MECHANICAL SEPARATION OF GROWTH AND 

 DIFFERENTIATION 



PUBPOSE : To inhibit growth (increase in mass) hy limitation of the physical environment 

 and the determination of the effect of such limitation upon differentiation. 



MAT^IAIS: 



Biological : Eggs and early emhryos of Anura and Urodela. 



Technical : Agar, 1^ to 55^ concentrations made up in appropriate culture media for the 

 forms used. Petri dishes and #2 Stenders. 



METHOD: 



Precautions : 



1. Bacterial contamination may appreciably shorten the duration of this experiment. 

 It might be effective to use 0.5^ sodium sulfadiazine in the agar mixture to cut 

 down on the incidence of certain bacteria. This drug is non-toxic to embryos. 



2. Avoid drying up of the agar through unnecessary exposure to warm or dry air. The 

 containers should be covered during the experiment, and the agar should be sub- 

 merged in the appropriate culture medium. 



Control : This consists of similar stage and age embryos kept under unrestricted con- 

 ditions in the normal culture medliim, in similar containers. 



Procedure : (Best results will be achieved with Amblystoma) 



1. Divest the eggs or early embryos of their membranes and place them In sterile, 

 slightly hypertonic culture medium. This will partially dehydrate them before 

 the Immersion in agar. 



2. Prepare several Petri dishes or #2 Stenders with the agar mixture (above), using 

 only enough agar to Just cover the embryos to be studied. When the temperature 

 reaches a tolerable level for the embryos and when the agar begins to gel, trans- 

 fer an embryo to the agar by means of a wide-mouthed pipette, and a minimum of 

 sterile medium. Gently press the embryo (with hair loop) into the agar until It 

 is submerged. 



5. As soon as the agar is Jelled, cover it with about ^ inch of the sterile culture 

 medium used to make up the agar mixture. This can also contain 0.5^ sodium sulfa- 

 diazine. 



k. With older embryos (I.e., neurula or tail bud) place the specimen so that the 

 gill, limb, or eye anlagen are uppermost and near the surface of the agar. When 

 Jelled, and submerged in culture medium, examine under a dissection microscope 

 and scrape away the agar immediately covering one of these organ anlage thereby 

 releasing it from mechanical restriction but retaining the balance of the embryo 

 under restriction. This should allow differential growth of the exposed area. 



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5. If the agar is sufficiently concentrated, it may be cut Into rectangular blocks, 

 each containing an embryo, parts of an embryo, or even Isolated cells. These 

 blocks may be transferred to larger volumes of the culture medium and are very 

 convenient to handle in that they may be turned over and the embryo be examined 

 from various aspects. With the tail bud or later stages the partial dehydration 

 by pre-treatment with hypertonic medium seems not to be so essential. 



OBSERVATIONS AJTD EXPERIMENTAL DATA: 



Record in the space on following page, by a series of drawings and/or photographs, 

 the changes that seem to occur during each day following the embedding of the embryo in 

 the agar. If free of bacteria some of these embryos may survive for 10-12 days, particu- 

 larly at temperatures slightly below those of the laboratory. 



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