154 MASS. EXPERIMENT STATION BULLETIN 167. 



(d) Indicators: (1) Phenolphthalein — dissolve 0.5 gram of phenolphthalein in 



500 c. c. alcohol and make it to a liter. (2) Litmus paper — a narrow strip 

 of Kahlbaum litmus paper. 



(e) Reagents: (1) Neutral formaldehyde — 50 c. c. of 40 per cent, commercial 



formaldehyde with 1 c. c. of the phenolphthalein is neutralized to rose color. 



(2) Bismark-brown, tropseolin and 00 and methyl violet — these reagents 



are prepared by dissolving 0.2 gram of the substance in one liter of water. 

 (/) Water; carbon dioxide and ammonia-free distilled water. This is prepared 



with usual precautions. 

 (g) Test solution. This is prepared with equal portions of m/15 KH._,PO< and 



m/15 Na,HPO^, and is perfectly neutral. 



Determination. 

 I. Right after the preparation of solution (o) : — 



Transfer 20 c. c. of the water (g) into a 50 c. c. flask and the same amount of the 

 solution (a) into another. Adjust the color of the water to the latter by adding about 

 2 drops of bismark-brown in this case, and use any one of the mixtures of tropseolin 

 and 00 or methyl violet to obtain a comparative coloration; neutralize it to 

 litmus, controlling the neutralization point by means of the test solution. Add 

 10 c. c. of the neutral formaldehyde. Titrate it to a dark red color with the stand- 

 ard alkali, and titrate it back to strong red color with the standard acids. Re- 

 cord the number of c. c. of standard acid and alkali used. Treat the flask with 

 the solution (a) in the same manner, except adjusting the coloration. After titrat- 

 ing it to dark red color titrate it back to its former color with (a). 



Results. 



Sample 

 Control (water), (peptone solution). 

 Number of c. c. used n/5NaOH 1.20 c. c. 3.35 c. c. 



Number of c. c. used n/5 HCl LIO ^33 



0.10 n/5 NaOH 3.02 



.10 (control) 



Since 



2.92 n/5 naOH 



1 c. c. n/5 NaOH =2.8 milligrams of amino nitrogen, 



2.92 X2.8 =8.176 milligrams amino nitrogen in 20 c. c. of the solution. This 

 is designated as Y in the above equation. 



II. After fifteen hours incubation at 40° C. : — 



The rest of the solution (a) was placed in incubator at 40° C. right after 20 c. c. 

 had been taken out for the above determination, and was kept there for sixteen 

 hours. At the end of that period 20 c. c. of the solution was drawn out for titra- 

 tion, and treated in the same manner. 



Results. 



Control. Sample. 



Number of c. c. NaOH 1.8 8.15 



Number of c. c. HCl . 0.1 .10 



1.7 n/5 NaOH 8.05 



1.70 (control) 



6.35 c. c. n/5 NaOH 

 6.35X2.8 = 17.780 milligrams of amino nitrogen. This is indicated as X. 

 X-Y=Z. 

 17.780—8.176= 9.604 milligrams of amino nitrogen was produced by proteolysis 

 during the sixteen hours' incubation. 



