64 PRELIMINARY COLD STORAGE STUDIES. 



eral times with hot water. The carbon insoluble in water, and the 

 small ashless filter paper required for the extraction, were returned 

 to the crucible and the ignition repeated, using ammonium nitrate, 

 if necessary, to oxidize the last particles of carbon. To the grayish 

 mass the aqueous extract was then added, dried on a water bath, and 

 ignited in a muffle until the ash was clean. 



NITROGENOUS COXSTITUEXTS. 



The study of the nitrogenous constituents was carried out as fol- 

 lows: The total nitrogen of the two kinds of muscle was determined 

 by the Gunning modification of the Kjeldahl method. For the study 

 of the water soluble nitrogen a portion of the finely divided red or 

 white meat, weighing 60 grains, was put into a tall slender bottle of 

 500 cc capacity, constructed to fit a centrifuge capable of carrying 1 

 liter of material; 300 cc of water were added and the flask gently 

 shaken for 15 minutes. The movement was merely sufficient to 

 keep the particles of meat in motion and the composition of the 

 extract homogeneous. Forcible shaking caused an emulsion to form, 

 as did the very fine grinding of the tissue. After shaking for the 

 required length of time the flask was rotated in an electric centrifuge 

 for 20 minutes, which caused the heavier particles to settle in a com- 

 pact mass and permitted the decantation of the supernatant liquid, 

 which was then filtered through paper. The extraction, as outlined, 

 was repeated with portions of 300 cc of water until the filtrate was 

 practically proteid free, as indicated by the biuret reaction. The 

 attainment of this result required ordinarily a volume of 1,500 cc to 

 2,500 cc. To guard against bacterial decomposition thymol was 

 added both to the flesh and to the extract, and to inhibit, so far as 

 possible, the action of the naturally occurring enzymes of the meat, 

 the solution and the meat itself were kept cold, ice being used when 

 necessary. 



The extraction of the white meat was a much simpler operation 

 than the extraction of the dark meat. The latter did not settle as 

 compactly in centrifuging, filtered more slowly, and persisted in show- 

 ing a distinct biuret reaction for a considerable time after the white 

 meat was free of water-soluble proteid. In fact, certain fowls, more 

 especially those which have been in cold storage for long periods of 

 time, never showed a red meat entirely free from water-soluble nitro- 

 gen. In such cases the question of the error due to long manipula- 

 tion and enzyme action, involving a rise in the actual quantity, had 

 to be considered. It was found by experiment that after long extrac- 

 tion of such tissue a point was reached when a very faint biuret reac- 

 tion, which did not apparently diminish, persisted indefinitely. Such 

 extractions were halted after about 26 hours, it being believed that a 

 greater error would result in the gain of what had been originally 



