20 CHEMICAL COMPONENTS OF CELLS (1) 



Exercise III 



molecules inside the sac, freeing them from all 

 contamination with small molecules. Dialysis 

 therefore is one of the most useful procedures, 

 not only for distinguishing large from small 

 molecules, but for purifying large molecules. 



Next week we shall work with an ultimate 

 fractionation method, paper chromatography. 



This can separate individual molecular species, 

 one from another, even when they differ only 

 slightly in structure. It requires also extremely 

 small amounts of material. Its sensitivity and 

 accuracy of resolution make it one of the most 

 useful procedures now available for biochemical 

 analysis. 



EQUIPMENT 



Per student 



4 small test tubes with stoppers 



8 test tubes (16 X 150 mm) 



4 50-ml plastic centrifuge tubes 



3 stirring rods 



400-ml (or 250-ml) beaker 



bunsen burner 



test-tube rack 



6" medicine dropper 



2 ft of f" dialysis tubing 



Per 2 students 



mortar and pestle 

 wax marking pencil 



Per 8 students 



matches 



5% trichloracetic acid (250 ml) 



2 vials of indicator paper 



2 dropping bottles of distilled water (with glass 



stopper) 



2 dropping bottles of 1-A' HCl (with glass stopper) 



2 dropping bottles of I-TV H2SO4 (with glass stopper) 



2 dropping bottles of l-A' NaOH (with rubber 



stopper) 



2 dropping bottles of 1-A' saturated Ba(OH)2 (with 



rubber stopper) 



dropping bottle of 0.5-M glucose (with rubber 



stopper) 



10% NaCl (200 ml) 



95% ethanol (500 ml) 



Per 30 students 



2 clinical centrifuges 



pair of scissors 



2 ink marking pencils ("Magic Markers") 



1 lb of purified sea sand 



phosphate butfer (0.1 M, pH 7.0) (200 ml) 



0.1% pangestin in phosphate buffer (0.1 M, pH 7.0) 



(200 ml) 



thymol (^ lb) 



Per laboratory 



molecular models (can be purchased from E. H. 



Sargent & Co., Chicago, III.) 



water baths at 100°C 



refrigeration space for 2 test tubes per student 



yeast preparations 



Commercial brewer's yeast contains very little gly- 

 cogen and is therefore unsatisfactory for this experi- 

 ment. Baker's yeast does contain glycogen, but the 

 cakes in which it is supplied are held together with 

 starch binder. The starch is in the form of grains 

 which are larger and denser than the yeast cells, so 

 that they are easily removed by sedimentation. 

 Suspend the yeast in water or dilute salt solution, 

 and centrifuge very briefly, for about 15 sec, at 500 

 to 1000 rpm; or let the suspension stand until the 

 starch grains settle. The upper layer of the suspen- 

 sion can then be decanted and centrifuged to pack 

 the yeast cells. A few repetitions of this procedure 

 should be enough to remove all starch. This point 

 is easily demonstrated by staining one drop on a 

 slide with iodine-KI (Lugol's) solution (see page 24). 

 Starch, of course, stains blue or purple, whereas the 

 yeast glycogen stains reddish brown, entirely within 

 the cells. 



Test tubes to be put into the water bath can be 

 labeled with masking tape. 



