]^ 5 6 Journal of Agricultural Research voi. 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: 



AdamUewicz reaction {SS, p. 917).— A suspension of healthy prepups 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 prepup* 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 of 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 prepupse 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 a 

 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 sufliciently 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 (SS, 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 FoMn and McCaUura (3S, p. 1093) for the determination of urinary ammonia 

 as described by Steel {42) . 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 ohlorid 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 Ph=2 to Ph=2.8 or 3. Also one culture out of 



