Apr. 12, 1924 
Development of American Foulbrood 
139 
added to each tube and it is then slanted. Series of agar slants varying in sugar 
concentration are inoculated with either vegetative cultures or diseased material 
containing only spores. To determine spore germination an approximately 
uniform amount, about one 2-mm. loopful of ropy material, when available, is 
used for inoculation of slants, otherwise a scale softened 
as described above. If no visible growth takes place 
after 48 hours’ incubation, stained smears are made, to 
determine whether any germination has occurred. In 
the case of the determination of growth from vegetative 
culture, a single uniform streak is made on the agar slant, 
using one 2-mm. loopful of growth from a 48-hour cul¬ 
ture of Bacillus larvae prevously isolated and cultivated. 
After 48 hours’ incubation, as well as after about one 
week, comparative observations are made of the relative 
amount and character j)f the growth. Where little or no 
growth has occurred, stained smears are made from the 
streak to see what has happened to the organisms. These 
experiments were carried out with a number of different 
strains of vegetative cultures and from a number of different samples of American 
foulbrood. 
Table I.— The effect of varying the sugar concentration in egg-yolk suspension 
medium ( 1) on germination and vegetative growth from spores; and (#) on vege¬ 
tative growth from vigorous vegetative cultures of Bacillus larvae. a 
Fig. 9.—Partially decom¬ 
posed American foulbrood 
larva at the stage of ropy 
consistency. (White ( 55 )} 
Per cent dextrose in medium 
Test material 
Spores. 
Vegetative cultures 
Control 
0.5 
0.7 
1.0 
1.3 
1.5 
1.75 
2.0 
++++ 
++ 
+++ 
++++ 
++++ 
4*++ 
+++ 
++ 
+4-4-4- 
+++ 
+++ 
++++ 
++++ 
++++ 
+++ 
+++ 
Per cent dextrose in medium 
x est material 
2.25 
2.5 
2.75 
3.0 
3.5 
4.0 
4.5 
5.0 
7.5 
io. a 
Spores..... 
Vegetative cultures... 
+ 
4* 
4- 
4* 
G 
G 
G 
G 
G 
G 
++ 
++ 
4-4- 
++ 
4- 
4- 
± 
— 
— 
— 
° The following symbols are used: 
+ Slight growth. 
+4* Fair growth. 
4-4*4- Good growth. 
++++ Heavy growth. 
dh Doubtful. 
— No evidence of growth. 
G Slight germination of spores. 
EXPERIMENTAL PROCEDURE, USING PLATE CULTURES 
The egg-yolk suspension agar is not entirely satisfactory for counting colonies 
in plate cultures, since-the egg yolk gives the medium a cloudy, semiopaque ap¬ 
pearance. However, by using the supernatant fluid from the egg-yolk suspen¬ 
sion or a somewhat smaller amount of the suspension for each tube of yeast extract 
agar (10 to 15 drops), a fairly satisfactory plate culture is obtained if the proper 
amount of inoculum is used. The following procedure is used: To a series of 
melted tubes of yeast-extract agar containing varying amounts of dextrose as 
described above (Table II) the egg-yolk suspension is added and the desired in¬ 
oculation of the tube made while the medium is still liquid. The tubes are agi¬ 
tated to mix the contents thoroughly and then poured into sterile Petri dishes- 
