136 
Journal of Agricultural Research 
Vol. XXVIII, No. 2 
spores of Bacillus larvae will germinate and grow feebly on an agar medium in the 
preparation of which healthy bee larvae 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 preparation, and 
particularly because of lack of material for its preparation except during the 
brood-rearing season. White (54) 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 50° C. Growth occurs 
on this medium quite abundantly, although with the technic described great 
care must be taken to prevent contamination. 
Maassen (28) 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. 
PREPARATION OF YEAST-EXTRACT 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_grams. _ 10 
Peptone_do_ 10 
Buffer (sodium glycero-phosphate)___do_ 5 
Water_cc_500 
This is heated in flowing steam for one-half hour, then adjusted to a hydrogen- 
ion concentration of P H =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 
