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2. THE HEMOCYTOMETER (COUNTING CHAMBER)

The hemocytometer (or haemocytometer or counting chamber) is a specimen slide which is used to
determine the concentration of cells in a liquid sample. It is frequently used to determine the concentration
of blood cells (hence the name “hemo-“) but also the concentration of sperm cells in a sample. The
hemocytometer (or haemocytometer or counting chamber) is a specimen slide which is used to determine
the concentration of cells in a liquid sample. It is frequently used to determine the concentration of blood
cells (hence the name “hemo-“) but also the concentration of sperm cells in a sample. The cover glass,
which is placed on the sample, does not simply float on the liquid, but is held in place at a specified height
(usually 0.1mm). Additionally, a grid is etched into the glass of the hemocytometer. This grid, an
arrangement of squares of different sizes, allows for an easy counting of cells. This way it is possible to
determine the number of cells in a specified volume.
For microbiology, cell culture and many of the applications that require use of cell suspensions, it is
necessary to determine the concentration of cells. The device used for determining the number of cells per
unit volume of a suspension is called a counting chamber. It is the most widely used type of chamber, since
it was mainly designed for performing blood cell counts. It is now used to count other types of cells and
other microscopic particles as well.

The hemocytometer was invented by Louis-Charles

Malassez. It is a special type of microscope slide
consisting of two chambers, which is divided into nine
(1.0mm x 1.0mm) large squares which are separated from
one another by triple lines. The area of each is
1mm². Cover glass is supported over the chambers at a
height of 0.1mm. Because of that the entire counting grid
lies under the volume of 0.9 mm² on one side. The cell
suspensions are introduced into the cover glass. The
hemocytometer is placed on the microscope stage and the
cell suspension is counted..

The glass microscope slide has a rectangular indentation that creates an 'H' shaped chamber at the centre.
This chamber is engraved with a laser-etched grid of perpendicular lines. Two counting areas with ruled
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grids are separated by the horizontal groove of the 'H'. There is also a very flat, reusable cover slip. The
glass cover slip is held at 0.1 mm above the surface of the counting areas by ground glass ridges on either
side of the vertical grooves of the H shape. The device is carefully crafted so that the area bounded by the
depth and lines of the chamber is also known. Because the height is constant, the volume of fluid above
each square of the grid is known with precision.

The hemocytometer is used by putting the cover slip on the device, and filling the space with a liquid
containing the cells you want to count. There is a "V" or notch at either end which is the place where the
cell suspension is loaded into the hemocytometer. The fluid is usually drawn into the space by capillary
action. A cover glass, which is placed on the sample, does not simply float on the liquid, but is held in place
at a specified height. In addition, the grid arrangement of squares of different sizes allows for an easy
counting of cells. It is possible to identify the number of cells in a specified volume by this method..
The ruled area of the hemocytometer consists of several large 1 x 1 mm (1mm² ) squares, which are
subdivided in three ways; 0.25 x 0.25 mm (0.0625 mm²), 0.25 x 0.20 mm (0.05 mm²) and 0.20 x 0.20 mm
(0.04 mm²). The central, 0.20 x 0.20 mm marked, 1 x 1 mm square is further subdivided into 0.05 x 0.05
mm (0.0025 mm²) squares. Hold the cover slip( 0.1 mm) at the raised edges of hemocytometer, which
gives each square a defined volume.
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A number of stains have been employed to distinguish between viable and nonviable cells. This is based on
the principle that live cells contain intact cell membranes that eliminate certain dyes, like trypan blue,
Eosin, or propidium. In dead cells, the stain enters the cytoplasm and the cells take on the stain. If more
than 25% of the cells are stained, the cell suspension is most likely not a viable one.

To prepare the counting chamber, the mirror-like polished surface is carefully cleaned with 75% ethanol
and the cover slip is also cleaned.. The cover slips used for counting chambers are specially made, and are
thicker than those cover slips used for conventional microscopy, since they must be heavy enough to
overcome the surface tension of a drop of liquid. A cover slip is placed on the counting surface prior to
putting on the cell suspension. Introduced any of the cell suspension into any of the V-shaped wells with
a micropipette. The area under the cover slip fills by capillary action. Sufficient liquid should be
introduced, so that the mirrored surface is just covered. The charged counting chamber is placed
under the microscope stage and the counting grid is brought into focus at low power.

Materials Required

1. Hemocytometer plus a supply of cover slips.

2. Uniform cell suspension.
3. 0.4% Trypan Blue stain (fresh & filtered) in phosphate buffered saline.
4. Tally Counter.
5. Cell Suspension.
6. Micropipettes.

Procedure

1. Obtain a uniform suspension of cells: Follow the typsinization/trypsin neutralization protocol for
the specific cell type. Place the cell suspension in a suitably-sized conical centrifuge tube. For an accurate
cell count to be obtained, a uniform suspension containing single cells is necessary. Pipette the cell
suspension up and down in the tube 5-7 times using a pipette with a small bore (5 ml or 10 ml pipette). For
cells thawed from cryopreservation (in 1ml cryopreservation medium), pipette up and down 7-10 times
using a one ml pipette.
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2. Prepare a 1:1 dilution of the cell suspension in trypan blue: Approximately 10 microliters of cell
suspension will be required to charge one chamber of the hemocytometer. In a conical microfuge tube, add
10 microliters of 0.4% trypan blue solution. Gently swirl (finger vortex) the cell suspension and remove 10
microliters of it using sterile technique. Combine the 10 microliters of cell suspension with the 10
microliters of trypan blue in the microfuge tube. Pipette up and down several times to ensure a uniform
cell suspension using the same pipette tip and allow to stand for 5-15 minutes.

3. Load the hemocytometer: Moisten and affix cover slip to the hemocytometer. Ensure the cover slip
and hemocytometer are clean and grease-free (use alcohol to clean). A small amount of trypan blue-cell
suspension is transferred to one of the chambers of the hemocytometer by carefully touching the cover
slip at its edge with the pipette tip and allowing each chamber to fill by capillary action. The chamber
should not be overfilled or underfilled.

4. Determine the number of cells (total and viable): View the cells under a microscope at 100x
magnification. Under the microscope, you should see a grid of 9 squares. Focus the microscope on one of
the 4 outer squares in the grid. The square should contain 16 smaller squares. Count all the cells in the four
1 mm corner squares. If there are too many or too few cells to count, repeat the procedure, either
concentrating or diluting the original suspension as appropriate.

For an accurate determination, the total number of cells overlying one 1 mm2 should be between 15 and
50. If the number of cells per 1 mm2 exceeds 50, dilute the sample and count again. If the number of cells
per 1 mm2 is less than 15, use a less diluted sample. If less dilute samples are not available, count cells on
both sides of the hemocytometer (8 x 1 mm2areas).

Keep a separate count of viable and non-viable cells. If more than 25% of cells are non-viable, the culture is
not being maintained on the appropriate amount of media. Reincubate the culture and adjust the volume
of media according to the confluency of the cells and the appearance of the media. Include cells on top and
left touching middle line. The cells touching middle line at bottom and right are not counted.
Estimating Cell Density Activity

The figure below represents the view of the hemacytometer through a microscope. The circles
represent cells that had previously been cultured in a Petri dish.

A 0.5 ml suspension of cells were removed from the Petri dish and mixed with 0.5 ml Trypan Blue
solution. Recall that Trypan Blue is a stain that selectively stains dead cells. The green dots in the
figure represent live cells and the blue dots are dead cells that have taken up the Trypan Blue.

Live cells Dead cells stained with Trypan Blue

Chamber A

Blank grids from: http://www.microbehunter.com/2010/06/27/the-hemocytometer-counting-chamber/

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Chamber B

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Procedure
1. Count the live and dead cells in each of the four quadrants in each of the counting
chambers (A & B) of the hemacytometer. Calculate the averages for each counting
chamber.
Chamber A
Quadrant #Live Cells #Dead Cells Total # Cells
1
2
3
4
Average
#cells:

Chamber B
Quadrant #Live Cells #Dead Cells Total # Cells
1
2
3
4
Average
#cells:

2. Calculate the average #cells from chambers A & B

Average # Average # Average #
Live Cells ____________ Dead Cells____________ Total Cells____________

3. Calculate the cell density.

# Cells/ml = Average # cells X dilution factor x 104

The dilution factor for this example is 2 because 0.5 ml of cell suspension
was diluted with 0.5 ml Trypan Blue.
# Cells/ml = _____________ X 2 x 104

# Cells/ml = ____________

4. Calculate cell viability

Average # live cells / Average # total cells x 100
______________ / ______________ x 100

Cell viability = ____________%

5. Why did only the dead cells take up the Trypan Blue stain and not the living cells? (Hint:
think about the structure and function of a cell)

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