138 



Journal of Agricultural Research voi. xxviii, No. 2 



entire apparatus is sterilized in the autoclave, using a temporary empty flask 

 into which the stopper for the culture flask is placed, and all is wrapped in paper 

 with a paper protective cap over the open end of the delivery bell. Before use, 

 the apparatus is removed from the paper and the stopper is carefully removed 

 from the empty flask so as to prevent contamination and is fastened firmly in 

 the fliask containing the egg-yolk suspension. After placing the pinch cock in 

 position, the apparatus is carefuUy inverted and hung 

 on a ring stand. The small-bore glass tube in the flask 

 now reaches a little above the surface of the hquid and 

 serves for an air inlet. By means of this apparatus, 

 sterile egg-yolk suspension can be added to tubes of 

 sterile base medium, with little danger of external con- 

 tamination, by inserting the tube under the protective 

 beU. 



METHOD OF ISOLATION OF PURE CULTURES 



OF BACILLUS LARVAE 



Fig. 7.— American foiUbrood j. . j 



scale. End view. (White When medium IS desired for the isolation or cultiva- 



(««)) tion of Bacillus larvae, tubes of the yeast-extract agar are 



melted in a water bath and cooled to 55° C, after 



which from 1 to 2 cc. of egg-yolk suspension is added for each 10 cc. of base, by 



means of the apparatus described above. The contents of the tubes are well 



mixed and then slanted. 



From a comb containing decaying material dead of the disease, a dried scale 

 (figs. 7 and 8) is removed with a sterilized needle scalpel (also used for removing 

 cappings) and dropped into the water of condensation in the culture tube to 

 soften. It is then smeared over the surface of the agar with an inoculating 

 needle. If ropy gluelike material is available it is more satisfactory (fig. 9). A 

 large loopful of this is removed from the cell, from which 

 the capping has been aseptically removed by means of 

 an inoculating needle, and is streaked over the surface 

 of the agar. A heavy initial inoculum gives best re- 

 sults, as it is often difficult to obtain growth with a small 

 amount. It is quite easy to obtain pure cultures by this 

 procedure, since almost never are secondary contam- 

 inations found associated with Bacillus larvae. Plating 

 may be carried out from these initial cultures if abso- 

 lute surety is desired, but initial growth is obtained 

 much more easily by the tube culture method. Germi- 

 nation of spores and some growth take place during the 

 first 24 hours' incubation at 37° C, but maximum growth 

 is not obtained much before 48 hours. 



EXPERIMENTAL PROCEDURE, USING AGAR SLANTS 



Fio. 8.— American foulbrood 

 scale. Side view. (White 



To determine whether there is a correlation between 

 germination of spores and vegetative growth of Bacillus 

 larvae SkuA the concentration of sugar in the culture 

 medium, a series of tubes is prepared with varying 

 percentages of dextrose, from 0.5 per cent to 10 per cent (Table I). These are 

 prepared by adding the required amounts of dextrose to 50 cc. portions of the 

 yeast-extract agar base, which is then tubed and sterilized at 10 pounds pres- 

 sure for 15 minutes. On cooling to 55° C, 1 cc. of sterile egg-yolk suspension is 



