room temperature. 
Good growth, no gas, and only slight changes in reaction occur in the sugar 
media. A slight coagulum forms in the milk which is slowly digested. Gelatin 
is rapidly fluidified. 
Bacillus orpheus is not pathogenic for the brood of bees when inoculated by 
feeding either by the direct or indirect method. Silkworm larve succumb fol- 
lowing inoculation by feeding and also by puncture. 
TECHNIQUE* 
Artificial conditions for the successful cultivation of Bacillus pluton 
have not yet been obtained. That this can be achieved by further 
study is not at all improbable. Without having accomplished this, 
it has been possible, however, to make the studies on the biology of 
the parasite that were most desired. This was done through ex- 
perimental work, using the larve of bees. The inoculations were 
made by feeding a suspension of the organism in sugar sirup. 
Two methods were employed in making the feedings, which will 
be referred to here as (a) the indirect method, in which the colony 
is inoculated, and (0) the direct method, in which only a few 
larvee are inoculated. Cane sugar and water were used in preparing 
the sirup in the proportion approximately of 3 to 2. This solution 
was then brought to the boiling point. 
From 5 to 10 diseased larve furnish sufficient infective material 
when the indirect method is followed. These, after being picked 
from the brood frame, are thoroughly crushed, added to about 300 
c. c. of the cooled sirup, and fed to a colony.?, When the suspension 
contains the living virus, symptoms of European foulbrood appear 
in 8 days following the inoculation. The earliest evidence of disease 
is manifested by sick rather than dead larve (p. 5). Often frag- 
ments of larvee (PL IV, B) are found upon examination of the 
brood nest. 
In the direct method Bacillus pluton is taken from the stomachs 
of infected bees. Sick rather than dead larve are preferred for ob- 
taining the virus free from the body tissues. By the use of dissect- 
ing needles and with a little care the stomach contents (Pl. VIII) 
can be pulled out of the blind end of the organ (15). The virus- 
containing material thus obtained is triturated with water and the 
aqueous suspension is added to sirup. The suspension of Bacillus 
pluton in a thin sirup is used in making the inoculation. Larve 
about 2 days old are especially desirable for the direct method. The 
inoculation is made by adding a small amount of the suspension to 
1The technique in general which was found to be satisfactory for bee-disease studies 
is detailed to some extent in the sacbrood paper (17). 
2'The experimental colony is described in earlier papers (17, 18). 
