Apr. 12, 1924 



Development of American Foulbrood 



153 



Table VI. — Test for diasiaiic action with alcoholic extracf* 



<* 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 taint lavender. 

 — No color or only iodln 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 (45) and by AUen (i). 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. 



