19*^ EFFECT OF CENTR I F UGAT I ON ON DEVELOPMENT 



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

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

 fugal pole, and the so-called microsome layer vd.ll he 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 5,000 B.P.M. 

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

 remove each with micropipette into separate homopathlc vials. Biochemical 

 tests should be applied to these isolated egg constituents, particularly 

 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 £ind centrifuge for 20 

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

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

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

 Embryo . ) 



ANALYSIS OF EGG CONSTITUENTS 



