GLYCOGEN 299 



Heatley Method for Total Glycogen 



SPECIAL REAGENTS 



All of the reagents used for the determination of reducing sugars 



(page 296) are required, in addition to: 



30% Potassium Hydroxide. 



Absolute Alcohol. 



0.6 N Hydrochloric Acid. 



0.1 N Hydrochloric Acid. 



0.05% Thymol Blue. 



1 N Sodium Hydroxide. 



Paraffin. 



PROCEDURE 



1. Place the tissue sample, which may be fixed in absolute 

 alcohol to stop enzyme action, in about 35 ju,l. 30% potassium hy- 

 droxide in a reaction tube. 



2. Cap the tube and heat in a steam bath for 20 min. 



3. With the pipette used for the alkali, add 35 /xl. water and then 

 105 ju,l. absolute alcohol. 



4. Mix well with the aid of a platinum wire. 



5. Immerse the tube in a steam bath for only a few sec. to 

 flocculate the glycogen without having the contents boil out. 



6. Centrifuge at 3500 R.P.M. for 15 min. and pipette off and 

 discard the clear supernatant. 



7. Wash the precipitate with about 100 /A. absolute alcohol; 

 centrifuge and again discard the supernatant. 



8. Repeat the washing process once more and remove the last 

 traces of alcohol by placing the tube in a steam bath for a few min. 



9. With about 35 ^1. 0.6 N hydrochloric acid wash down the 

 deposit on the upper walls of the tube. 



10. Add a 20-30 mg. piece of paraffin and place the tube in the 

 steam bath for 2.5 hr. 



11. While the tube is still hot, invert and rotate it to spread an 

 even film of paraffin over the upper walls of the tube. 



12. Add a "flea" and use it to break through the thin film of 

 paraffin on the surface of the liquid. 



