156 
Journal of Agricultural Research 
Vol. XXVIII, No. 2 
Since tryptophan is probably one of the principal constituents of the protein 
molecule in the healthy prepupa as well as in solution in diseased material, 
certain tests were made to determine its presence, because this amino-acid is 
easily utilizable by bacteria and gives decomposition products indicating the 
nature of bacterial action. The following tests are specific tryptophan reactions: 
Adamkiewicz reaction {32, p. 917 ).—A suspension of healthy prepupsc or of 
diseased material in glacial acetic acid, treated by pouring concentrated sul¬ 
phuric acid down the side of the inclined tube, causes a violet ring to form at 
the junction of the two liquids, indicating the presence of tryptophan, either 
as part of the complex molecule or in solution. 
•Rhodes reaction (41 )-—To a suspension of healthy prepupae or of diseased 
material in water, a few drops of a weak solution of dimethyaminobenzaldehyde 
is mixed and concentrated sulphuric acid poured down the side bf the inclined 
tube. This produces a violet ring at the junction of the two liquids which, if 
shaken, produces a reddish violet coloration in the mixture. 
PROTEIN DECOMPOSITION PRODUCTS 
It is therefore evident that the composition of the nitrogenous material in the 
healthy prepupae is more or less complex but that certain amino-acids are avail¬ 
able for bacterial metabolism, or are produced as a result of bacterial action. 
In the decomposition of nitrogenous material, however, certain bacteria have 
the power of breaking down these amino-acids, such as tryptophan, to more 
simple compounds, some of them alkaline in nature, and often more or less 
foul smelling, or even to break them up into ammonia, the final product of 
nitrogenous decomposition. Indol is one of the products of such action of 
bacteria on material containing tryptophan. Its determination is largely used 
in the characterization of various organisms {36). Two indol tests were used, 
Ehrlich’s aldehyde test {19) and the vanillin test {19), using for both suspen¬ 
sions of diseased material as well as cultures. Test of suspensions of diseased 
material gave positive results for the presence of indol, both with the Ehrlich 
method and even more definitely with vanillin. For testing in pure culture $ 
broth consisting of 2 per cent peptone, 10 per cent yeast extract, and a few 
cubic centimeters of egg-yolk suspension was inoculated, incubating at 37° C. 
for about one week. Growth took place in this broth sufficiently to give a slight 
positive pink color with the Ehrlich aldehyde test, increasing on standing, and 
a much more positive result with the vanillin test. 
AMMONIA PRODUCTION 
Test of a suspension of diseased material as well as some of the above culture 
broth with Nessler’s reagent {32, p. 1084) for presence of ammonia gave indica¬ 
tions, from the resulting slight production of characteristic yellowish color, that 
the decomposition had passed even to the ammonia stage. A more delicate 
qualitative test was devised, using the modification of the microchemical method 
of Folin and McCallum {32, p . 1093) for the determination of urinary ammonia 
as described by Steel {43). To 25 cc. of a suspension of diseased material, or to 
broth culture similar to the above, 1 gram of sodium hydroxid and 15 grams of 
sodium chlorid are added and ammonia-free air bubbled through into 20 cc. of 
an approximately N/20 sulphuric acid, to which 10 drops of the indicator thymol 
blue are added. This showed the sulphuric acid solution to have a primary 
hydrogen-ion concentration of about Ph=2. After bubbling air through for an 
hour or more, in the case of the decomposed ropy material, sufficient ammonia 
had been carried over to neutralize part of the acid and change the hydrogen-ion 
concentration reaction from P H =2 to P H =2.8 or 3. Also one culture out of 
