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 (S). 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 Pn=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 

 Ph = 6.8, varying to slightly more acid with the amount and type of pollen in 

 the food material. Intestines were dissected out from the larvis 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 (SS) 

 found this value to be Pa =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 earlier unpublished work by the writer, as well as by the above-mentioned inves- 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 Ph=6.8. The yeast extract medium is 

 tubed, sterilized in the autoclave at 15 pounds pressure for 15 minutes, and 

 stored until needed. 



^ 



PKEPARATION OF EGG-YOLK SUSPENSION 



The egg yolk can be diluted much more than was directed in the original 

 formula with even better results, the more dilute suspension giving a more 

 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 



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 egg-yolk suspension can be stored or withdrawn at 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 

 aU 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 

 fltted 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 outward about IJ inches. A piece of 

 rubber tubing 5 inches long is fitted to this tube, closed with a pinchcock. To 

 this rubber tube is attached a delivery tube which passes through another 

 rubber stopper placed in one end of a glass cylinder 1\ inches in diameter and 

 4 inches long, to about half its length. This forms a protective beU for tha 

 delivery tube similar to that used In filling vaccine or antitoxin ampules. The 



5095— 24t 2 



Fig 6. — Apparatus to replace pipet- 

 ting of egg-yolk suspension 



