EUROPEAN FOULBROOD. 15 



of the rod with the protoplasm distributed uhnig the opposite side and the two 

 ends (fig. 5; PI. VII, H). The rod, together with the spore within It, measures 

 about 2.4(U, in length and 1.2^1 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 tlie sugar 

 media. A slight coagulum forms in the milk which is slowly digested. Gelatin 

 is rapidly lluidified. 



Bacillus orpheus is not pathogenic for the brood of bees when inoculated by 

 feetling either by the direct or indirect method. Silkworm larvae succuml) fol- 

 lowing inoculation by feeding and also by puncture. 



TECHNIQUE* 



Artificial conditions for the successful cultiviition of Bacillus fluton 

 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 larvae 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 (&) the direct method, in which only a few 

 larvae 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 larvae 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 3 days following the inoculation. The earliest evidence of disease 

 is manifested by sick rather than dead larvae (p. 5). Often frag- 

 ments of larvae (PI. IV, B) are found upon examination of the 

 brood nest. 



In the direct method Bmillus pluton is taken from the stomachs 

 of infected bees. Sick rather than dead larvae 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 (PI. VIII) 

 can be pulled out of the blind end of the organ (15). The virus- 

 containing material thus obtained is triturated Avith water and the 

 aqueous suspension is added to sirup. The suspension of Bacillus 

 fluton in a thin sirup is used in making the inoculation. Larvae 

 about 2 days old are especially desirable for the direct method. The 

 inoculation is made by adding a small amount of the suspension to 



* The technique in general which was found to he satisfactory for hee-disease studies 

 is detailed to some extent in tlie sacbrood paper (17). 



2 The experimental colony is described in earlier papers (17, 18). 



