22. THE EFFECT OF CENTRIFUGATION 

 ON DEVELOPMENT 



PURPOSE: To determine the susceptibility of various stages (from the unfertilized egg to 

 the neurula stage) to centrifugal force, and the types of abnormalities produced by a 

 shifting of the egg or embryo contents. 



MATERIALS: 



Biological: 



Technical: 



Ovulating female frogs (Rana pipiens), sexually mature males (Rana 

 pipiens) and early developmental stages of Amblystoma. 



Centrifuge with large tubes, controlled speed, and brake. 



Nujol. 



METHOD: 



Precautions : 



1. Calculate centrifugal force in terms of gravity, using the formula 



F = ^^ 



r 



where F is the gravitational force; r is the radius from the center of the cen- 

 trifuge to the rotational position of the biological material; m is the total 

 mass in grams; and v is the velocity as determined by ZTTx revolutions per 

 minute. In general the R. P. M. figure is somewhat greater than the force 

 times gravity. In every instance, record exactly the value for r and m and 

 the number of revolutions per minute so that computations can be checked if 

 necessary. If the same apparatus is used throughout, the relative values of 

 R. P. M. will be adequate. (See Costello, Science, May 2, 1947, p. 474 for 

 a criticism of centrifugation experiments. ) 



2. Crowding must be limited to the duration of centrifugation, and the controls 

 should be similarly crowded for a corresponding period. As soon as possible 

 after treatment, the eggs or embryos should be given optimum conditions of 

 volume, and temperature. 



Control: Eggs from the same source, untreated by centrifugation but otherwise kept 

 under conditions identical with the conditions of the experimentals. 



Procedure : 



A. CONSTITUENTS OF THE AMPHIBIAN EGG 



I. Apply a very thin coat of albumen-water to several microscope slides. Open 

 a sexually mature female Rana pipiens and excise several ovarian eggs. 

 Place a single egg (within its capsule) on each of the five slides, and rupture 

 it with watchmaker's forceps, allowing the contents to flow freely over the 

 dried albumen-water. With the edge of a coverslip, the egg contents may be 

 spread evenly and thinly over the egg-albumen base. 



a. Examine one of the egg smears under the microscope before it has dried. 

 Note the yolk granules of various sizes. Can you identify any other 

 formed structures? 



b. Expose two of the slides (after they have become dried) to formaldehyde 

 or osmic vapors to fix the egg contents, and then stain with an alcoholic 

 solution of Sudan III. This dye is specific for fat. 



c. When thoroughly dried, apply the plasmal test to the other two slides 

 (see section on Chemistry of the Embryo). 



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