42 



THE BACTEKIA OF THE APIAKY. 



spores was secured in an agar (bee-larvae agar) made from this special 

 bouillon when Liborius's method for cultivating anaerobes was used. 



The technique for making cultures successfully from the diseased 

 material is not difficult if the following method is used: Place a 

 loopful of the decayed tissue of the larvse into a tube of bouillon; 

 heat to 65° C. for 10 minutes to kill any vegetative forms which might 

 be present; incubate for 12 hours, and heat again to 65° C. for 10 

 minutes. This is usually sufficient, but it may be necessary to repeat 

 the same process. Liquefied bee-larvse agar in a test tube is then in- 

 oculated and incubated. The successive heating will destroy the veg- 

 etative stage of any spore-producing species which is common about 

 the apiary, e. g.^ members of the group represented by Bacillus A, as 

 described on pp. 13-14 of this paper. Agar slant and bouillon, when 

 inoculated from this source, remain sterile ; but when bee-larvse agar 

 is used a slow but abundant growth takes place. Under certain con- 

 ditions the growth appears very near or at the surface when cultures 

 are made in the above manner. A surface growth can be obtained 

 after a few generations by reinoculating slant agar of this same 

 medium. 



The above method was used successfully in diagnosing the follow- 

 ing samples from different apiaries: 



Results of examination of specimens of American foul irood, formerly called 



simply " foul brood." 



The results of these examinations show that BaciUus larvae- was 

 present in all the specimens examined, which suggests that it very 

 probably figures as an etiological factor in this disease. Other bac- 

 teria of different species are occasions^^lly found associated with this 

 bacillus. 



Baoillus larvse. 



Occurrence. — Constantly present in diseased brood from colonies affected with 

 American foul brood. 



Gelatin. — ^There is no growth. 



Morphology. — It is a slender rod; having a tendency to form In chains. This 

 is especially true when grown in bee-larvae bouillon. 



Motility. — The bacillus is rather sluggishly motile. 



Spores. — Spore formation talies place. This can be observed best in the dif- 

 ferent stages of the disease and decay of the larvse. 



Oxygen requirements. — When Liborius's method is used, the best growth 

 usually appears near to but not on the surface. After a few generations a 

 surface growth may be obtained. 



