138 
Journal of Agricultural Research voi. xxviii, no. 2 
entire apparatus is sterilized in the autoclave, using a temporary empty flask 
into which the stopper for the culture flask is placed, and all is wrapped in paper 
with a paper protective cap over the open end of the delivery bell. Before use, 
the apparatus is removed from the paper and the stopper is carefully removed 
from the empty flask so as to prevent contamination and is fastened firmly in 
the flask containing the egg-yolk suspension. After placing the pinch cock in 
position, the apparatus is carefully inverted and hung 
on a ring stand. The small-bore glass tube in the flask 
now reaches a little above the surface of the liquid and 
serves for an air inlet. By means of this apparatus, 
sterile egg-yolk suspension can be added to tubes of 
sterile base medium, with little danger of external con¬ 
tamination, by inserting the tube under the protective 
bell. 
METHOD OF ISOLATION OF PURE CULTURES 
OF BACILLUS LARVAE 
Fig. 7.—American foulbrood 
scale. End view. (White 
( 55 )) 
When medium is desired for the isolation or cultiva¬ 
tion of Bacillus larvae , tubes of the yeast-extract agar are 
melted in a water bath and cooled to 55° C., after 
which from 1 to 2 cc. of egg-yolk suspension is added for each 10 cc. of base, by 
means of the apparatus described above. The contents of the tubes are well 
mixed and then slanted. 
From a comb containing decaying material dead of the disease, a dried scale 
(figs. 7 and 8) is removed with a sterilized needle scalpel (also used for removing 
cappings) and dropped into the water of condensation in the culture tube to 
soften. It is then smeared over the surface of the agar with an inoculating 
needle. If ropy gluelike material is available it is more satisfactory (fig. 9). A 
large loopful of this is removed from the cell, from which 
the capping has been aseptically removed by means of 
an iiioculating needle, and is streaked over the surface 
of the agar. A heavy initial inoculum gives best re¬ 
sults, as it is often difficult to obtain growth with a small 
amount. It is quite.easy to obtain pure cultures by this 
procedure, since almost never are secondary contam¬ 
inations found associated with Bacillus larvae. Plating 
may be carried out from these initial cultures if abso¬ 
lute surety is desired,- but initial growth is obtained 
much more easily by the tube culture method. Germi¬ 
nation of spores and ‘some growth take place during the 
first 24 hours' incubation at 37° C., but maximum growth 
is not obtained much before 48 hours. 
EXPERIMENTAL PROCEDURE, USING AGAR SLANTS 
Fig. 8.—American foulbrood 
scale. Side view. (White 
( 56 )) 
To determine whether there is a correlation between 
germination of spores and vegetative growth of Bacillus 
larvae and the concentration of sugar in the culture 
medium, a series of tubes is prepared with varying 
percentages of dextrose, from 0.5 per cent to 10 per cent (Table I). These are 
prepared by adding the required amounts of dextrose to 50 cc. portions of the 
yeast-extract agar base, which is then tubed and sterilized at 10 pounds pres¬ 
sure for 15 minutes. On cooling to 55° C., 1 cc. of sterile egg-yolk suspension is 
