EXPERIMENTAL EQUIPMENT AND PROCEDURES 11 



OPERATING PROCEDURES 



Most operations will be carried out in Syracuse dishes or salt cellars. Since embryonic 

 tissues often adhere to glass, the base of the Syracuse dish is lined with one or another 

 plastic substance. The following are satisfactory: 



a. Permoplast - American Art Clay Co. , Indianapolis, Indiana. 



b. Beeswax to which has been added lampblack, to take glare off background. 



c. Rainbow Wax - American Art Clay Co. , Indianapolis, Indiana. 



d. White refined beeswax. 



e. Pieces of cellophane or Pyralin. 



f. Agar, 2% or more concentrated. 



The Permoplast is probably best for delicate operative procedures, as it is easily molded 

 without heating. However, under solution it tends to fragment. It may be necessary to 

 compensate for this by melting it with about 20% low melting point paraffin. 



For frog work, melted beeswax to which enough lampblack has been added to give it a 

 dark gray appearance, will prove to be satisfactory. Occasionally, after the dishes are 

 prepared, the wax base will, under water, float off the bottom. To prevent this, place a 

 few pieces of 2 mm. glass rod in the bottom of each dish before adding the melted bees- 

 wax. These will add sufficient weight to hold the wax in place. Have at least 10 operating 

 dishes available at all times. Grooves to hold embryos may be made with ball tips before 

 the beeswax hardens. 



OPERATING SOLUTIONS 



The operating medium will vary with the age, the condition of the embryo (or tissue) and 

 the species used. Operations are generally performed in more concentrated solutions, 

 and, after healing, are returned to weaker salt solutions (see exercise on "Wound Heal- 



ing"). 



For operations on Urodeles the Urodele Operating Medium is used and the enibryos will 

 heal normally if left in these solutions. After the operation wound has healed the Urodele 

 embryo is transferred to Urodele Growing Solution. For the Anura, double strength and 

 then Normal Standard Solutions are used. Brief boiling of the solutions to sterilize them 

 may be necessary. If convenient, large volumes could be autoclaved, filtered and stored 

 as stock solutions. Controlled salt solutions are more satisfactory than Spring Water or 

 conditioned tap water because there is greater uniformity. 



In all cases remember that surface rather than volume is important. The embryos need 

 be just covered in the solution and no more, providing evaporation is reduced to a mini- 

 mum. 



GLASSWARE 



Ovulating animals should be kept in fish bowls or small battery jars, properly covered. 

 If eggs are to be layed in these containers, it will be necessary to provide them with ap- 

 propriate solutions. The Urodeles generally attach their eggs to vegetation. 



For fertilization of frog's eggs, finger bowls or Petri dishes are used and all eggs from 

 a single female may be inseminated in a single container providing they are flooded in 1 5 

 minutes and separated to lots of about 25 per finger bowl before the first cleavage. De- 

 velopment will continue to hatching in finger bowls but beyond hatching a different pro- 

 cedure will be necessary (see section on "Culturing of Embryos"). 



