Apr. 12, 1024 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 Ph =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 larvje 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 Americafi foulbrood contains nitrogenous 

 substances as shown by the Kjeldahl nitrogen determination equivalent to 1.45 

 per cent nitrogen (4S) . The source of this nitrogen is mainly albuminous mate- 

 rial, one of the constituents of the larval fat body. 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 (3S, p. 

 1067). A suspension of healthy prepupse in water, treated by pouring about 

 4 cc. of concentrated 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 

 (SS, 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 (SS 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 (S2, 

 p. 916). A few cubic centimeters of a suspension of healthy prepupae, 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 

 simDarly, 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. 



