Part V: Counting cells
Welcome to the fifth of a series of articles providing useful hints for
culturing animal cells. This article contains considerations for counting
This protocol has been adapted from references 13. Protocols in this
series are examples of methods for general cell culture and have not been
rigorously validated and optimized by QIAGEN. There are many alternative
protocols in current use.
Cell counting using a hemocytometer
It is often necessary to count cells, for example, when plating cells for
transfection experiments. One method for counting cells is to use a hemocytometer.
A hemocytometer contains 2 chambers (Figure 1). Each chamber is ruled into
9 major squares (volume of 0.1 mm3 or 1 x 104 ml each). Cell concentration
is determined by counting the number of cells within a defined area of known
1. Clean the surface of the hemocytometer with 70% ethanol or another suitable
disinfectant, taking care not to scratch the surface of the central area.
Dry with lens paper.
2. Clean the coverslip, wet the edges very slightly, lay it over the grooves
and central area of the hemocytometer and gently press down.
Tip - It is important that the coverslip is properly attached to obtain
the correct chamber depth. The appearance of Newtons rings (bright
and dark rings caused by interference in the air between the coverslip and
the glass surface of the hemocytometer) will confirm that the coverslip
is attached properly.
3. Harvest the cells, either by trypsinization (adherent cell cultures;
see QIAGEN News 2002 No. 3, 20) or by centrifugation at 200 x g for 5 min
(suspension cell cultures). Resuspend the cells in an appropriate volume
of prewarmed growth medium. At least 106 cells/ml are required for accurate
Tip - It may be necessary to centrifuge cells and resuspend in a smaller
volume to obtain the desired cell concentration for counting. For adherent
cells, it is important to produce a single-cell suspension after trypsinizing.
Cell clumping will make counting difficult and inaccurate.
4. Mix the cell suspension sample thoroughly. Using a pipet, immediately
transfer 20 l to the edge of one side of the coverslip to fill one
chamber of the hemocytometer. Repeat for the second chamber.
Tip- The cell distribution should be homogeneous in both chambers.
Tip - The cell suspension is drawn under the coverslip and into the chamber
by capillary action. The cell suspension should just fill the chamber. Blot
off any surplus fluid without disturbing the sample underneath the coverslip.
5. Transfer the slide to the microscope, and view a large square ruled by
3 lines using a 10x objective and 10x ocular. Count the total number of
cells in 5 of the 9 major squares.
Count cells that overlap the top and left border of squares but not those
overlapping bottom and right borders (Figure 1). This prevents counting
overlapping cells twice. If the cell density is too high, the cell suspension
should be diluted, noting the dilution factor.
6. Repeat the counting for the second chamber to give a total of 10 squares.
7. Add the number of cells counted in all 10 squares together to give the
number of cells in 1 x 103 ml. Multiply by 1000 to give the number
If the original cell suspension was diluted for counting,
multiply by the dilution factor to obtain the number of cells/ml.
8. Clean the hemocytometer and coverslip by rinsing with 70% ethanol and
then with distilled water. Dry with lens paper.
Counting Cells Using a Hemocytometer
Figure 1 A
One chamber of a hemocytometer slide under 10x objective
and 10x ocular. The chamber is divided into 9 major squares. B
view of one
of the 9 major squares. Only cells that overlap the top and left borders
of squares should be counted to avoid overestimating the cell concentration.
O: cells that should be counted; : cells that should be ignored.
1. Freshney, R.I. (1993)
Culture of Animal Cells:
a Manual of Basic
Technique. 3rd ed. New
2. Ausubel, F.M. et al., eds.
(1991) Current Protocols
in Molecular Biology.
New York: Wiley
3. Spector, D., Goldman,
R.R., and Leinwand, L.A.,
eds. (1998) Cells: a
Laboratory Manual. Cold
Spring Harbor, NY: Cold
Spring Harbor Laboratory
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