Apr. 12,1924 
Development of American FouTbrood 
153 
Table VI .—Test for diastatic action with alcoholic extract a 
Test material 
Control tubes 
Color with iodin after incubation of— 
0 hour 
$ hour 
2£ hours 
5£ hours 
18 hours 
Glycogen 
Starch 
Glycogen 
Starch 
Glycogen 
Starch 
’ 
Glycogen 
Starch 
Glycogen 
Starch 
Glycogen 
Starch 
Extract of healthy pre¬ 
pupae. 
Extract of decomposed 
ropy remains. 
Extract of vegetative 
cultures. .. 
*M-++ 
(brown) 
++++ 
++++ 
++++ 
(blue) 
+++ 
++ 
++ 
++ 
j_i_L. 
+ 
+ 
++ 
+ 
+ 
++ 
± 
± 
+ 
■ 1 1 r 
j_|_L _l 
i 1 P t 
i 1 r 
° The following symbols are used: 
++++ Deep color, brown or blue. 
+-|-+ Slightly lighter brown than check or wine 
color. 
++ Light coffee brown or lavender. 
+ Trace faint brown or trace faint lavender. 
— No color or only iodin color, showing com 
plete diastatic action. 
To further determine the production of diastase by Bacillus larvae , a series of 
Petri dishes were poured, using yeast-extract egg-yolk suspension agar, to which 
had been added respectively 0.25 per cent and 1 per cent of glycogen and 0.25 per 
cent and 1 per cent of starch, this being an adaptation from methods described 
by Vedder (48) and by Allen ( 1 ). After solidification of the media in the Petri 
dishes, smears were made upon the surface of the agar from 48-hour cultures of 
various previously isolated strains of Bacillus larvae. After several days the plates 
were examined, first by holding up to the light and then later by flooding with 
iodin solution, and comparing with control plates containing no starch or glycogen. 
In nearly all the plates good growth had occurred, causing clear areas to be pro¬ 
duced in the cloudy culture medium extending slightly beyond the edge of the 
area of growth. When flooded with iodin the halo around the culture growth, 
although not wide, was more prominently differentiated from the surrounding 
medium, showing in both glycogen and starch plates. These results, in con¬ 
junction with those of the extraction experiments, demonstrate that weak dia¬ 
static action is produced by Bacillus larvae. 
ACID PRODUCTION 
It has been shown that there is still an appreciable amount of sugar (reducing 
sugars in the food remaining in the intestines and dextrose available from gly¬ 
cogen) present in the larva after sealing and in the prepupa at the age when 
American foulbrood attacks, available for fermentation (Tables IV and V). In 
the various cultural investigations both by others and by the present writer, there 
is no evidence of carbon dioxid production. It would be expected, however, that 
at least some acid would be produced from the bacterial fermentation of these 
sugars, which is known to be present. To determine this more definitely than 
heretofore, a culture medium was devised for the qualitative determination of 
acid production, which gave good vigorous growth of Bacillus larvae. 
The method used is an adaptation of the method of using agar slants for detect¬ 
ing acid formation, instead of liquid medium, described by Conn and Hucker (18 ), 
in which the change in reaction can readily be seen. The regulation yeast- 
extract egg-yolk suspension agar was prepared for this purpose by adding to the 
yeast extract base before sterilization an indicator in the proper amount both to 
the plain medium and also to a portion to which was added 1 per cent of dextrose. 
