MICROBIAL DISEASES OF INSECTS 923 



CAUSAL ORGANISM. Bacillus larva is a slender rod with ends slightly rounded 

 and with a tendency to grow in chains. The length varies greatly, depending for 

 the most part upon the medium used for its cultivation. It varies from 2.5 to SM 

 in length, and is about 0.5/1 in breadth when grown on the surface of brood-filtrate 

 agar. In a liquid medium it is usually much longer, frequently becoming filamen- 

 tous. Giant whips occur in large numbers, especially in the condensation water of 

 brood-filtrate agar slant cultures. They are also present in decaying larvae dead of 

 American foul brood. The flagella are peritrichic: when twisted into giant whips, 

 these corkscrew-like structures vary widely in their dimensions from scarcely visible 

 coiled filaments to bodies several microns in diameter. Motility moderate in young 

 cultures from the surface of brood-filtrate agar, sluggish in liquid cultures. Spores 

 formed about the third day on brood-filtrate agar; median, causing a spindle-shaped 

 enlargement of the rod; free spores measure about 0.6 by 1.3^. Few or no spores 

 are formed in liquid media, deep in solid media, and on media containing glycerin, 

 mannit, or dextrose. Some of the other sugars, and also honey inhibit spore forma- 

 tion. The rods stain readily with ordinary anilin dyes, and are Gram positive. 



B. larva is cultivated with difficulty, growing best on media made as the ordinary 

 laboratory media, substituting bee larvae for meat, or on egg-yolk-suspension agar. 

 Bee larvae agar, however, is limited in its usefulness on account of the large amount of 

 brood required in its preparation. The unheated egg-yolk agar is prepared as 

 follows: immerse fresh eggs in a disinfecting solution, break the shell, pour off the 

 white, and drop the yolk into a flask containing about 70 cc. of sterile water; agi- 

 tate the flask to make a homogeneous suspension of the yolk, and with a sterile 

 pipette transfer the aqueous suspension to sterile tubes and store until needed. For 

 use : melt tubes of agar and cool to about 50, add about i cc. of egg-yolk-suspension 

 to each 5 c.c. of melted agar, and either incline and allow to harden, or use im- 

 mediately for plating as desired. A more detailed description of the technic em- 

 ployed in making both of the special media necessary for the cultivation of B. larva 

 will be found in the bulletin by White. Bacillus larva is present in practically pure 

 cultures in brood dead of American foul brood, so this organism can be readily ob- 

 tained from brood dead of this disease by heating the spore-containing material in 

 aqueous suspension at 100 for one or two minutes, and plating in bee-larvae agar or 

 egg-yolk suspension agar. When bee-larvae agar alone is employed and inoculations 

 are made with spores, following Liborious' method for anaerobes, growth as a rule 

 appears more often near to than on the surface, indicating partial anaerobiosis. 

 Sub-cultures on brood-filtrate agar and egg-yolk-suspension agar or their combina- 

 tion yield abundant surface growth. 



Brood-filtrate agar slant, growth rapid, being moderate to heavy in twenty-four 

 hours, somewhat spreading, grayish white and slightly viscid; has a more or less 

 uniform border, a smooth surface, and a ground glass appearance. Older cultures 

 are less prominent than the younger ones. Brood-filtrate agar plates, surface colonies 

 vary in size depending upon the number present. When well isolated they not in- 

 frequently spread, attaining a diameter of i cm. or more; growth only slightly raised, 

 smooth surface, ground glass appearance, with a clearly defined, uniform border. 

 Deep colonies vary from lenticular to irregular in form with filamentous outgrowths 

 from portions of their surface. No visible gas, but slight acidity in carbohydrate 



