15. MECHANICAL SEPARATION OF 

 GROWTH AND DIFFERENTIATION 



PURPOSE: To inhibit growth (increase in mass) by limitation of the physical environ- 

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



MATERIALS: 



Biological : Eggs and early embryos of Anura and Urodela. 



Technical : Agar, 1% to 3% concentrations made up in appropriate culture media for 

 the forms used. Petri dishes and #2 Slenders. 



METHOD: 



Precautions : 



1. Bacterial contamination may appreciably shorten the duration of this experi- 

 ment. It might be effective to use 0. 5% sodium sulfadiazine in the agar mix- 

 ture 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 submerged in the appropriate culture medium. 



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

 conditions in the normal culture mediuin, in similar containers. 



Procedure : (Best results will be achieved with Amblystoma) 



1. Divest the eggs or early embryos of their membranes and placa' 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 be- 

 gins to gel, transfer 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. 



3. 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% so- 

 dium sulfadiazine. 



4. 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. 



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. 



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