PRESSURE EFFECTS ON CLEAVAGE 



PURPC6E : To determine the effect of altering the yolk- cy top laemlc axis on cleavage and 

 on the subsequent development of the embryo. Specifically, to attempt to shift the 

 third cleavage from the equatorial to the meridional plane by the application of un- 

 equal pressure. 



MATER lAIB : 



Biological : Fertilized eggs of any Amphibian. 



Technical : Petri dishes, glass tubing 2.0 mm. in diameter. 



METHOD : 



Precautions : 



a. Do not crowd the eggs; allow sufficient medium for appropriate aeration. 



b. Separate and remove the eggs whose cleavage plane has been altered, placing 

 them in #2 Stenders where they may be given special care. 



Controls : Eggs fertilized at the same time, from the same female, but not subjected 

 to any pressure. 



Procedure : 



A. Strip some uterine eggs into a sperm suspension in an Inverted cover of a Petri 

 diah. Gently shake them so that they spread out into a single layer of eggs. 

 Flood with Spring Water or Standard Solution in 5 minutes. Mark the time of 

 insemination on the dish. 



Ey 2^ hours after insemination these eggs should be in the 2-cell stage, 

 and 1 hour later (Ja hours after insemination) most of them should be in the 

 four cell stage. The first two cleavages are normally vertical (meridional) and 

 generally bisect each other in the center of the animal pole. The third cleav- 

 age is horizontal but slightly above the true equator of the egg, at right 

 angles to both the first and the second cleavages. 



As soon as most of the eggs are in the l+-cell stage, place the 

 bottom of the same Petri dish over the eggs and, while observing them under low 

 power magnification, add water to the upper dish until pressure is exerted on 

 the eggs to such an extent that they are definitely distorted but not ruptured. 

 The bottom of the Petri dish partially filled with water provides the pressure, 

 and this pressure can be controlled by adding or removing water. The dish also 

 acts as a pseudo-lens so that the eggs bene&th can be observed directly and at 

 all times. The pressure must be maintained from before the initiation of and 

 through the time of the third cleavage of both the experlmentals and controls. 



B. A second method of applying pressure is to draw up the eggs with their Jelly in- 

 to glass tubing which has a d-lameter slightly less than that of both the egg and 

 its Jelly- This will be about 2.0 mm. for Sana pipiens eggs. The eggs should 

 be drawn up by suction at the l+-cell stage, and observed through the side of the 

 tubing, under water. The eggs will be considerably distorted and sketches should 

 be made while the eggs are under pressure and Immediately thereafter. 



OBSERVATIONS AND TABULATION OF DATA : 



The observations here are purely qualitative and a series of sketches or photographs 

 should be made of several eggs whose cleavage planes have been altered. Then the eggs, 

 properly identified with their sketches, should be isolated In #2 Stenders and allowed to 

 develop as far as they will normally. Any variations from the controls should be Indi- 

 cated by parallel sketches of experlmentals and controls. 



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