ANALYSIS OF TISSUES 579 



II. Total Non-Protein Nitrogen, Ammonia, Urea, Creatinine, 

 Creatine, Uric Acid. 



{a) Blood. 



(1) Drawing of Blood. 



The blood is drawn through hypodermic needles into 2 or 5 c.c. pipettes, 

 i mm. in diameter and about 25 mm. long. The needles are immersed in a 

 dilute solution of vaselin in ether and allowed to drain on clean paper for a 

 few minutes. They are sterilised before drawing human blood. 



The needle is attached to the pipette by rubber tubing. A pinch of 

 finely powdered potassium oxalate (to prevent clotting) is put into the upper 

 end of the pipette and allowed to drop into the tip. The upper end of the 

 pipette is fastened by rubber tubing to a mouthpiece and a pinch cock is put 

 upon the rubber near the pipette. One person inserts the needle into the artery 

 or vein ; another person regulates the blood flow by means of the pinch cock and 

 by suction so that the exact quantity is obtained. 



(2) Removal of Proteins. 



The blood is transferred into 25 c.c. (for 2 c.c. blood) or 50 c.c. (for 5 c.c. 

 blood) measuring flasks half-filled with pure methyl alcohol, free from acetone ; 

 the flasks are filled to the mark and thoroughly shaken. 



After 2 or more hours the contents of the flasks are filtered through dry 

 papers into dry flasks and 2 or 3 drops of a saturated solution of zinc 

 chloride in alcohol are added. After a few minutes they are again filtered 

 through dry papers. The zinc chloride precipitates the remainder of the 

 proteins and colouring matter. 



The same volume of the filtrate is taken for each analysis. 



In the case of cat's blood 2 c.c. are diluted to 25 c.c. : 5 c.c. filtrate are used 

 = -4 c.c. blood. 



In the case of human blood 5 c.c. are diluted to 50 c.c. : 10 c.c. filtrate 

 are used = i c.c. blood. 



(3) Estimation of Total Nitrogen (p. 558). 



5 or 10 c.c. are placed in a Jena glass test tube, i drop of sulphuric acid, 

 i drop of paraffin and a pebble are added and the methyl alcohol is removed 

 by placing the test tube in a boiling water- bath for 5-10 minutes. 



The oxidation is effected with i c.c. of sulphuric acid, i gm. of K 2 SO 4 , 

 i drop of CuSO 4 . 



The ammonia is collected in i c.c. of *iN acid + 2-3 c.c. of water and 

 estimated by the colorimetric method with 7-8 c.c. of diluted Nessler, or 

 more if much ammonia be present. 



The standard containing i mgm. N is set at 20. 



The results are expressed in mgm. N per 100 c.c. (or 100 gm.) blood and 

 are obtained from the following formulae : 



5 ^ x D is the figure with 2 c.c. blood and 5 c.c. filtrate, 



20 



x D ,, 5 c.c. ,, ioc.c. 



where R is the reading of the colorimeter and D is the volume to which the 

 ammonia has been diluted. 



