GLYCOGEN 249 



and, when completely dissolved, add 60 ml. cone, hydrochloric 

 acid, with stirring. Store in a glass-stoppered dark bottle in a cool 

 place, and discard when the reagent begins to acquire a bluish 

 color. Add a few crystals of sodium hyposulfite to improve the 

 keeping quality. 

 Standard Glycogen Solutions. Prepare from glycogen reprecipi- 

 tated from aqueous soln. by alcohol. 



PROCEDURE 



1. To the glycogen which has been centrifuged down and washed, 

 add water to make a solution of suitable concentration — this will 

 have to be determined for the particular case. 



2. Add 5 vol. diphenylamine reagent to 2 vol. glycogen soln. 

 Mix and centrifuge to eliminate any insoluble material. Avoid get- 

 ting the reagent on the sides of the tube where it will evaporate 

 during the heating and leave a film that will not go into solution. 



3. Heat the tubes exactly 40 min. in boiling water and plunge 

 into cold water for at least 3 min. 



4. Run glycogen standards parallel with the unknowns. 



5. Read color intensities (filter No. 635 with the Evelyn colorim- 

 eter) within 1 hr. after removal from the bath, using a water blank, 

 and obtain the values of the unknowns from a calibration curve de- 

 rived from the standards. 



Van Wagtendonk, Simonsen, and Hackett Method for Glycogen 



SPECIAL REAGENTS 



Lugol Solution. Dissolve 1 g. iodine in a soln. containing 2 g. potas- 

 sium iodide in 20 ml. water. Store in a well-stoppered dark bottle. 



Standard Glycogen Solution. Dissolve 25 mg. glycogen {Eastman 

 Kodak Co., White Label) in 25 ml. 35% potassium hydroxide. 

 (See note below.) 



PROCEDURE 



1. To a given vol. glycogen soln., diluted to an appropriate de- 

 gree as determined in advance, add 0.01 vol. Lugol soln., and mix 

 well. 



2. Read the color at once (filter No. 54 with the Klett-Summer- 

 son colorimeter) using a blank consisting of the Lugol soln. diluted 

 100 times with water. 



