CYTOCHEMICAL TESTS ON EMBRYOS 341 



The method of Serra and Lopes (1945) 



1. Fix small pieces of tissue in a mixture of 2 volumes of 95% alcohol, 1 volume 

 of formol, and a few drops of glacial acetic acid per 10 cc. of total volume. 

 Carnoy's fixative is also satisfactory. 



2. Wash in running water, then in distilled water. 



3. Hydrolyze for 3 days or more at 10 to 12 C. and in darkness, if possible. Use 

 3 cc. of the following reagent: to 20 cc. of distilled water add 0. 5 gms. ammon- 

 ium molybdate and 10 cc. of 30% HCl, dilute the mixture to a total of 50 cc. with 

 distilled water. 



4. Transfer to temperature of 20° to 25° for several days. 



5. Add 1 drop of acetic benzedine. (Dissolve 25 mgms. of benzedine in 5 cc. of 

 pure glacial acetic acid. Dilute to 50 cc. with distilled water. Stir or agitate 

 for 3 minutes. ) 



6. Add 2 drops of pure saturated sodium acetate. The tissue, if phosphorus is 

 present, will rapidly develop an intense blue color. The color is durable. 



7. Mount in glycerine to which has been added a few crystals of sodium acetate. 



THE WINKLER METHOD OF MEASURING OXYGEN CONSUMPTION* 



This is a titrimetric method of measuring dissolved oxygen, first used in 1908 by Warburg 

 in a study of the change in rate of oxygen consumption of the Arbacia egg following fertil- 

 ization. It has been used recently by Barth (1942) in a study of the oxygen consumption of 

 fragments of the amphibian gastrula. 



STOCK SOLUTIONS: (For Class Use) 



1. Manganese chloride (40%, iron-free) 500. cc. 



2. Potassium iodide (15%) in NaOH (36%) kept in dark 500. 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. 



3. Hydrochloric acid (C. P. cone. , no free CI-,) 500. cc. 



4. Sodium thiosulphate (N/100) 5000. cc. 



For each liter dissolve 2.482 grams of C. P. grade 

 Na2S202" 5H2O in distilled water. If solution is to 

 be kept several days, add 4 cc. of In-NaOH per liter. 



5. Starch solution (0. 5%) 300. 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, decant 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 (Rana pipiens) and secure several mature males. 



2. Prepare 3 respiration bottles consisting of 125 cc. glass-stoppered bottles. 

 Mark them A, B, and C. 



3. Prepare 3 Erlenmeyer flasks, 250 cc. capacity, and into each introduce an 

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

 and cork them. 



* This is a modification of the method used by Dr. A. Tyler in the course in Marine Embryology given at the Marine Biological 

 Laboratory, Woods Hole, Mass. 



