342 CYTOCHEMICAL TESTS ON EMBRYOS 



4. Prepare 3 finger bowls, mark them A, B, and C, and into each introduce exactly 

 10 cc. of Spring Water (Standard Solution) or any medium in which frog's eggs 

 are normally inseminated. Into finger bowl "C" only, introduce and macerate 

 one pair of adult frog testes. Allow these bowls to stand for 10 minutes. 



5. By stripping, remove a few eggs from an ovulating female and discard them. 

 Then strip about 200 eggs into finger bowl "B" (no sperm) and "C" (sperm sus- 

 pension), and see that the eggs are completely covered with the medium. Avoid 

 transfer of any sperm from bowl "C" to bowl "B". Allow them to stand for 2 

 minutes, then add to each of the three finger bowls exactly 250 cc. of the same 

 medium (i. e. , Spring Water). This will provide a total volume in each bowl of 

 260 cc. , plus eggs in two of the bowls. 



6. Using a clean section lifter, gently separate the eggs from the bottom of bowl "B" 

 and then bowl "C" after 6 minutes. (Avoid possible insemination of eggs in bowl 

 "B" by washing off and drying the section lifter each time it is used. ) Allow the 

 jelly on the eggs to expand another 5 minutes. 



7. Fill Erlenmeyer flask "A" with the medium from finger bowl "A" to overflowing, 

 and add the cork stopper without introducing any air. Fill flask "B" with the 

 supernatant fluid from finger bowl "B", then carefully count out 100 eggs from 

 bowl "B" and add them to the flask, then cork without introducing any air. Sim- 

 ilarly fill Erlenmeyer flask "C" with the supernatant fluid from finger bowl "C", 

 count out 100 eggs from finger bowl "C" and add them to this flask, insert the 

 cork without introducing any air. Retain finger bowls "B" and "C" for the dura- 

 tion of the experiment to determine the developmental changes in the eggs. Dur- 

 ing the subsequent 3 hour period there should be variable oxygen consumption in 

 "B" and "C" as compared with "A" in which there should be no oxygen change. 



8. Make the stoppers in the Erlenmeyer flasks secure by placing a heavy rubber 

 band lengthwise around the entire flask and stopper. Note the time and tempera- 

 ture and place the three flasks on a standard shaker, agitating about 5 to 25 round 

 trips per minute at 2 to 10 inches amplitude. This will facilitate oxygen con- 

 sumption. Agitate for 3 hours, or until after the control eggs (in finger bowl "C" 

 have completed the first cleavage). 



9. Determination of the oxygen consumption: 



a. Transfer with minimum agitation and exposure to air, supernatant medium 

 from each of the flasks to 50 cc. calibrated bottles, marked A. B. and C. 



b. Using 1 cc. measured pipettes, transfer 0. 2 cc. of the Manganese chloride 

 solution (listed above) and 0. 2 cc. of the KI-NaOH solution into each of the 

 calibration bottles, inserting the tip of the pipette about halfway down the 

 bottle. Avoid air bubbles in replacing the glass stopper. 



c. Agitate the bottles for several minutes, then allow the precipitate to settle so 

 that there is some clear fluid at the top. 



d. Carefully remove the stopper and introduce 0. 4 cc. of HCl just below the sur- 

 face of fluid in each of the calibrated bottles. Stopper (without air) and shake 

 until the precipitate is dissolved. 



e. Transfer the fluid from each of the bottles to similarly marked (clean) 125 cc. 

 capacity Erlenmeyer flasks for titration. Solution "C" at least contains some 

 free iodine which must be titrated soon in order to avoid loss due to volatility. 



f. Titration procedure for each sample (A, B, and C): 



(1) Add sufficient sodium thiosulphate to cause most of the yellow (iodine) 

 color to disappear. 



(2) Add 4 or 5 drops of the starch solution to give a distinct blue color. Con- 

 tinue the titration until the blue color just disappears. Each cc. of N/100 

 sodium thiosulphate corresponds to 0. 0025 millimoles of Oo- The rela- 

 tive values of solutions A, B, and C should be determined, and since the 

 number of eggs in B and C is known, the oxygen consumption per fertilized 

 and unfertilized egg can be deterrnined. 



