THE COUNTING OF RED CELLS 175 



with suction on the rubber tube a column of blood to exactly 0.5 or i. The variation 

 of 1/25 of an inch from the mark would make a difference of almost 3%. If the 

 column goes above 0.5, it can be gently tapped down on a piece of filter-paper until 

 the 0.5 line is cut. Now insert the tip of the pipette into some diluting fluid and, 

 revolving the pipette on its long axis while filling it by suction, you continue until 

 the mark 101 is reached. A variation of 1/25 of an inch at this mark would only 

 give an error of about 1/30 of i%. After mixing thoroughly by shaking for one 

 or two minutes, the fluid in the pipette below the bulb is expelled (this, of course, is 

 only diluting fluid). A drop of the diluted blood of a size just sufficient to cover 

 the disc when the cover-glass is adjusted, is then deposited on the disc and the cover- 

 glass applied by a sort of sliding movement, best obtained by using a forceps in 

 one hand assisted by the thumb and index-finger of the other. 

 Among diluting fluids Toisson's is probably the best: 



Sodium chloride, i gram 



Sodium sulphate, 8 grams 



Glycerine, 30 c.c. 



Distilled water, 160 c.c. 



Dissolve the sodium chloride and the sodium sulphate in the glycerine water and 

 add sufficient methyl or gentian violet to give a rich violet tint. 



A 2 1/2% solution of potassium bichromate makes a very satisfactory diluting 

 fluid in the counting of red cells. 



A salt solution of about i% strength, tinged with about i drop of a saturated 

 alcoholic solution of gentian violet to about 50 c.c., is a good substitute, or the salt 

 solution alone will" answer when no white count is to be made at the same time as the 

 red one. 



It is important to work quickly in adjusting the cover-glass, or there will be cells 

 settling in the center of the drop from a greater depth than the one which the apposi- 

 tion of the cover-glass makes (i/io millimeter deep). 



A good preparation should show: 



1. Presence of Newton's rings. 



2. Absence of air bubbles. 



3. Entire surface of ruled disc covered. 



4. Equal distribution of cells. 



Before counting, about five minutes should be allowed for the set- 

 tling of the cells. 



It will be remembered that the small squares are 1/20 millimeter square. The 

 depth of fluid from upper surface of shelf to lower surface of cover-glass is i/io mm. 

 Hence each space embraced by the small square and the depth of fluid is 1/4000 of 

 the unit used in estimating number of corpuscles in blood, or i cubic millimeter 

 (1/20X1/20X1/10=1/4000). Count 100 of the small squares .(this enables one 

 to use decimals). There are nine squares between triple-ruled lines, each containing 

 sixteen small squares. Count the number of corpuscles in the sixteen small squares 

 contained in upper left-hand triple-ruled square. Put down this count. Next 



