5U6 CYTOCHEMICAL TESTS ON EMBRYOS 



without introducing any air. Betain finger bowls "B" and "C" for the duration of 

 the experiment to determine the developmental changes in the eggs. During the 

 suhsequent 5 hour period there should he 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 temperature 

 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 



30 that there Is some clear fluid at the top. 



d. Carefully remove the stopper and introduce O.h cc. of HCl Just below the sur- 



face of fluid in each of the calibrated bottles. Stopper (without air) £ind 

 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 con- 

 tains 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 U or 5 drops of the starch solution to give a distinct blue color. 



Continue the titration until the blue color Just disappears. Each cc. 

 of N/100 sodium thiosulphate corresponds to 0.0025 mllllmoles of Og. 

 The relative values of solutions A, B, and C should be determined, 

 and since the number of eggs In B and C is known, the oxygen consump- 

 tion per fertilized and unfertilized egg can be determined. 



This procedure seems at first a bit crude and yet very accurate results can be ob- 

 tained. The reactions in the procedure are as follows: 



CI2 



Therefore, for each molecule of Og present, two molecules of Ig are liberated. In 

 titrating, the free iodine reacts with the sodium thiosulphate to form tetrathionate and 

 sodium Iodide as follows: 2Na2S20j ■>- Ig = NagSi^Og + 2 Nal, which are both colorless, 

 allowing the end point to be determined by the disappearance of the blue color that forms 

 when iodine reacts with the starch indicator. 



Once this procedure is stabilized, and the student has achieved reproducible results, 

 it is suggested that the following additional tests be made: 



a. Oxygen consumption at various stages of development, particularly during a 



series of early cleavages and at gastmlatlon. 



b. Effect of cyanide, dlnitrophenol, iodoacetate, colchicine. 



c. Effect of low and of high pH. 



d. Effect of temperature over definite time Interval. 



e. Oxygen consumption of artificially activated eggs (parthenogenetic) and of 



androgenetlc eggs. 



