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Counting Cells With A Hemacytometer: Procedure

The document provides instructions for counting cells with a hemacytometer. A hemacytometer has etched grids that divide its chambers into squares of known volume. To count cells, a diluted and killed sample is loaded and allowed to settle before counting the cells within squares. The number of cells counted is then used to calculate the concentration of cells in the original sample volume based on the dilution factor and hemacytometer specifications.

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64 views3 pages

Counting Cells With A Hemacytometer: Procedure

The document provides instructions for counting cells with a hemacytometer. A hemacytometer has etched grids that divide its chambers into squares of known volume. To count cells, a diluted and killed sample is loaded and allowed to settle before counting the cells within squares. The number of cells counted is then used to calculate the concentration of cells in the original sample volume based on the dilution factor and hemacytometer specifications.

Uploaded by

asfand yar
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 DOCX, PDF, TXT or read online on Scribd
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Counting Cells with a Hemacytometer

Hemacytometers were developed for counting blood cells, but can also be used to count
spermatozoa. A hemacytometer has two chambers and each chamber has a microscopic
grid etched on the glass surface. The chambers are overlaid with a glass coverslip that
rests on pillars exactly 0.1 mm above the chamber floor. Thus, the volume of fluid above
each square of the grid is known with precision.

Procedure
The semen must be killed to prevent movement and diluted before loading into the
hemacytometer. This can be done by diluting the semen into a buffer containing a small
quantity of formaldehyde. The dilution factor must be recorded to allow calculating the
concentration. When there are 20-25 cells per large square (see below) the sample is at the
proper dilution.

Loading the Hemacytometer: The tip of the pipette is


placed in the V-shaped groove on the hemacytometer
to load the sample into the chamber (about 15
microliters.) Capillary action will draw the fluid into the
chamber. It is important not to overload the chamber,
as doing so will give an inaccurate count. The same is
true if the cover slip is moved after the sample is
loaded.

The sample is allowed to settle for 2 or 3 minutes so


that the cells stop drifting around the chamber and
most will be in the same plane of focus. It is important
not to allow the sample to settle too long or it will dry
out, concentrating the cells over the grid. To avoid
drying, the hemacytometer can be placed on straws
within a petri dish containing a moistened filter paper.

Counting: The full grid on a hemacytometer contains nine squares, each of which is 1 mm


square (see figure below). The central counting area of the hemacytometer (as it will be
called here) contains 25 large squares and each large square has 16 smaller squares.
When counting, count only those cells on the lines of two sides of the large square to avoid
counting cells twice.

The example at right shows red lines


where cells on the line would be
counted. If red dots represent cells, one
would count 3 cells in the top middle
large square.

All 25 large squares can be counted, or a counting pattern using fewer squares can be used
like the ones below. It is important to distribute the counting areas in a non-biased manner
since cells can be more concentrated on one side of the chamber. If you count over only 5
of the 25 large squares, then multiply that value by 5 to obtain the number of cells per
central counting area.
At least two chambers should be counted, including at least 100 cells within each central
counting area of each chamber. For higher precision, additional sperm can be counted and
the average used to calculate cell concentration.

Calculating Concentration
Each of the nine squares on the grid, including the central counting area of 25 large
squares, has an area of 1 square mm, and the coverglass rests 0.1 mm above the floor of
the chamber. Thus, the volume over the central counting area is 0.1 mm 3 or 0.1 microliter.
You can thus multiply the average number of sperm over each central counting area by
10,000 to obtain the number of sperm per ml of diluted sample.

In other words, to calculate the number of sperm per ml of original sample:

1. Calculate the mean number of sperm counted for each chamber (i.e. for each of the
central counting areas of each chamber).
2. Multiply the mean obtained in (1) by 10,000 to obtain the number of cells per ml of
diluted sample.
3. Multiply the count obtained in (2) by the dilution factor.

Example: Assume that you dilute the original semen sample by adding 0.1 ml of semen to
9.9 ml of diluent (1:100 dilution factor). You then count the number of sperm in 5 of the 25
large squares within the central counting area of two chambers, obtaining counts of 132 and
128 cells.

1. The mean number of sperm per chamber is thus 130 x 5 or 650 cells per counting
area (650 cells per 0.1 microliter).
2. Multiply the 650 cells per counting area by 10,000 to obtain the number of cells per
ml of diluted sample (answer = 6,500,000)
3. Multiply 6,500,000 cells per ml of diluted sample by 100 (the dilution factor) to obtain
650,000,000 per ml of original semen sample.

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