480 Journal of Agricultural Research voi. xiii. no. 10 



The mixtures were contained in wide-mouthed loo-c. c. bottles, and 

 were closed with rubber stoppers tied firmly in place. The mixtures 

 were always saturated with chloroform and kept in an incubator room 

 at 37.5° C. Culture tests made from time to time showed that the mix- 

 tures were sterile. 



ANALYTIC DATA • 



At suitable intervals, usually the day before or after the guinea pigs 

 were inoculated, chemical analyses of the mixtures were made for the 

 purpose of ascertaining the extent of protein digestion. The determi- 

 nations made were coagulable protein and amino nitrogen. Inasmuch 

 as details on the preparation and analyses of digestion mixtures have 

 been published elsewhere (5), they will be omitted here. 



Total coagulable protein.— The protein was precipitated, co- 

 agulated, centrifuged, dried, and weighed in centrifuge tubes (heavy 

 glass bacteriological test tubes), the outer dimensions of which were: 

 Length 95 mm., diameter 17 mm. Eight such tubes, cleaned, dried, 

 and weighed to o.i mgm., were used at a time, two for each of the mix- 

 tures A, B, C, and D. No analyses were made of mixture E. Into each 

 tube 7 c. c. of water and 2 c. c. of digestion mixture or 9 c. c. of water 

 and I c. c. of serum or antitoxin were measured, with Ostwald pipettes 

 of I and 2 c. c. delivery for the latter. Each tube was warmed over a 

 Bunsen burner, and sufficient dilute acetic acid or sodium-hydroxid 

 solution was added to precipitate completely the protein, after which 

 the contents of the tube were brought almost to a boil. The tubes were 

 then centrifuged for 20 to 25 minutes at about 2,400 revolutions per 

 minute. When the flocculation had been properly performed, the 

 coagulated protein packed firmly to the bottom, leaving a water-clear 

 supernatant fluid which may be poured off without appreciable loss of 

 precipitate, even if the tube is inverted. The tubes were then immersed 

 in a sulphuric-acid-potassium-dichromate cleaning mixture, rinsed, and 

 dried on the outside to make certain that there was no adherent dirt. 

 The tubes containing the moist precipitates were dried to constant 

 weight at room temperature in a sulphuric-acid desiccator evacuated to 

 about 10 mm. of mercury. The drying was not difficult; usually a 

 single drying of 24 hours was sufficient, provided the acid was renewed 

 very frequently — that is, after two dryings. In every case the drying 

 was continued until the loss in weight of a precipitate did not exceed a 

 few tenths of a milligram. The difference between the weight of the 

 tube empty and the weight with the precipitate gave the weight of 

 coagulable protein. 



These tubes, which had been numbered with hydrofluoric acid, after 

 being used were cleaned with hot dichromate mixture, washed, dried in 

 the hot-air oven, placed in the desiccator, and weighed, ready for the 

 next determinations. This method for total coagulable protein gives 



