CYTOCHEMICAL TESTS ON EMBRYOS 3j^ 



THE WINKLER METHOD OF MEASURING OXYGEN CONSUMPTION* 



Thia Is a tltrimetric method of measuring dissolved oxygen, first used in I908 ty 

 Warburg in a study of the change in rate of ojtygen consumption of the Arbacia egg follow- 

 ing fertilization. It has been used recently by Earth ( 19^+2) in a study of the oxygen con- 

 sumption of fragments of the amphibian gastrula. 



STOCK SOLUTIONS : (For Class Use) 



1. Manganese chloride {kCf^, iron-free) 500.0 cc. 



2. Potassium iodide (15^) in NaOH (56^) kept in dark 500.0 cc. 



Dissolve 180 grams of NaOH in distilled water, 

 cool, add 75 grams of KI, make up to 500. cc and 

 keep in cool dark place. 



5. Hydrochloric acid ( C. P. cone, no free Clg) 500.0 cc. 



h. Sodium thiosulphate (n/iOO). 5000. Occ. 



For each liter dissolve 2.1*82 grama of C, P. grade 

 Na2S20i'5^0 in distilled water. If solution is 

 to be kept several days, add h cc. of In-NaOH per 

 liter. 



5. Starch solution (0.5^) 500.0 cc. 



Emulsify 1 gram of potato starch with 25 cc. of 

 water and pour slowly into 175 cc. of boiling 

 water, boil a few minutes, allow to settle, de- 

 cant off the clear supernatant fluid. If it is 

 to be kept for several days add a few drops of 

 chloroform. 



OXYGEN CONSUMPTION DURING THE FIRST CLEAVAGE OF THE FROG'S EGG 



1. Prepare an ovulating female ( Bana pipiens) and secure several mature males. 



2. Prepare 5 respiration bottles consisting of 125 cc. glass -stoppered bottles. Mark 



them A, B, and C. 



5. Prepare 5 Erlenmeyer flasks, 250 cc. capacity, and into each introduce an equiva- 

 lent number of glass beads or small marbles. Mark them A, B, and C, and cork 

 them. 



k. 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 suspension), 

 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 5 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 super- 

 natant 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. Similarly 

 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 



*Thls is a modification of the method used by Dr. A. lyier in the course in Marine 

 Embryology given at the Marine Biological Laboratory, Woods Hole, Mass. 



