INOCULATION EXPERIMENTS WITH BACILLUS ALVEI. 87 



Oelatin colonies. — Gelatiu is a medium in which it develops slowly. The col- 

 ony becomes very irregular in outline, owing to thread-like outgrowths which 

 take place in curves from its border. Growth is better when 5 per cent glycerin 

 is added. From the small, white, spherical colonies which form along the line 

 of puncture gray, thread-like growths shoot out thru the medium. In about 2 

 months the gelatin is changed to a thick liquid, holding gray flocculeut masses 

 of organisms which gradually settle, forming a strong, slightly viscid sediment. 



Indol. — In old cultures a decided indol reaction is obtained. 



Power to resist disinfectants. — Preliminary observations give the following 

 results : The spore form resists drying for a considerable time. Spores which 

 have been drying for 1 year germinate promptly when introduced into bouillon. 

 The vegetative form : One per cent carbolic acid kills in 10 minutes ; 3 per 

 cent carbolic acid kills in 2 minutes ; mercuric chlorid solution, 1 to 1,000. 

 kills in 1 minute ; mercuric chlorid solution, 1 to 2,000, kills in 2 minutes. 



Spore form. — Mercuric chlorid, 1 to 1,000, kills in 30 minutes. 



Pathogenesis in vertebrates. — Inoculations into guinea pigs and frogs have 

 not proven this organism to be pathogenic to these animals. 



Inoculation Experiments. 



That part of the investigation which involves the producing of the 

 disease experimentalh^ by inoculating with pure cultures of the 

 organism is usually the most dilRcult one. Very rarely indeed is one 

 able to produce the disease with symptoms closely simulating those 

 found in nature. The experimental production of a disease involves 

 many variable factors, such as attenuation of the organism, methods 

 of inoculation, resistance of the host, and the immediate environment. 



On August 4, 1902, vre inoculated a hive containing nothing but 

 healthy brood, free from bacteria, by feeding with sirup (sugar and 

 water in equal parts) to which was added the growth from the sur- 

 face of the plate cultures containing spores and bouillon cultures of 

 Bacillus alvei. Similar feedings were given to these bees from one 

 to three times a week until September 28, but symptoms of foul 

 brood did not develop. On August 6 cultures were made from a 

 few of the hive larvae. They were foinid to contain the bacilli. 



Inoculation experiments were again made in 1903. Because of a 

 failure to produce a diseased condition with cultures of Bacillus alvei 

 in the experiment of 1902, the variable factors above mentioned were 

 carefully considered in the experiment of this year. The inocula- 

 tions were made when climatic conditions were such as seemed to 

 favor the ravages of the disease in the apiaries; namely, low tem- 

 perature, dampness, and cloudiness. A colony of black bees was 

 used, as they were almost universally considered more susceptible. 

 Cultures of Bacillus alvei were freshly isolated from foul-brood 

 specimens and kept in stock on bee-larvae agar (described under 

 American foul brood, pp. 41-42). All cultures were incubated at 34° 

 C., which temperature is observed to be slightly below that of the 

 hive. The spore form of Bacillus alvei was used. 



Inoculations were made in different ways. A diseased condition 



