EXPERIMENTAL EQUIPMENT AND PROCEDURES 



Tor fertillEation 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 15 minutes and separated to lots of about 25 per finger bowl before the first cleavage. 

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

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



Operations are performed in dishes described above. Following the operation, when 

 the transplant has "taken", the embryo may be transferred to a covered stender, the bottom 

 of which has a thin layer of U-5^ agar. This provides a softer base than glass so that 

 there is less danger of injury. Such operated animals should be kept at constant tempera- 

 ture toward the lower levels of tolerance for the particular species under investigation. 



ASEPSIS 



The amphibian embryo is relatively immune to infection, or bacterial contamination. 

 Operated embryos are, of course, exposed to such conditions but the wounds heal so rapid- 

 ly that simple aseptic precautions are generally sufficient to protect the embryo. 



In recent years Detwiler, Copenhaver, and Robinson (19^7) have demonstrated that 

 sodium sulfadiazine (0.5^) in any of the various operating or culture media is perfectly 

 harmless to the embryo, and will reduce the operative casualties on the central nervous 

 system from 95^ to 5^. The sulfadiazine may partially crystallize out, but these crystals 

 are harmless except as they may mechanically pierce the ectoderm of the embryo. 



At later larval stages, or just after metamorphosis, amphibia may become susceptible 

 to fungus or other skin infections. These can be treated locally with dilute mercuro- 

 chrome, neo-sllvol, or very concentrated salt solutions painted onto the affected area. 



MEMBRANES 



The eggs of eJ.1 amphibians are surrounded by secondary (jelly) membranes secreted by 

 the oviduct in addition to the vitelline membrane. These membranes presumably protect the 

 embryo from bacteria and injury during development. Any operations on such embryos re- 

 quires that such membranes be removed. It must be remembered, therefore, that the unpro- 

 tected embryo is rather easily injured and that it is more susceptible to physical and 

 chemical changes in the environment, as well as to bacterial infection. 



URODELA 



The Urodele jelly is rather tough and may be peeled off of the eggs with sharp 

 pointed watchmaker's forceps. The vitelline membrane will be resistant to puncturing so 

 that it may be necessary to use glass micro-needles to make the Initial break. Urodele 

 egg capsules should be opened in Urodele Growing Solution. 



ANURA 



The Anuran egg is surrounded by loose jelly which is adherent to the vitelline mem- 

 brane. Attempts have been made to remove this jelly by Ultra-violet light, or by dilute 

 KCN, but there is no reliable chemical method of Jelly removal which allows the embryo to 

 survive. Should someone discover a method of fertilizing body cavity eggs (devoid of 

 jelly) this would be a boon to experimental embryology. The Anuran egg jelly can be re- 

 moved to some extent by rolling the egg on filter paper or paper toweling, pushing it 

 along with the flat side of a scalpel. The danger in this method is, of course, excessive 

 mechanical injury to the egg so that the correct rate and amount of absorption (by filter 

 paper) and the exact amount of rolling will have to be determined empirically. With prac- 

 tice it is possible to pierce the jelly with one prong of the forceps, slide a prong of a 

 second pair of forceps along the first, and cut through the jelly with an outward move- 

 ment. Then the split jelly capsule can be shelled off of the egg. The enzyme hyaluronl- 

 dase, so effective In denuding the mammalian egg, may prove of similar value with the 

 amphibian egg. (Personal communication from D. C. Metcalfe and reference to Kurzrok I9I18: 

 Am. Jour, Clin. Pathology, p. '+9I. ) 



