 
Summary: Counting Cells with the Hemocytometer
1. Vortex culture vigorously. If densely saturated, dilute culture 10fold.
2. Center hemocytometer cover slip between the outside railings over the two counting grids. Use
only a hemocytometer cover slip, which is thick to prevent flexing due to capillary action.
3. Place a 10  20 µl droplet of a wellmixed cell suspension at each notch. Add droplet all at once to
ensure rapid and even distribution of cells. Alternatively, add droplet to grid, then add cover slip.
4. Count cells using 400X magnification (40X objective + 10X eyepiece). Use push button counter.
· For dilute cultures, count cells in 1 mm squares (= 0.1 µl volume). Ideally, count eight 1 mm
squares on 2 grids. If counting multiple samples, count four 1 mm squares on 1 grid. Calculate the
average. Multiply the average by 1 X 104
to determine the number of cells per milliliter.
· For concentrated cultures, count eight of the 1/16 sq. mm squares (2 in each of the 4 corners).
Calculate the average. Multiply the average by 1.6 X 105
to give the number of cells per milliliter.
· For E. coli, count ten of the smallest squares in middle section of the hemocytometer. These
squares are 1/400 sq. mm (1/25 X 1/16 sq. mm). Calculate the average. Multiply the average by
4.0 X 106
to determine the number of cells per milliliter.
