THE EFFECT OF CENTRIFUCATION ON DEVELOPMENT 



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

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

 shifting of the egg or embryo contents. 



MATERIAI£ : 



Biological : Ovulating female frogs (Sana pipiens), sexually mature males (Rana 

 pipiens) and early developmental stages of Amblystoma . 



Technical : Centrifuge with large tubes, controlled speed, and brake. 

 Nujol. 



METHOD: 



^''recautlons : 



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



_, mv 



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 2 tt x revolutions per 

 minute. In general the B.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 ade- 

 quate. (See Costello, Science, May 2, 19*+?, p. '+?'+ for a criticism of centrl- 

 fugation experiments.) 

 b. Crowding must be limited to the duration of centrifugatlon, azii 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 centrifugatlon but otherwise kept 

 under conditions Identical with the conditions of the experlmentala.- 



Procedure : 



A. CONST ITUEtlTS OF THE AMPHIBIAN EGG 



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

 sexually mature female Eana 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 plaamal teat to the other two alides (see 

 section on Chemistry of the Embryo). 



2. Dissect the ovaries from a sexually mature female frog and crush them in a mor- 

 tar, (in an ice bath If available). The crushing may be accomplished the batter 

 with a small amount of clean sand. 



a. To half the egg brel add 10 volumes of cold phosphate buffer (m/200 at 

 pH 7), mix well, and centrifuge for 10 minutes at 5,000 R.P.M. The am- 



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