1 62 BACTEEIOLOGICAL METHODS 



attempted if less than loo grams are available, and an amount greater than 500 grams 

 is never taken. 



Extraction with Alcohol. — The sample is melted and poured into a flat-bottomed 

 flask of i-liter capacity which is closed with a rubber stopper perforated with three 

 holes. This flask is set on the top of the steam bath and connected to a reflux 

 condenser and to a 700 cc. round-bottomed flask containing 500 cc. of 95 per cent, 

 alcohol. A glass tube which is adjusted so that its lower end is about one-fourth of 

 an inch above the surface of the fat and whose upper end is bent at a right angle and 

 closed by means of a short piece of rubber tubing and a pinchcock fills the third hole in 

 the stopper. The distilling flask is set down in the steam so that the alcohol boUs 

 briskly. The outlet tube reaches down to the bottom of the flask containing the 

 sample so that the alcohol vapor as it distills over bubbles up through the fat and 

 keeps it in a state of vigorous agitation. The alcohol vapor is condensed in the 

 reflux condenser and returned to the flask containing the fat. The distillation is con- 

 tinued until all of the alcohol has collected in the flask containing the fat. The dis- 

 tilling flask is now disconnected. The alcohol in the flask immediately ceases to boil 

 and soon separates from the fat. The empty distilling flask is next connected to the 

 bent tube by a piece of glass tubing of sufiicient length, the pinchcock opened, and the 

 alcohol layer siphoned off into the distilling flask. This is then connected as before 

 and the distillation continued until the alcohol has again collected in the first flask. 

 It is then siphoned into the distilling flask as before, and a third extraction made. 

 After the third extraction the alcohol layer is again siphoned off into the distilling 

 flask and the fat is discarded. The alcohol now contains practically all of the choles- 

 terol and phytosterol originally present in the fat. 



Saponification and Extraction with Ether. — The alcohol in the distilling flask is 

 next concentrated by boiling to about 250 cc, and 20 cc. of a concentrated potassium- 

 hydrate solution (100 grams KOH dissolved in 100 cc. water) added to the boiling 

 liquid. It is boiled for 10 min. to insure complete saponification of all the fat and 

 is then removed from the steam bath and allowed to cool almost to room temperature. 

 After it has cooled sufSciently it is poured into a large separatory funnel containing 

 500 cc. of warm ether and shaken to insure thorough mLxing. The mixture may be 

 clear, but is more often opalescent. There is now poured in 500 cc. of distilled water, 

 and the funnel is rotated gently. Shaking must be avoided, as it leads to the forma- 

 tion of extremely stubborn emulsions, but the water should be mixed with the alcohol- 

 ether-soap solution. Separation takes place at once and is clear and sharp. The 

 soap solution is drawn off and the ether layer washed with 300 cc. of distilled water, 

 shaking being still avoided. After this washing it is washed repeatedly with small 

 quantities of water until all soap is removed. The ether layer is then transferred to 

 a flask and the ether distilled off. Distillation is stopped when the contents of the 

 flask have been reduced to about 25 cc, and the concentrated ether solution contain- 

 ing the cholesterol, phytosterol, and all other unsaponifiable matter is transferred to a 

 tall so cc. beaker. The evaporation is continued until all ether is driven off and the 

 residue is perfectly dry. If desired, a tared beaker may be used and the weight of the 

 unsaponifiable matter determined at this point. 



