Apr. 12, 1924 



Development of American Foulbrood 



139 



added to each tube and it is then slanted. Series of agar slants varying in sugar 

 concentration are inoculated with either vegetative cultures or diseased material 

 containing only spores. To determine spore germination an approximately 

 uniform amount, about one 2-mm. loopful of ropy material, when available, is 

 used for inoculation of slants, otherwise a scale softened 

 as described above. If no visible growth takes place 

 after 48 hours' incubation, stained smears are made, to 

 determine whether any germination has occurred. In 

 the case of the determination of growth from vegetative 

 culture, a single uniform streak is made on the agar slant, 

 using one 2-mm. loopful of growth from a 48-hour cul- 

 ture of Bacillus larvae prevously isolated and cultivated. 

 After 48 hours' incubation, as well as after about one 

 week, comparative observations are made of the relative 

 amount and character of the growth. Where little or no 

 growth has occurred, stained smears are made from the 

 streak to see what has happened to the organisms. These 

 experiments were carried out with a number of different 

 strains of vegetative cultures and from a number of different samples of American 

 foulbrood. 



Fig. 9.— Partially decom- 

 posed American foulbrood 

 larva at the stage of ropy 

 consistency. (White C55)) 



Table I. — The effect of varying the sugar concentration in egg-yolk suspension 

 medium (1) on germination and vegetative growth from spores; and {2) on vege- 

 tative growth from vigorous vegetative cultures of Bacillus larvae. <» 



Test material 



Per cent dextrose in medium 



Control 0.6 0.7 1.0 



1.3 



Spores _ 



Vegetative cultures - 



++++ 



++ 

 +++ 



+++ 

 +++ 



++++ 

 ++++ 



++++ 

 ++++ 



+++ 

 ++++ 



+++ ++ 

 +++ +++ 



Per cent dextrose in medium 



Test material 



2.2i 2.6 2.75 3.0 3.S 4.0 4.6 5.0 7.6 10.0 



Spores ._ 



Vegetative cultures - 



+ 

 ++ 



+ 

 ++ 



+ 

 ++ 



+ 

 ++ 



o Tbe following symbols are used: 

 + Slight growth. 

 ++ Fair growth. 

 +++ Good growth. 

 ++++ Heavy growth. - 



± Doubtful. 



— No evidence of growth 



O Slight germination of 



EXPERIMENTAL PROCEDURE, USING PLATE CULTURES 



The egg-yolk suspension agar is not entirely satisfactory for counting colonies 

 in plate cultures, since the egg yolk gives the medium a cloudy, semiopaque ap- 

 pearance. However, by using the supernatant fluid from the egg-yolk suspen- 

 sion or a somiewhat smaller amount of the suspension for each tube of yeast extract 

 agar (10 to 15 drops), a fairly satisfactory plate culture is obtained if the proper 

 amount of inoculum is used. The following procedure is used: To a series of 

 melted tubes of yeast-extract agar containing varying amounts of dextrose as 

 described above (Table II) the egg-yolk suspension is added and the desired in- 

 oculation of the tube made while the medium is still liquid. The tubes are agi- 

 tated to mix the contents thoroughly and then poured into sterile Petri dishes. 



