Exercise XVII 



PERMEABILITY AND ACTIVE TRANSPORT 89 



room temperature for 60 to 90 min (the longer, 

 the better). Arrange for air to bubble through 

 the solution during this time. This should be 

 done gently so as not to mar the intestinal sac. 

 Note the exact length of time that each sac is 

 incubated. 



Prepare the other sections of intestine exactly 

 as above. One (or two) sacs should be incubated 

 with aeration, as above, to allow metabolic 

 processes to continue normally. The other sec- 

 tions of intestine are to have their metabolism 

 stopped by adding an inhibitor of the respiratory 

 production of ATP, dinitrophenol (DNP), and/ 

 or by stopping the aeration. To the tubes con- 

 taining the sacs whose metabolism is to be in- 

 hibited, either add 1 drop of DNP solution, or 

 stop aerating, or do both. 



After the incubation is complete (60 to 90 

 min), remove the sac from the tube, blot it dry 

 on a paper towel, and carefully cut open the end 

 so as to collect the liquid inside in a small test 

 tube or in the depression of a spot plate. This 

 is a tricky operation, so use extreme care or you 

 may lose the results of the experiment ! Save the 

 solution also in which the sac has been incubated. 



Glucose analysis 



Three drops of the solution to be tested are 

 placed in a test tube, and 2 ml of Benedict's solu- 



tion are added. Mix, and place in a boiling water 

 bath for 5 min. The approximate concentration 

 of glucose can be determined by comparison 

 with known glucose standards run similarly. The 

 following analyses should be carried out: 



(1) solutions inside each of the intestinal sacs 

 (4 tubes), 



(2) solutions outside each of the intestinal sacs 

 (4 tubes), 



(3) the original Krebs phosphate Ringer solu- 

 tion with 20 mM glucose (1 tube), 



(4) tubes containing 0, 1,2, and 3 drops of the 

 standard 50 mM glucose solution (4 tubes). 



Be sure each tube is carefully labeled in such a 

 way that the boiling water will not obliterate the 

 label! Masking tape high up on the tube is 

 convenient for this. 



Benedict's reaction depends upon the reduc- 

 tion of blue cupric (Cu++) to red cuprous (Cu+) 

 ions by the aldehyde group of the sugar. CU2O 

 is the red product formed. The variation in color 

 is a measure of the amount of glucose originally 

 present. A clear blue solution indicates none; a 

 deep red indicates a high concentration. (Re- 

 call a previous use of the Benedict test in Exer- 

 cise IV, p. 23.) 



Report your results as the approximate ratio 

 of glucose inside the sac to that outside. 



EQUIPMENT 



Per 2 students 



hamster (killed immediately before use, preferably 

 without anesthesia, or by injecting a fatal dose of 

 nembutal) 



dissecting instruments 

 2 petri dishes 



6 or 8 droppers (some or all should be long) 

 sewing cotton 



glass rod (1.5 mm X 15 to 20 cm) 

 2 pipets (2 ml and 5 ml of solution have to be de- 

 livered) 

 18 test tubes 

 spot plate 



paper towels 



400-ml (or 250-ml) beaker for boiling water bath 



bunsen burner 



ring stand 



metal gauze 



Per laboratory 



Benedict solution; prepare as directed in Exercise IV 

 Krebs phosphate Ringer solution 

 Stock solutions : 



(1) 0.9% NaCl (0.154 M) 



(2) 1.15% KCl (0.154 M) 



