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P.J. Hansen: Use of A Hemacytometer

This document describes procedures for using a hemacytometer to count cells. It details how to fill the hemacytometer, stain cells, and use two counting methods to calculate cell concentration based on the number of cells counted in specific areas of the hemacytometer grid.

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86 views2 pages

P.J. Hansen: Use of A Hemacytometer

This document describes procedures for using a hemacytometer to count cells. It details how to fill the hemacytometer, stain cells, and use two counting methods to calculate cell concentration based on the number of cells counted in specific areas of the hemacytometer grid.

Uploaded by

Santi Wilujeng
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as PDF, TXT or read online on Scribd
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Laboratory Procedures, PJ Hansen Laboratory - University of Florida

Use of a Hemacytometer

P.J. Hansen1
1
Dept of Animal Sciences, University of Florida

A hemacytometer (also spelled hemocytometer) is an etched glass chamber with raised sides that will
hold a quartz coverslip exactly 0.1 mm above the chamber floor. The counting chamber is etched in a
2
total surface area of 9 mm (see Figure 1).

Figure 1. Dimensions of a hemacytometer.

Calculation of concentration is based on the volume underneath the cover slip. One large square (see W
in Figure 2) has a volume of 0.0001 ml (length x width x height; i.e., 0.1 cm x 0.1 cm x 0.01 cm).

In both methods, the hemacytometer is filled by capillary action - place the pipette filled with a well-
suspended mix of cells at the notch at the edge of the hemacytometer and then slowly expel some
contents so that the fluidis drawn into the chamber by capillary action.

Staining of cells often facilitates visualization and counting. Either mix cells with an equal volume of
trypan blue [0.4% (w/v) tyrypan blue in PBS] to determine live/dead count (dead cells are blue) or kill cells
with 10% formalin and then stain with trypan blue or other stain (to improve visualization of all cells.

Here are two simple methods for counting cells based on the surface area of the hemacytometer used to
determine cell count. Other counting schemes are accetable also. The choice of methods depends upon
the cell concentration - the accuracy of the procedure depends upon the number of cells counted. When
cell concentration is low, one should count more grids.

Method A
Count the number of cells in the 4 outer squares (see the left panel of Figure 2).

The cell concentration is calculated as follows:

Cell concentration per milliliter = Total cell count in 4 squares x 2500 x dilution factor

Example: If one counted 450 cells after diluting an aliquot of the cell suspension 1:10, the original cell
concentration = 450 x 2500 x 10 = 11,250,000/ml

1 | Hemacytometer
Laboratory Procedures, PJ Hansen Laboratory - University of Florida

Method B
Estimate cell concentration by counting 5 squares in the large middle square (see the right panel in
Figure 2).

The cell concentration is calculated as follows:

Cell concentration per milliliter = Total cell count in 5 squares x 50,000 x dilution factor

Example: If one counted 45 cells after diluting an aliquot of the cell suspension 1:10, the original cell
concentration = 45 x 50,000 x 10 = 22,500,000/ml

Figure 2. Counting procedure for Methods A (left panel) and B (right panel).

Text, PJ Hansen, 2000


written 9-90; prepared for website, 8-31-00; modified 10-30-01

2 | Hemacytometer

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