230 CUVETTE COLORIMETRY 



2. Digest at 38° for 30 min., and then place in ice water and add 

 0.5 ml. 0.02 A^ sodium hydroxide with sufficient force to mix the solns. 



3. Transfer to a cuvette and measure the color intensity using 

 light at 400-420 m/^. 



4. Add 2-4 ix\. cone, hydrochloric acid and take a second colori- 

 metric reading. The difference in the optical densities gives the 

 corrected density of the unknown. 



5. Run standards and blanks by treating 5 /xl. vol. of the 

 standards and distilled water in the same manner as the serum. 

 Construct a standard curve from the corrected optical densities. If 

 the same pipettes are used for both the standards and unknowns, the 

 exact pipette vol. need not be known. 



Bessey et al. employ a "millimole unit" which is defined as the 

 phosphatase activity which will liberate 1 milf nitrophenol/liter/hr. 

 1 millimole unit is approximately equal to 1.8 Bodansky units. For 

 sera weaker in phosphatase, such as those from adults, the vol. serum 

 and reagent may be doubled without increasing the vol. alkali; this 

 will yield a color of nearly double the intensity. 



NITROGEN AND AMMONIA 



General 



As in the more macro methods, the determination of nitrogen in 

 histochemical investigations involves conversion of the total nitro- 

 gen to ammonium sulfate by digestion. The digest may be Ness- 

 lerized directly, or it may be alkalized and the liberated ammonia 

 absorbed in acid and measured either colorimetrically or titrimetri- 

 cally. However, the determination of the very small quantities in- 

 volved requires unique treatment. Since the preliminary procedures 

 are common to both the colorimetric and titrimetric methods, it 

 will perhaps contribute to greater integration and clarity if the 

 chief developments in both forms of analysis are considered together 

 in chronological order at this point. 



At about the same time, Linderstr0m-Lang and Holter ( 1933b) 

 and Conway and Byrne (1933) reported methods for the micro- 

 estimation of ammonia which depended on the transfer, by diffu- 

 sion, of the ammonia from an alkalized solution to one of standard 

 acid followed by titration. Linderstr0m-Lang and Holter employed a 



