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Journal of Agricultural Research voi. xxvin. No. 2 



Brom thymol blue was first used, as it covers the range of the supposed optimum 

 reaction for Bacillus larvx 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 Uter. 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 

 Ph=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 Ph=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 



' Doubtful growth. 



* Beyond end point, no growth. 



' No change in color, good growth. 



'' Beyond end point, good growth. 

 « No change in color, no 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 eventuaUy practicaUy 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 Ph=6.6 to Ph=6.8 (Table VII). In 

 the case of the medium to which 1 per cent dextrose had been added, the final 

 reaction averaged about Ph=6.0 for brom thymol blue and from Ph=5.8 to 

 Ph=6.0 for brom cresol purple (Table VII). WhUe, therefore, only a sUght 

 change in reaction occurred in media without sugar, a marked production of 



