Apr. 12, 1924 
Development of American Foulbrood 
137 
(3 per cent) solution of agar, washed and filtered by the method described by 
Ayers, Mudge, and Rupp (8 ). The final hydrogen-ion concentration reaction is 
adjusted so that upon addition of 1 cc. of the egg-yolk suspension to 10 cc. of 
the yeast-extract agar the reaction is about P H = 6.8. The normal hydrogen-ion 
concentration value of the contents of the larval intestine at various ages during 
the active feeding period with honey and pollen and just after sealing averages 
P H =*6.8, varying to slightly more acid with the amount and type of pollen in 
the food material. Intestines were dissected out from the larvse and macerated 
in 10 cc. of neutral distilled water and compared colorimetrically with known 
buffer solutions, using brom thymol blue as an indicator. Fabian and Parks {22) 
found this value to be P H =* 6.6 by macerating the entire larva in water. From 
earlier unpublished work by the writer, as well as by the above-mentioned inves¬ 
tigators, the optimum hydrogen-ion concentration for the growth of Bacillus 
larvae was found to be approximately P H =6.8. The yeast extract medium is 
tubed, sterilized in the autoclave at 15 pounds pressure for 15 minutes, and 
stored until needed. 
PREPARATION OF EGG-YOLK SUSPENSION 
The egg yolk can be diluted much more than was directed 
formula with even better results, the more dilute suspension 
transparent medium with fully as profuse growth. 
A wide-mouthed flask containing 200 cc. water, 
sterilized with a cotton plug protected by a paper 
cap, is used for each egg yolk. At times, from 0.5 
per cent to 1 per cent of a neutral buffer salt is 
added to the water previous to sterilization. This 
holds in check the slow increase in acidity observed 
on long standing. A small amount of normal so¬ 
dium hydroxid (2 to 3 cc.) is also added to the flasks 
before sterilization to bring the resulting reaction 
of the egg suspension nearer to the desired reaction 
for the final medium. 
APPARATUS TO REPLACE PIPETTING 
in the 
giving 
original 
a more 
An apparatus was devised (fig. 6) which to a 
great extent eliminates the danger of contamination 
of agar tubes when adding egg-yolk suspension, 
and also makes possible the preparation of a large 
quantity of medium in a short time. As a rule 
egg-yolk suspension can be stored or withdrawn at 
any time after the apparatus has been set up, until 
all used up, unless the egg yolk itself is not sterile. 
A two-holed rubber stopper, of correct size to fit 
the flask containing the egg-yolk suspension, is 
fitted with two tubes, one of small bore to reach nearly to the surface of the 
liquid when placed in the egg-yolk flask, and a second larger tube fitted flush to 
the inner surface of the stopper, protruding ouwartd about 1J inches. Apiece of 
rubber tubing 5 inches long is fitted to this tube, closed with a pinch cock. To 
this rubber tube is attached a delivery tube which passes through another 
rubber stopper placed in one end of a glass cylinder li inches in diameter and 
4 inches long, to about half its length. This forms a protective bell for the 
delivery tube similar to that used in filling vaccine or antitoxin ampules. The 
Fig 6.—Apparatus to replace pipet¬ 
ting of egg-yolk suspension 
