7. Immediately prior to cell counting, mix again by
gentle inversion, taking care to cover the upper
opening of the overflow chamber with your
8. Place the coverglass on the hemacytometer
counting chamber, making sure coverglass is
clean and free of grease. (Fingerprints must be
9. Remove the pipette from the reservoir. Squeeze
the reservoir and reseat the pipette in the reverse
position, releasing pressure to draw any fluid in
the capillary tube into the reservoir. Invert and
fill the capillary pipette by gentle pressure on the
reservoir. After discarding the first 3 drops, load
(charge) the counting chamber of the
hemacytometer by gently squeezing the
reservoir while touching the tip of the pipette
against the edge of the coverglass and the
surface of the counting chamber (fig. 7-13). A
properly loaded counting chamber should have
a thin, even film of fluid under the coverglass
(fig. 7-14, view A). Allow 3 minutes for cells to
settle. If fluid flows into the grooves (moats) at
the edges of the chamber or if air bubbles are
seen in the field, the chamber is flooded and
must be cleaned with distilled water, dried with
lens tissue, and reloaded (fig. 7-14, view B). If
the chamber is underloaded, carefully add
additional fluid until properly loaded.
10. Place the loaded hemacytometer into a petri dish
with a piece of dampened tissue to keep the
hemacytometer from drying out (fig. 7-15).
Allow 5 to 10 minutes for the cells to settle.
11. Once the cells have settled, place the
hemocytometer on the microscope. Use the
low-power lens to locate the five small fields (1,
2, 3, 4, and 5) in the large center square bounded
by the double or triple lines. See figure 7-16.
Each field measures 1/25 mm2, 1/10 mm in
depth, and is divided into 16 smaller squares.
These smaller squares form a grid that makes
accurate counting possible.
12. Switch to the high-power lens and count the
number of cells in field 1. Move the
hemacytometer until field 2 is in focus and
repeat the counting procedure. Continue until
the cells in all five fields have been counted.
Note the fields are numbered clockwise around
the chamber, with field 5 being in the center.
Figure 7-12.Mixing blood sample and diluent.
Figure 7-13.Loading the counting chamber.