248 CUVETTE COLORIMETRY 



cuvettes, will enable the corresponding refinement. The chief diffi- 

 culty is the erratic behavior of the diphenylamine reagent, which 

 necessitates a new calibration each time the determinations are car- 

 ried out. 



The method of van Wagtendonk et al. ( 1946j is based on the color 

 development which occurs when Lugol solution is added to the gly- 

 cogen isolated from the tissue. A Klett-Summerson photoelectric 

 colorimeter was used with filter No. 54 and the measurements were 

 made in the range 0.05-2 mg. glycogen in a total volume of 5 ml. 

 Morris (1946) has pointed out that the color developed with iodine 

 varies considerably with temperature, and therefore temperature 

 control is required for accurate work. The concentration of iodine 

 is also a factor that affects the color intensity, and the importance 

 was stressed for standardization with glycogen obtained from the 

 same source as the material to be analyzed. Morris is of the opinion 

 that the precautions required to render the iodine method sufficiently 

 accurate constitute a major disadvantage. Nevertheless, if tem- 

 perature is controlled and care is taken to maintain a constant iodine 

 concentration the method should yield reproducible results. If, in 

 addition, the standard solution is prepared from glycogen native to 

 the tissue to be analyzed, sufficient accuracy should be obtained. 



For work on the histochemical level, the tissue may be digested 

 with alkali and the glycogen precipitated with alcohol according to 

 steps 1-7 in Heatley's procedm'e (page 299). The glycogen thus iso- 

 lated can be treated in the manner used by Boettiger or van Wag- 

 tendonk et al. Some preliminary work will be required, no doubt, to 

 obtain the proper color intensities for the apparatus employed. 



Boettiger Method for Glycogen 



SPECIAL REAGENTS 



Diphenyla7nine Reagent. The purest diphenylamine must be used. 

 Oxidized crystals are brownish and impart a blue color to the re- 

 agent. The compound can be purified by dissolving in alcohol at 

 55°, and crystallizing out by cooling and adding a little water. The 

 product is dried and stored in a glass-stoppered dark bottle in a 

 cool place. Glassware used for the reagent must be free of all 

 organic matter, hence it must be cleaned without soap and kept 

 dust-free. Add 100 ml. of glacial acetic acid to 3 g. diphenylamine, 



