THE CULTURE OF ISOLATED AMPHIBIAN ANLACEN 



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

 organ anlagen of the early amphibian embryo. 



MATERIAlS : 



Biological : Anuran (stage #l8) and Urodele (stage #28) embryos. 



Technical: 



Culture media: 



1. Amphibian Ringer's solution. 



2. Standard ( Holtfreter' s) solution. 



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



coelomic fluid, or crushed embryo extracts. 

 '+. Urodele growing medium plus Urodele coelomic fluid. 



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



Medium #5 should be made up with sterile Standard Solution 

 and ^k with sterile Urodele growing medium. Sodium sul- 

 fadiazine 0.5^ may be added to give further protection 

 against bacterial contamination.) 

 Depression slides, watchglasses, Syracuse and Petri dishes. Circular 

 coverslips, cellophane tubing (Visking cellulose sausage casings of 

 minimum 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 951^ alcohol. Large 

 glassware may be autoclaved. Culture media which do not contain body fluids 

 (lymph, plasma, etc.) may also be autoclaved. Othexvise, 0.55^ sodium sulfa- 

 diazine can be added without any effect but an aid to asepsis. 



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

 centration of the constituents of the medium. 



5. If embryos which are to contribute the anlagen are divested of their membranes 

 and are then passed throijgh several changes of sterile Standard Solution (for 

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

 bacteria will be removed. 



h. 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 in- 

 cidence 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 autoclavJng, 

 for it is not altered by this treatment. 



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

 mediate Investigation. Jbr Instance, the experimental Isolate might be the gill or 

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

 anlage is removed from an embryo, the bilaterally located mate anlage may be con- 

 sidered the control organ, or embryos of identical stage may be cultured under 

 parallel conditions of temperature, etc. for direct comparison of the isolate with 

 the intact anlage. 



Procedure : 



1. Prepare 2 culture media at least. For Anura use Standard Solution and for 

 Urodela use Growing Medium, each of which should be sterilized by boiling or 

 autoclaving. Boiling must be breif to avoid changing the salt concentration. 

 The second type of culture medium should be either of the above (depending upon 

 whether Anura or Urodela are used) to which is added peritoneal fluid. This can 

 be done by injecting about 5 cc of the salt solution into the body cavity of 

 adults of the same species, preferably females and then removing it, with the 

 same hypodermic, after a few minutes in the body cavity. This second medium can- 

 not be boiled, but the 0.5^ sodium sulfadiazine should be added. 



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