136 Journal of Agricultural Research - voi. xxvin. No. 2 



spores of Bacillus larvae will germinate and grow feebly on an agar medium in the 

 preparation of which healthy bee larvse are used as is meat in ordinary culture 

 media, sterilizing as usual by heat in an autoclave (49). However {51), if a 

 broth made by macerating healthy bee larvae in several times their volume of 

 water is sterilized without heating by filtering through sterile bacteria-proof 

 filters and then is pipetted aseptically into tubes of previously sterilized liquefied 

 agar cooled to 50° C, the resulting medium gives much better growth. This 

 medium is nevertheless unsatisfactory, owing to difficulties of prepara,tion, and 

 particularly because of lack of material for its preparation except during the 

 brood-rearing season. White {5f) therefore devised a medium which consists of 

 a suspension of the yolk of an egg aseptically in 70 cc. of sterile water, 1 cc. of 

 which suspension is added by sterile pipette to each 5 cc. of ordinary sterilized 

 tubed agar medium which has been melted and cooled to 60° C. Growth occurs 

 on this medium quite abundantly, although with the technic described great 

 care must be taken to prevent contamination. 



Maassen {2S) has also devised a medium made from a mixture of equal parts 

 of a broth from calf or pig brain and a solution of egg albumin in water, to which 

 1.8 per cent agar and 1 per cent each of Witte's and Chapoteaut's peptone are 

 added, after which it is filtered, tubed, and sterilized. This medium gives an 

 almost neutral or weakly acid reaction to blue litmus paper. Maassen also 

 found that the vegetative forms develop abundantly if grown on a meat and 

 water medium if it is acid in reaction and if 0.25 per cent of pollen and 1.5 per cent 

 of Aschmann's or Chapoteaut's peptone are added, but that the former medium is 

 more favorable. Both media are found to deteriorate on too much heating. It 

 is also stated that in acid peptone bouillon, in bouillon of bee larvae, and in the 

 brain bouillon, the bacillus may be cultivated, although growth is slow, the bouillon 

 becoming weakly turbid and a thick slimy deposit gradually being formed. 



For the purpose of the present experiments, after consideration of the advan- 

 tages or disadvantages of the various media so far described, a modification of the 

 egg-yolk suspension medium of White was adopted as the most satisfactory 

 general medium. During the course of the experiments some modifications were 

 made both in the medium and in the technic of preparation. 



PREPAEATION OF YEAST-EXTEACT AGAR BASE 



Because of most satisfactory results in other work with various brood disease 

 cultures, a yeast-extract agar described by Ayers and Rupp (2) was used instead of 

 beef infusion agar as a base, because of the ease of preparation and the uniformity 

 of the medium. Spores of Bacillus larvae on the surface of a slant of this agar 

 germinate to some extent on this medium alone, and vegetative cultures from egg- 

 yolk suspension agar transferred to the yeast medium grow fairly vigorously. 

 The addition of egg-yolk suspension to the yeast-extract agar increased the vigor 

 of growth and longevity of cultures. 



One liter of the yeast extract agar is prepared as follows: 



Dried yeast '. grama.. 10 



Peptone do 10 



Buffer (sodium glycero-phosphate) do 5 



Water cc "^ gOo 



This is heated in fiowing steam for one-half hour, then adjusted to a hydrogen- 

 ion concentration of Pb=7.6 to 7.8 by the colorimetric method of Clark and 

 Lubs {16, 17). The broth is then boiled for one minute over an open flame and 

 filtered through filter paper on a perforated porcelain funnel, using siliceous earth 

 to clarify. To this broth is added an equal amount (500 cc.) of double strength 



