190 EFFECT OF CENTRIFUGATION ON DEVELOPMENT 



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

 mortar, (in an ice bath if available). The crushing may be accomplished the 

 better with a small amount of clean sand. 



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

 pH 7), mix well, and centrifuge for 10 minutes at 3, 000 R. P. M. The 

 amphibian egg contains fat, translucent protoplasm, heavy yolk, pigment 

 granules, and a germinal vesicle. The pigment will be found at the cen- 

 trifugal pole, and the so-called microsome layer will be found between 

 the fat and the pigment, as a cloudy layer. The translucent protoplasm 

 comprises the middle layer and the centripetal pole will have the whitish, 

 opaque cap of hyaloplasm. With micropipette, remove material from 

 each of these layers and examine immediately under high magnification of 

 the microscope. (Do not expect to find an intact germinal vesicle. ) 



b. To the other half of the egg brei add 10 volumes of cold phosphate buffer 

 (M/200 at pH 7), mix well, and centrifuge for 10 minutes at 3, 000 R. P. M. 

 Before there has been any opportunity for mixing of the various layers, 

 remove each with micropipette into separate homopathic vials. Bio- 

 chemical tests should be applied to these isolated egg constituents, par- 

 ticularly to the microsome layer which can be identified as the cloudy 

 layer between the fats and pigment. 



Place the microsome layer in a centrifuge tube and centrifuge for 20 

 minutes at 12, 500 R. P.M. (ultracentrifuge). Note the supernatant fluid 

 and the pellets. To the latter apply the following tests: indophenolox- 

 idase; peroxidase; -SH; and plasmal. (See section on Biochemistry of 

 the Embryo. ) 



B. RESISTANCE OF EMBRYONIC STAGES TO CENTRIFUGATION DAMAGE 



There are two aspects of this study: (A) The ability of various stages to survive centrif- 

 ugation damage and (B) The variety of abnormalities produced by standard centrifugation 

 at different stages of development. 



The stages that are to be used are: Uterine eggs, recently fertilized (but uncleaved) 

 eggs; blastulae; and gastrulae. With the large International Centrifuge the approximate 

 speed to be used should range from about 1500 to 3000 R. P. M. , but the data should be 

 recorded in terms of the value times gravity (see forniula on preceding page). The dura- 

 tion should be from 1 to 10 minutes, the shorter interval at the higher speeds. (For ex- 

 ample, a speed of 180 times gravity for 10 minutes to 1800 times gravity for 1 minute 

 might be the extremes tested. ) If but one speed and time are used, the lower force for 

 the longer interval is recommended for all stages. 



It has been suggested (Brachet) that if the eggs are centrifuged immediately after fertil- 

 ization, the eggs that do not develop fail because all of the ribonucleic acid is carried to 

 one pole, opposite that of the yolk. Centrifugation at later stages (e. g. , blastula) may 

 produce triploid embryos because of the excessive concentration of ribonucleic acid in 

 specific areas. 



The effect of fertilization can be tested very simply by stripping several hundred eggs 

 from an ovulating female into a concentrated sperm suspension. The female is then to 

 be opened and the uteri tied off above and below, and removed as a double sack full of 

 eggs. The two uterine sacks may then be separated and placed directly into a centrifuge 

 tube, previously coated (internally) with Nujol (paraffin oil). In a balancing tube, place 

 the fertilized eggs, and centrifuge simultaneously. All eggs will be from the same fe- 

 male and the only difference will be that one group are fertilized. The unfertilized eggs 

 should be fertilized immediately upon removal from the centrifuge, by cutting open the 

 uteri and stripping the eggs into a concentrated sperm suspension. 



