METHODS 199 



2. Seal off the larger end of the pipette in a minute gas flame 

 and centrifuge at once. 



3. Cut the tube a little above the juncture of the cells and plasma, 

 let the plasma flow back from the cut end by gravity, and then seal 

 both ends of the tube taking care not to heat the plasma. 



4. Attach a large capillary tube (0.6 mm. inside diameter) to 

 the water manipulator and fix it on the stage of the microscope. 

 Draw back 5 mm. from the end of a column of Vis N sulfuric acid 

 5.0 mm. long. 



5. Introduce a 4 mm. column of plasma and add 10% sodium 

 tungstate to it until the column becomes 5 mm. long. 



6. The two columns now in the tube are made to oscillate back 

 and forth several times by means of the water manipulator in order 

 to effect thorough mixing of the plasma and tungstate. 



7. Break off the distal part of the tube and seal the ends. The 

 end nearest the acid is sealed last and held in the flame long enough 

 to make a small bulb. 



8. Centrifuge the tube, bulb end down. Reverse and recentrifuge 

 at least six times for complete precipitation. The final centrifuga- 

 tion should be thorough and the material should be left in the nar- 

 row end of the tube. 



9. Break the tube about 5 mm. above the surface of the fluid and 

 draw off the protein-free liquid into a pipette. Should a zone of hazi- 

 ness exist between the clear fluid and the precipitate, too much oxa- 

 late was used. 



Trichloroacetic Acid Supernatants 



1. For frog plasma phosphates a 4 mm. column of plasma is 

 placed in a larger capillary tube followed by 1 mm. of 90% trichloro- 

 acetic acid ( by weight ) . Seal the tube and centrifuge with the plasma 

 end down. 



2. Immerse in hot water for a moment and centrifuge several 

 times inverting the tube each time. 



3. Should the supernatant fluid be turbid, separate from the pre- 

 cipitate by cutting the tube, draw into another pipette, seal the large 

 end, and centrifuge at high speed. 



4. Separate the sediment by cutting off the tube. The protein- 

 free liquid is ready for transfer to a mixing capillary for color devel- 

 opment. 



