CALCIUM 221 



PROCEDURE 



1. Mix 5 vol. distilled water with 1 vol. serum in a 12-15 ml. 

 centrifuge tube; run in duplicate. Add 6 vol. distilled water to 

 another centrifuge tube to be treated in a parallel manner as a 

 standard; run in duplicate. 



2. Add 1 vol. saturated ammonium oxalate to each tube, stir by 

 tapping, cover tubes to keep out dust, and let stand at least 16 hr. 



3. Centrifuge for 5 min. at 2600 R.P.M. and carefully siphon 

 off all but about 0.2 ml. of the supernatant with an upturned capil- 

 lary, the tip of which is kept immersed. 



4. Wash the entire inner surface of each tube with 3 ml. 2% 

 ammonia added slowly from a pipette moved around the top of the 

 tube. 



5. Tap the tubes until the precipitates just begin to move up; 

 then again centrifuge and withdraw the supernatant. 



6. Add 1 ml. of the water-alcohol-ether mixture; stir and mix 

 well. Add 3 ml. more of the mixture and mix gently to keep a mini- 

 mum of the precipitate in the upper portion of the liquid. Centrifuge 

 and withdraw the supernatant. 



7. Repeat the washing with the water-alcohol-ether mixture as 

 in step 5. 



8. Place the tubes in an oven at 100-110° at an angle of about 

 15° for 0.5-1.0 hr. to dry completely. 



9. To each of the two standard tubes add different vol. of the 

 standard oxalate soln. (See Table VI.) Add the sulfuric acid to 

 all the tubes (see table) and heat for 5 min. in a beaker of water 

 kept below boiling. 



10. Remove tubes, let cool to room temperature and add the eerie 

 bisulfate (Table VI). Mix well, cover the tubes, and let stand at 

 room temperature for 30 min. or in a water bath at 70° for 10 min. 



11. Transfer solns. to vessels in which color development and 

 dilution to final vol. (Table VI) are carried out. To facilitate trans- 

 fer, coat a part of the outer rim of the tube with a thin film of 

 paraffin. Wash out tubes with 4 ml. portions of water to make the 

 transfer quantitative. 



12. Add potassium iodide (Table VI) with a minimum of 

 agitation necessary to mix well. After 60 sec. add filtered alcohol 

 and then water to bring to final vol. 



