heterophils and eosinophils take this dye; there- 

 fore, the extrapolation to give a total cell count 

 from a differential count must include both cell 

 types. If this is kept in mind, accurate results 

 can be obtained with the Wiseman table. For 

 convenience, the taJjle was increased to half 

 units so that the calculations could be made on 

 the basis of the average of cells counted in both 

 chambers of the hemocytometer. 



The following formula, which calls for less 

 than half as much phloxine as recommended by 

 Wiseman, was used: 



Phloxine 20 mgm. 



Formalin 5 cc. 



Ringer's solution 95 cc. 



To each 100 cc. .of dye was added 0.5 cc. of 

 0.1 N HCl, which gave a pH of 5.7 to the solu- 

 tion. The dilution in the red-cell pipette was 1 

 to 200. The pipette was held in the refrigerator 

 1 to 3 hours and then placed on a slow-moving 

 Bryan-Garrey (1935) rotating cylinder, which 

 does not damage the cells. The fluid in the tip 

 of the pipette was drawn off onto a piece of gauze 

 before the chambers of the hemocytometer were 

 filled. Heterophils and eosinophils in all 

 squares of both chambers were counted and di- 

 vided Ijy 2. If one chamber gave results con- 

 siderably different from the other in the number 

 of eosin-stained cells present, a new preparation 

 was made. The volume of fluid under all the 

 squares of one side of the counting chamber is 

 0.9 cubic millimeters. Therefore, if only one 

 eosin-stained cell were seen, this would repre- 

 sent — 



1X200 , ,., . , , 

 ^r-^r (dilution tactor) 



or 222.2 cells (heterophils and eosinophils) per 

 cubic millimeter of blood. If in the differential 

 count these two granulocytes were the only cells 

 present, the leukocytes would also total 222.2; 

 but if these two types represented 50 percent of 

 the leukocytes present, the white cells would total 

 twice as much — 444.4 cells. If the eosin-stained 

 cells represented 1 percent, the total cell count 

 would be 22,222.2 cells. It is possible to pre- 

 pare a table by which the total number of white 

 cells per cubic millimeter can be determined, or 

 the table given by Wiseman can be used for this 

 purpose. The number of eosin-stained cells 

 counted in one side of the chamber multiplied by 

 the factor in the table for the percentage of com- 



bined heterophils and eosinophils found in the 

 differential count gives the total number of white 

 cells per cubic millimeter. 



When the total number of heterophils and 

 eosinophils counted in the chamber is high and 

 the percentage level in the differential count is 

 also high, the Wiseman method has reliable ac- 

 curacy; but when both these variables are low, the 

 probability of error is increased proportionately. 



The Rees-Ecker method probably is the best of 

 the direct methods for estimating total white-cell 

 counts. It is an adaptation of a method designed 

 for counting platelet number in human blood 

 ( Wintrobe, 1952) and has been used by students 

 in the Poultry Department at Cornell University. 

 (Ram, 1949; Goodwin, 1950; and Machado, 

 1951 ). The method was first used for making 

 cell counts on avian blood by DeEds (1927) who 

 based his technic on the pujjlication of Rees and 

 Ecker (1923 ) . The only difference between this 

 method and the one given here, based on Win- 

 trobe's procedure, is in the amount of brilliant 

 cresyl blue. Rees and Ecker (1923) used only 

 0.1 grams of the dye instead of 0.5 grams. 

 The diluent is composed of: 



Sodium citrate 3. 8 gms. 



Neutral formalin 0.2 cc. 



Brilliant cresyl blue 0.5 gms. 



Distilled water 100.0 cc. 



Keep in a glass-stoppered bottle in the refrigera- 

 tor and filter before using. If formic acid is 

 produced from the breakdown of formaldehyde, 

 erythrocytes will be hemolysed and the solution 

 should be discarded. 



Blood is drawn to the 0.05-cc. mark on the 

 pipette and the diluent added to bring the fluids 

 up to the mark above the bulb. The tube is 

 shaken in the Bryan-Garrey pipette rotor to give 

 an even distribution of cells; then a suitable 

 quantity of the mixture is placed on each side of 

 the counting chamber. 



Coates (1929) adapted a different direct 

 method, using brilliant cresyl blue, for the count- 

 ing of chicken leukocytes. He used two solutions : 

 Brilliant cresyl blue in water, and potassium 

 cyanide in water. We have not tested the Coates 

 method. 



One of the chief difficulties in using some of 

 the direct methods for avian blood was the con- 

 fusion that came in separating thrombocytes and 

 lymphocytes. The brilliant cresyl blue stains 



233 



