2 
been examined by him. No inoculation experiments were made, for 
the reason that sufficient cultures in suitable condition could not be 
obtained from any medium then known. 
Since the media used in former investigations are not suitable for 
obtaining cultures for purposes of inoculation, in taking up the further 
study it has been necessary to devise a medinm which would be satis- 
factory in this respect. Such a medium has been discovered, and 
large amounts of the culture suitable for experimental inoculations have 
been obtained. This medium is prepared and used asfollows: Healthy 
bee larvee or young pup are picked from the comb, crushed, strained 
through cheese cloth, diluted with 20 to 50 times their volume of water, 
filtered through ordinary filter paper, and then passed through an earthen- 
ware filter (the Berkefeld filter is satisfactory) to remove any bacteria 
which are present. ‘The sterile filtrate thus obtained may be pipetted 
into tubes or flasks and stored until needed. When Bacillus larve is 
to be isolated, a tube of the ordinary agar of the laboratory is liquefied 
and cooled to 45° or 50° C. Then about 2 c.c. of the filtrate men- 
tioned above is added to it. A very small amount of the decaying 
larvee affected with American foul brood is then added. The procedure 
from this point is as usual in making agar plate cultures; these plates 
are afterward incubated. When a large amount of culture is desired for 
experimental purposes it is convenient to use the ordinary agar medium 
in large test tubes to which has been added, as above, about 2 c.c. of 
the sterile larvee filtrate. These agar tubes are then inclined and the 
surface of the congealed agar is inoculated. Inno case should the larvee 
or filtrate reach a high temperature. The object, of course, is to obtain 
a medium which contains the food constituents which are afforded the 
bacteria in the living larve. 
Inoculation experiments have been made by feeding to a healthy 
colony the scales from combs which had contained brood affected with 
American foul brood. The result of the feeding was that the colony 
became affected by disease, the symptoms of which were the same as 
those observed in the apiary where American foul brood is found. Like 
symptoms have been produced by feeding scales which had been put 
into ordinary meat bouillon, incubated for twenty-four hours, and then 
heated to 65° C. for twenty minutes. 
On microscopic examination of the decaying larve dead from the 
disease thus produced experimentally, the same large number of spores 
and rods are seen as when samples are examined which are taken from 
an apiary affected with American foul brood. From these dead larvee 
pure cultures of Bacillus’ larve were obtained from plates. using the 
new medium described above. These experiments show that by the 
feeding method the disease may be produced and that the contagion is 
found in the scales. The second experiment tends to indicate that the 
