31. WOUND HEALING IN EMBRYOS 



DEFINITION: The ability of the interrupted living surface to close. 



PURPOSE: To determine the ability and rate of wound closure of the egg, blastomere, 

 and embryonic epidermis under various environmental conditions. 



MATERIALS: 



Biological: Ovulating females and early developmental stages of any Anura or 

 Urodela. 



Technical : Stock solutions (see below) 



Glass needles, pointed lancet 

 Vital dyes: Nile blue sulphate (1/7500) 

 Neutral red (1/2500) 



METHOD: 



Precautions : 



1. Avoid infection. Sterilization of the instruments and solutions will not be 

 necessary if ordinary care is observed. 



2. Keep eggs and embryos in separate #Z Stenders with covers, at room temper- 

 atures or below. Provide 10 to 20 cc. of solution per egg or embryo. This 

 relatively large volume will reduce any effects from accumulating metabo- 

 lites. 



3. In Ca-free experiments, wash the egg or embryo in several changes of Ca- 

 free solution prior to making the incision. Adherent calcium or calcium 

 diffused from the embryo may alter the results. 



Controls : Holtfreter (1943) says: "In an optimal medium, as represented by our 



Standard Solution, the healing faculty is extraordinary. " We may therefore con- 

 sider the healing process in this solution as the control situation with which the 

 process, in other media, is to be compared. 



Procedure : 



Observations are to be made on wound healing of the following embryonic stages: 



1. Body cavity eggs, having vitelline inembranes only. 



2. Recently fertilized eggs - 1 hour after insemination. 



3. The 4-cell stage, a single blastomere to be injured. 



4. Animal pole of early gastrula. 



5. Epidermis of neurula. 



The effect on wound healing of the following solutions is to be determined: 



1. Distilled water (glass distilled water preferred), - hypotonic. 



2. Standard Solutions diluted to 10%, - hypotonic. 



3. Standard Solution - isotonic. 



4. Standard Solution x 2: - hypertonic. 



5. Standard Solution plus 0. 1% KOH - alkalinized. (Determine the pH. ) 



(Standard Solution for this should be made without the buffer. ) 



6. Standard Solution plus 0. 1% HCl - acidified. (Determine the pH. ) 



(Standard Solution for this should be made without the buffer. ) 



Prepare an ovulating female frog, Rana pipiens, about 48 hours before the time of the 

 experimental work. The various solutions should be made up and distributed to labelled 

 #2 Stenders ready for the introduction of eggs or embryos. 



* This exercise has been organized with the generous aid of Dr. J. Holtfreter. 



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