Apr. 12,1924 
Development of American Foulbrood 
155 
acid was indicated in the tubes to which 1 per cent dextrose had been added. 
The maximum production of acid, however, required approximately 48 hours 
or more, the fermentation of the sugar apparently being relatively slow. As 
has been stated, however, the reaction of diseased material in various stages of 
decomposition and drying down is never found to reach a hydrogen-ion concen¬ 
tration of more than P H =6.6, and usually averages Ph =6.8. 
PROTEIN DECOMPOSITION 
It is known that certain organisms have the ability to break down protein 
material under proper conditions, with the production of amino acids and alka¬ 
line decomposition substances, which latter tend to neutralize any acid produced 
from fermentation of sugar. If it can be shown that Bacillus larvae has this 
ability, it will explain the fact that the remains of larvae dead from American foul- 
brood do not show a greater acid reaction resulting from the fermentation of the 
sugar of the intestinal contents. A series of experiments was devised to demon¬ 
strate whether such is the case with Bacillus larvae. 
The prepupa at the age attacked by American foulbrood contains nitrogenous 
substances as shown by the Kjeldahl nitrogen determination equivalent to 1.45 
per cent nitrogen (4#). The source of this nitrogen is mainly albuminous mate¬ 
rial, one of the constituents of the larval fat bqdy. Its exact composition has 
not been determined, but without doubt it is complex in nature. There are 
certain color reaction tests by means of which the constitution of this nitrogenous 
material may be indicated. 
A delicate test for the presence of coagulable protein is that of Heller (82, p. 
1067). A suspension of healthy prepupse in water, treated by pouring about 
4 cc. of concentrateed nitric acid down the side of the.inclined test tube, causes a 
white ring to form at the junction of the two liquids. Decomposed ropy material 
tested in this way gives no indication of such a ring, indicating that the complex 
protein has disappeared. 
One of the most characteristic reactions for complex protein is the biuret test 
(82, p. 915). If some healthy prepupse are suspended in a few cubic centimeters 
of 10 per cent sodium hydroxid and are treated with a few drops of a 0.5 per 
cent copper sulphate solution, a distinct pinkish-violet color is formed, again 
indicating the presence of complex protein material. Decomposed ropy material 
tested in this way gives no indication of this color, again indicating the complete 
disappearance of the complex protein. 
There is also the xantho-proteic reaction (32 p. 916), which is given both by 
solid and by dissolved protein, and indicates the presence of the amino-acids, 
tryptophan, tyrosin, or phenylalanin in the protein molecule, or in solution. 
Tryptophan gives the reaction most intensely. Both healthy prepupse and 
ropy material, boiled with concentrated nitric acid, produce a lemon-yellow 
color which on cooling and neutralizing with sodium hydroxid changes to an 
orange, denoting a positive reaction. 
An even more delicate reaction for protein is that with Millon’s solution (32, 
p. 916). A few cubic centimeters of a suspension of healthy prepupse, treated 
with a few drops of Millon’s reagent and boiled, cause a brick-red precipitate to 
form, leaving the liquid practically clear. A solution of decomposed ropy 
material, treated in the same way with Millon’s reagent and boiled, causes a 
somewhat similar reddish precipitate, but the solution is also distinctly colored 
similarly, indicating that the protein has been changed in some way, part at 
least being soluble in water. Tyrosin is the only amino acid in protein that 
gives this reaction. 
