Introduction to Counting Cells How to Use a Hemacytometer

With a lab partner, discuss and answer the following questions:

Why might an investigator want to determine the total number of cells growing in a Petri
dish, in a flask, or in a test tube?
How could you estimate the number of cells in a Petri dish? What are some
limitations of your proposed method?

Today you will learn one method that is used to determine the amount of cells growing in a
culture vessel using a counting chamber or slide called a hemacytometer. View the following
video before you continue with this activity: http://www.youtube.com/watch?v=wPeNK0pRIpA

The Hemacytometer
Figure 1: Hemacytometer Slide http://en.wikipedia.org/wiki/File:Hemocytometer.jpg#filelinks

*Counting Grids:
2 grids/hemacytometer

Figure 2: The counting grids are located on the reflective portions* of the hemacytometer
and are visible using the microscope.
http://www.microbehunter.com/2010/06/27/the-hemocytometer-counting-chamber/

Note the 4 quadrants in the 4 corners of the

counting grid indicated by the arrows.
Each is composed of 16 squares.

Preparing Sample
To obtain an accurate estimate of the cell density of your sample the cells need to be evenly
suspended in your culture vessel before you remove an aliquot for counting. The method will
vary depending on your culture method. For algae, you might be sampling from a flask or test
tube. For cell cultures you probably are sampling from a Petri dish. To resuspend cells in a flask
or test tube you can simply swirl the flask or invert the test tube and then quickly remove your
aliquot of cells. For a cell culture growing in a Petri dish, you need to resuspend the cells growing
on the bottom of the dish by gently using a pipette to remove cells and media from a dish and
then gently expelling them back into the dish. This aspiration of the contents of the Petri dish
should be completed several times, each time expelling the cells and media while moving the
pipette across the bottom of the dish to gently discharge cells growing on the entire surface. After
resuspension the aliquot is quickly removed from the vessel before the cells settle to the bottom
again.
For cell culture applications cells are often stained with Trypan Blue so that dead cells can be
distinguished from live cells. For example, a 0.5 ml suspension of cells would be removed from
the Petri dish and mixed with 0.5 ml Trypan Blue solution in an Eppendorf or small test tube.
Trypan Blue is a stain that selectively stains dead cells. For algal cells, Lugols solution (KI3) is
commonly used to stain an aliquot of cells in a small test tube. The Lugols solution will
immobilize and kill the algal cells. The amount of stain used to dilute the cell suspension must be
measured and recorded so that you can apply it as your dilution factor in the final calculations.

Loading Samples
The goal when loading your sample of cells is to obtain an even distribution of cells on the
hemacytometer slide. In order to achieve this result the slide has to be very clean and you must
load your sample quickly and smoothly. It is important to handle the hemacytometer and the
cover slip carefully. Never place your fingers on the reflective surface of the slide.

Always clean the slide before you load the sample by rinsing the slide and cover slip with
70-95% ethanol. Air dry or gently wipe the slide and cover slip with lens paper or
Kimwipes. Never use paper towels or soaps.
Place the clean and dry slide on your work surface and place the coverslip on top to cover
the reflective surfaces.
Figure 3: Hemacytometer with cover slip
http://www.vivo.colostate.edu/hbooks/pathphys/reprod/semeneval/hemacytometer.html

If you have diluted your cells in a test tube, invert the tube several times to resuspend the
cells.
Using a micropipette, quickly and smoothly without interruption, add 10 l of your cell
suspension (or 1 drop from a transfer pipette) to the v-shaped groove on each side of the
hemacytometer., If the slide is clean, the suspension should move quickly under the cover
slip covering the entire reflective surface of the hemacytometer. The suspension should
not flow into the channels or gutters along the slides of the reflective surface. If your
sample moves into the gutters you may not have loaded the sample in the correct location
or you may have used too large of an aliquot. Practice first and make any adjustments
that are necessary. If the sample does not flow quickly across the surface the
hemacytometer may not be clean or you may not have expelled
the solution quickly enough.

Figure 4: Loading the cells on the hemacytometer using a

micropipette
http://www.vivo.colostate.edu/hbooks/pathphys/reprod/semeneval/hemacytometer.html

-2-
Estimating Cell Density using a Hemacytometer
You should be able to visualize the grid of the hemacytometer when viewing through a compound
microscope. You will need to utilize your good microscope skills taking care to focus well and
adjust the iris so that you have good contrast to view the grid. The hemacytometer is much
thicker than a normal slide so you need to be careful and note the reduced working distance. It is
a good idea to examine a hemacytometer without a sample using the microscope before you begin
your counts.

Consistency is important in science. In using a hemacytometer it is important use a consistent

counting methods. Different laboratories might use different patterns of counting but you will
utilize the method figured below today. You will count all of cells within each of the four large
quadrants in the four corners of each counting chamber on the hemacytometer (see Figure 2).
Count all of the cells within each quadrant except those on the far right edge and lower bottom
edge (see Figure 5 below). If the number of cells is high, you should use a tally counter to keep
track of your counts. View the hemacytometer at the highest magnification that allows you to see
an entire quadrant and then focus to visualize the cells.

Figure 5: Cells in one quadrant of the hemacytometer as viewed through a microscope.

http://www.hpacultures.org.uk/technical/ccp/cellcounting.aspx

-3-
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/

-4-
Chamber B

-5-
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)

-6-