154 
Joumal of Agricultural Research voi.'xxvm, No. 2 
Brom thymol blue was first used, as it covers the range of the supposed optimum 
reaction for Bacillus larvae as described earlier. Baker (4) also has shown that 
brom thymol blue, used in about a 0.0024 per cent concentration in culture media> 
gives the most desirable color for comparison, without inhibiting acid fermentation. 
This concentration was obtained by using 12 cc. of a 0.2 per cent alcoholic solu¬ 
tion of the indicator per liter. After marked acid production in the dextrose 
tubes was demonstrated with brom thymol blue, brom cresol purple was used as 
suggested by Conn and Hucker (18) in a 0.001 per cent concentration as a check 
on the end point. This concentration was obtained by using 8 cc. of a 0.2 per 
cent alcoholic solution of the indicator per liter. The yeast-extract base, both 
with and without dextrose, was adjusted so that after the addition of the egg- 
yolk suspension the final medium would have a primary reaction of approximately 
P h = 7.2, a definite blue grass green in the case of brom thymol blue and a marked 
purplish tinge with brom cresol purple, except in one series, where the primary 
reaction of the plain medium was P H =7.6. These tubes after being slanted were 
inoculated as usual, both with vegetative cultures and with diseased material 
containing spores. The change in reaction was noted after different lengths of 
incubation, and the final reaction was determined by comparison with standard 
buffer tubes used in combination with tubes of plain egg-yolk suspension media 
slanted in the same manner. The approximate increase in hydrogen-ion con¬ 
centration was determined by this comparison (Table VII). 
Table VII .—Acid production by Bacillus larvae 
Culture No. 
Brom thymol blue indicator 
Brom cresol purple indicator 
Plain medium 
1 per cent dextrose 
Plain medium 
1 per cent dextrose 
Control 
Inocu¬ 
lated 
Control 
Inocu¬ 
lated 
Control 
Inocu¬ 
lated 
Control 
Inocu¬ 
lated 
Ph 
Ph 
Ph 
Ph 
Ph 
Ph 
Ph 
Ph 
9693-1. 
7.6 
6.8 
7.2 
6.0 
( 6 ) 
(•) 
( d ) 
5.8 
9834-1_ 
7.2 
6.6 
7.2 
6.0 
9834-2. 
7.6 
0 7.4 
7.2 
6.2 
(») 
(•) 
(<0 
6.0 
9853.... 
6.8-7.0 
±6.6 
6.6 
6.0 
9857... 
7.2 
6. 6-6.8 
7.2 
6.0-6.2 
9867.. 
7.6 
“7.4 
7.2 
6.4 
(*) 
<•) 
( d ) 
6.2 
9869... 
7.6 
“7.4 
7.2 
6.0 
(>) 
(«) 
( d ) 
5.8 
9874___ 
7.6 
7.0-7.2 
7.2 
6.0 
(>) 
(•) 
( d ) 
5.8 
“ Doubtful growth. d Beyond end point, good growth. 
b Beyond end point, no growth. « No change in color, no growth. 
e No change in color, good growth. 
OBSERVATIONS 
Several interesting facts were observed from these experiments. Addition of 
buffer salts to the media delayed the approach to the final hydrogen-ion con¬ 
centration reaction somewhat, but eventually practically the same end point 
was reached. Also, in one series of media in which the plain medium was 
adjusted to about Ph=7.6, little if any growth occurred in these tubes except 
with two strains of Bacillus larvae , indicating that the alkaline limit for growth 
is about at this point. In cases where the initial reaction of the plain medium 
was Ph=7.2, the final reaction averaged P H =6.6 to P H =6.8 (Table VII). In 
the case of the medium to which 1 per cent dextrose had been added, the final 
reaction averaged about P H =6.0 for brom thymol blue and from Ph = 5.8 to 
Ph=6.0 for brom cresol purple (Table VII). While, therefore, only a slight 
change in reaction occurred in media without sugar, a marked production of 
