268 Journal oj Agricultural Research voi. 45, No. s 



days' incubation where no growth was observed after 10 days' incu- 

 bation. Work on this phase of the problem was continued during 

 the summer and fall of 1930. Several sets of cultures were made in 

 which Bacillus larvae from eight different localities were used in a 

 series of seedings with a decreasing number of spores for each lot of 

 the organism and all incubated for 30 days. (Table 4, Group 2.) 



methods op procedure 



Culture Media 



A culture medium was used similar to that employed by the writer 

 in the preliminary experiments (^7) and also in earlier cultural work 

 with Bacillus larvae (26} — -that is, a combination of the medium made 

 of yeast-extract and egg-yolk suspension and the carrot-extract 

 medium of Lochhead (18). The yeast-carrot extract medium was 

 prepared as follows: 



(A) Dried yeast grams__ 10 



Peptone do 10 



Buffer (sodium glycerophosphate) do 2. 5 



Water (distilled) cubic centimeters ._ 500 



This solution was heated in flowing steam for one-half hour and, after a table- 

 spoonful of siliceous earth had been added to assist in the filtration and clarifica- 

 tion, it was filtered through filter paper on a perforated porcelain funnel with 

 suction. 



(B) Two hundred grams of cleaned carrots was macerated in a meat grindei, 

 added to 500 c c of distilled water, and allowed to stand for at least 30 minutes, 

 preferably longer. The macerated carrot was removed by filtration through 

 fine muslin, as much liquid as possible being squeezed from the mass. The 

 filtrate was then clarified by the addition of siliceous earth and filtration in the 

 same manner as the yeast-extract medium. 



(C) The final base medium was prepared by mixing 500 c c of A with 200 c c 

 of B and adding 700 c c of a 3 per cent solution of washed agar. 



The reactidn of the medium was so adjusted that when 2 c c of 

 sterile egg-yolk suspension, prepared as described in a previous paper 

 (26), was added to 10 c c of the yeast-carrot extract base medium by 

 means of the apparatus shown in Figure 1, and described previously 

 (26), the pH value was 6.8. The medium was then sterilized in the 

 Autoclave at 15 pounds' pressure (sea level) for 15 minutes. After it 

 iad cooled to 45° C, 20 drops, or about 2 c c, of the sterile egg-yolk 

 suspension was added to each tube of medium, mixed by shaking, 

 and the medium was then allowed to solidify in a slanting position. 



The Lochhead yeast-extract medium was tried without the addition 

 of egg-yolk suspension, but although it gave good growth with the 

 heavier seedings of spores, the combination medium was found to give 

 more uniform germination and heavier vegetative growth with the 

 more dilute seedings. The addition of the carrot extract, while pos- 

 sibly adding somewhat to the growth-producing qualities of the med- 

 ium, served m these experiments as an indicator for vegetative growth 

 because of the abiUty of Bacillus larvae to produce nitrite in the carrot- 

 extract medmm without the addition of potassium nitrate (18). 



Pkeparation op Dilutions of Spores 



The stock suspensions of spores of Bacillus larvae were made up as 

 described earlier m this paper. A series of primary dilutions, each 

 one-tenth of the preceding dilution, was then made up in sterile 125 



