(1) Draw well-mixed capillary or venous blood exactly to the 0.5 mark in a
white blood cell diluting pipet. This blood column must be free of air bubbles.
(2) Wipe the excess blood from the outside of the pipet to avoid transfer of
cells to the diluting fluid. Take care not to touch the tip of the pipet with the gauze.
(3) Immediately draw diluting fluid to the "11" mark while rotating the pipet
between the thumb and forefinger to mix the specimen and diluent. Hold the pipet
upright to prevent air bubbles in the bulb.
(4) Mix the contents of the pipet for 3-5 minutes to ensure even distribution
of cells. Expel unmixed and relatively cell-free fluid from the capillary portion of the
pipet (usually 4 drops).
(5) Place the forefinger over the top (short end) of the pipet, hold the pipet
at a 450 angle, and touch the pipet tip to the junction of the cover glass and the counting
(6) Allow the mixture to flow under the cover glass until the chamber is
completely charged. Similarly, fill the opposite chamber of the hemacytometer.
If the mixture overflows into the moat or air bubbles occur, clean and dry the
chambers, remix the contents of the pipet, and refill both chambers.
(7) Allow the cells to settle for about 3 minutes. Under low-power
magnification and reduced light, focus on the ruled area and observe for even
distribution of cells.
(8) Count the white cells in the four 1 sq mm corner areas corresponding to
those marked A, B, C, and D of Figure 5-1 in each of two chambers.
(9) Count all the white cells lying within the square and those touching the
upper and right-hand center lines. The white cells that touch the left-hand and bottom
lines are not to be counted. In each of the four areas, conduct the count as indicated by
the "snake-like" line in figure 5-1. A variation of more than 10 cells between any of the
four areas counted or a variation of more than 20 cells between sides of the
hemacytometer indicate uneven distribution and require that the procedure be repeated.