210 CAPILLARY TUBE COLORIMETRY 



PROCEDURE 



1. Pipette 3 fA. plasma into 21 /xl. water in a reaction tube of 

 250 /xl. capacity, and add 7 /xl. Veronal buffer with magnesium and 

 7 /xl. substrate soln. Mix with a magnetic stirring "flea" (page 179). 



2. Set up a control experiment in which the substrate and buffer 

 are placed as a separate drop on the side of the tube where it 

 cannot touch the enzyme soln. 



3. Place tubes in a rack in a desiccator containing water in the 

 bottom and a small bottle of chloroform to produce a vapor inhibit- 

 ing growth of microorganisms. The desiccator is kept at 37° and 

 the digestion is allowed to proceed for 4 hr. 



4. Stop the reaction by setting the tubes in ice water, and add 

 10 /xl. 10% trichloroacetic acid to each. 



5. Centrifuge, and pipette 15 /xl. of the supernatant into another 

 tube. 



6. Add 7 /xl. of the molybdic-sulfuric acid reagent to the super- 

 natant. 



7. Pipette 5 /xl. aminonaphtholsulfonic acid soln. on the side of 

 the tube as a separate drop. 



8. Set up standards with 15 /xl. of the known phosphate solns., 

 following steps 5-7. 



9. Mix the drops on the side of the tubes with the rest of the 

 liquid using stirring "fleas." 



10. Draw the solns. into capillary tubes of uniform lumen (inside 

 diameter 0.65 mm., length 30 mm.) and seal the ends with Duco 

 cement. 



11. Compare the colors as in the Walker method. 



REDUCING SUBSTANCES 



Sumner's ( 1925) dinitrosalicylic acid method was adapted to 

 capillary tube colorimetry by Walker and Reisinger ( 1933) . In 

 this manner, quantities of glucose of the order of 0.1 /xg. in 0.2 /xl. 

 liquid (50 milligram per cent) can be determined with a maximum 

 error of 3 milligram per cent in duplicate measurements of solutions 

 of known concentration. For other methods see page 296. 



