blood, is multiplied by a factor of 2.3736, the 

 product is the number of grams of hemoglobin 

 per 100 cc. of Idood. The derivation of the con- 

 stant is given by Bankowski (1942). 



Method for heniatocrit determination 



Van Allen hematocrit tubes were found suitable 

 for embryos, small chicks, and the adult fowl. 

 They are simple to use, give accurate results, and 

 are easy to clean. From a small puncture about 

 one drop of blood was drawn into the tube. The 

 column of blood should be stopped exactly at the 

 100-percent mark; then the open tip of the tube 

 should be quickly immersed in the diluent — 

 1.6-percent solution of sodium oxalate in distilled 

 water — which is sucked into the tube until the 

 bulb is half full. Then the springclip cap is 

 placed over the open end; fluid is not allowed to 

 fall out of the open end as this is done. The 

 springclip cap must be firmly and carefully 

 seated. 



The tubes are placed in the brass shields of 

 the centrifuge and the shields are filled to near 

 the top with water and are l^alanced by the amount 

 of water added. They are spun at 2000 r. p. m. 

 for 20 minutes. The radius to the middle of the 

 shield was 7V-2 inches. The level of packed cells 

 and the thickness of the buffy layer were read 

 with a hand lens. 



After they are used, the tubes must be carefully 

 cleaned and dried. The use of acid was avoided 

 because, if not thoroughly washed out of the tube, 

 it will hemolyse tlie red cells the next time the 

 tube is used. 



It must be rememljered that the buffy coat is 

 composed of both leukocytes and thrombocytes, 

 and the latter constitute half or more than half of 

 the buffy coat. 



Method for making thrombocyte counts 



Although an indirect method was used to de- 

 termine the number of thrombocytes per cubic 

 millimeter, it seemed reasonably accurate. At 

 the time blood was taken for use in erythrocyte 

 counts, a dried smear was made. After the 

 counts had been recorded, the stained slide was 

 examined with an ocular reticle that had been 

 scribed with two concentric squares. The inner 



square had one-twentieth the area enclosed by 

 the larger square. The latter was of such size 

 that it would fit within the opening made by the 

 diaphragm ring of the eyepiece. 



The thrombocytes in tlie large square and the 

 erythrocytes in the small square are counted. 

 Cells that touch two of the sides are counted; 

 those that are crossed by the lines on opposite 

 sides are not counted. If the small square is 

 exactly one-twentieth of the large square, a tabu- 

 lation of 250 erythrocytes in that square is the 

 equivalent of 5,000 erythrocytes in the large 

 square. The figures now collected are substi- 

 tuted in the following formula: 



„, , , Throm. counted X ery./mm.^ 

 Throm./mm. = ^^^ 



The values for erythrocytes per culnc millimeter 

 were determined from the counts made with the 

 hemocytometer. 



If the inner square is not exactly one-twentieth 

 of the area of the larger one, differences can be 

 compensated for jjy varying the counts of ervthro- 

 cytes above and below 250 by an amount needed 

 to give the equivalent of 5,000 in the large square. 



Methods for white-cell counts 



The relative merits of three methods — direct, 

 semi-indirect, and indirect — were discussed by 

 Denington and Lucas (1955) but no conclusion 

 as to which is the best was given. Many authors 

 have suggested technics that fall into these three 

 categories. The indirect method is the sim- 

 plest — after a total erythrocyte count has been 

 made, the number of white cells relative to the 

 nuniiier of erythrocytes is counted on the stained 

 slide and from that ratio, the number of white 

 cells per cubic millimeter can be estimated. The 

 same reticle and procedure that were described 

 for thrombocytes can be used. 



The Wiseman (19.31a) method is classed as a 

 semi-indirect method. It is based on the prin- 

 ciple that the total number of white cells in a 

 cubic millimeter can be calculated if (1) the 

 eosin-staining cells are counted directly in the 

 counting chamber of a hemocytometer and (2) 

 these values are used in conjunction with the per- 

 centage of eosin-staining cells tabulated in differ- 

 ential counts obtained from a stained slide. 



Wiseman based his method on the affinity of 

 phloxine for cells he called eosinophils, but both 



232 



