292 PEACTICAL PHYSIOLOGY 



The flask is closed with a cork, having a wide glass tube about five feet long 

 passing through it to serve as a reflux condenser, and it is then immersed in 

 a boiling water bath and left there for three hours, with occasional shaking. 

 (Less time than this suffices to completely destroy the protein of liver.) On 

 removal from the water bath, the contents of the flask are allowed to cool, 

 and are then thoroughly shaken, with 200 c.c. water (thus bringing the 

 percentage of KOH to fifteen). 800 c.c. of ordinary (96%) alcohol are then 

 added to the solution, the mixture shaken and allowed to stand for several 

 hours (preferably overnight). 



The more or less white precipitate of glycogen will by this time have settled 

 down, so that the supernatant reddish fluid can with care be poured off into a 

 beaker, after which it is filtered through a filter paper of suitable size, so as 

 to collect on the filter any particles of glycogen which the decanted fluid may 

 contain. The precipitate of glycogen is now thoroughly shaken with about 

 ten times its volume of 66% alcohol (about 700 c.c. alcohol and 300 c.c. water) 

 containing 1 c.c. per litre of a saturated solution of NaCl. This washing fluid 

 removes many of the impurities which adhere to the glycogen. 



After settling, the wash fluid is decanted into the same beaker as was 

 employed for receiving the original supernatant fluid, and filtered through the 

 same filter. This process is repeated at least once again, after which the 

 precipitate is shaken with ordinary alcohol (about 10 times its volume), and 

 the suspension thrown on to the same filter paper as used above. 



When the alcohol has all drained off, the precipitate is washed on the filter 

 paper with ether. All the washed glycogen has thus been collected on the 

 filter paper and must now be dissolved, for which purpose the filter is filled up 

 with boiling water, and the solution of glycogen allowed to filter through into 

 a clean Erlenmeyer flask. When the first added water has completely drained 

 through the filter, the filter is filled up with boiling water a second and a 

 third time. It is essential to allow the filter to drain completely before adding 

 more water. To be certain that all the glycogen has been dissolved, some of 

 the final filtrate should be tested with alcohol for glycogen. 



The resulting opalescent solution can now be employed either for the 

 preparation of pure glycogen or for its quantitative estimation. For the 

 former purpose the glycogen is precipitated by alcohol ; for the latter purpose 

 the glycogen solution is made up to a litre in volume, and of this 200 c.c. are 

 taken, mixed with 10 c.c. HC1 (cone.) (i.e. 5 c.c. HC1 to a 100 c.c. of glycogen 

 solution), and heated in a flask on the water bath for three hours. 1 Complete 

 hydrolysis of the glycogen is certain within this time, although the resulting 

 solution often contains a flocculent precipitate which is probably of some 

 protein body. The solution, after cooling, is neutralised with 20% KOH and 

 filtered into a 250 c.c. measuring flask through a small filter' (10 cm.) paper. 



The flask used for inversion is rinsed three times with distilled water, the 

 washings being each time poured on to the filter and added to the contents of 

 the measuring flask. In this way the volume of the dextrose solution is 

 brought exactly to 250 c.c. 



Where only 10 or 20 gr. of liver were originally employed, the above 

 measurements must of course be altered, it being usually best to take all of 



1 If the glycogen be reprecipitated and redissolved in a known volume of water 

 the resulting solution can be examined in the polarimeter and its glycogen con- 

 tent calculated according to the formula on p. 284. (a) D = 196 '63. 



