27. THE CULTURE OF ISOLATED 

 AMPHIBIAN ANLAGEN 



PURPOSE: To test the self-differentiating capacities of the constituents of the various 

 organ anlagen of the early amphibian embryo. 



MATERIALS: 



Biological: Anuran (stage #18) and Urodele (stage #28) embryos. 



Technical : 



1. Culture media : 



a. Amphibian Ringer's solution. 



b. Standard (Holtfreter' s) solution. 



c. Standard (Holtfreter ' s) solution plus frog blood plasma, lymph, coelomic 

 fluid, or crushed embryo extracts. 



d. Urodele growing medium plus Urodele coelomic fluid. 



(Note: Media #1 and #2 may be autoclaved and kept in ampoules. Medium 

 #3 should be made up with sterile Standard Solution and #4 with 

 sterile Urodele growing medium. Sodium sulfadiazine 0. 5% may be 

 added to give further protection against bacterial contamination. ) 



2. Depression slides, watchglasses, Syracuse and Petri dishes. Circular 

 coverslips, cellophane tubing (Visking cellulose sausage casings of minimunn 

 diameter). 



METHOD: 



Precautions : 



1. While amphibian tissues do not require the asepsis required by avian tissues, 

 aseptic conditions will undoubtedly prolong the development in isolation. 



The operating instruments may be boiled (glassware) or dipped in 95% alco- 

 hol. Large glassware may be autoclaved. Culture media which do not con- 

 tain body fluids (lymph, plasma, etc. ) may also be autoclaved. Otherwise, 

 0. 5% sodium sulfadiazine can be added without any effect but an aid to asepsis. 



2. Moist chamber conditions should be provided since evaporation changes the 

 concentration of the constituents of the medium. 



3. If embryos which are to contribute the anlagen are divested of their mem- 

 branes and are then passed through several changes of sterile Standard 

 Solution (for the Anura) of Urodele Growing Medium (for the Urodela) most 

 of the adherent bacteria will be removed. 



4. The isolates should be kept at temperatures near the lower limit of viability 

 of the species under investigation. This will retard development but also 

 the incidence of infection. 



5. Large amounts of culture medium may be placed in ampoules and autoclaved, 

 to be used when needed. The 0. 5% sodium sulfadiazine may be added before 

 autoclaving, for it is not altered by this treatment. 



Controls : There are two types of controls: (1) Isolates other than those under im- 

 mediate investigation. For instance, the experimental isolate might be the gill 

 or limb anlagen and the control could be tail ecto- and mesoderm. (2) If but a 

 single anlage'is removed from an ennbryo, the bilaterally located mate anlage 

 may be considered the control organ, or embryos of identical stage may be cul- 

 tured under parallel conditions of temperature, etc. for direct comparison of 

 the isolate with the intact anlag^. 



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