Exercise 1.6 - Measuring Volume
Figure 1.8 Improved Neubauer hemacytometer
- Hemacytometer and coverslip
- Suspension of yeast 13
- Make a serial dilution series of the yeast suspension,
from 1/10 to 1/1000.
- Obtain a hemacytometer and place it on the desk before
you. Place a clean coverslip over the center chamber.
- Starting with the 1/10 dilution, use a pasteur pipette
to transfer a small aliquot of the dilution to the
hemacytometer. Place the tip of the pipette into the V
shaped groove of the hemacytometer and allow the cell
suspension to flow into the chamber of the hemacytometer by
capillary action until the chamber is filled. Do not
overfill the chamber.
- Add a similar sample of diluted yeast to the opposite
side of the chamber and allow the cells to settle for about
1 minute before counting.
- Refer to the diagram of the hemacytometer grid in
Figure 1.8 and note the following:
- The coverslip is 0.1 mm above the grid, and the lines
etched on the grid are at preset dimensions.
- The four outer squares, marked 1-4, each cover a a
volume of 10 ml.
- The inner square, marked as 5, also covers a volume of
10 ml, but
is further subdivided into 25 smaller squares. The volume over each
of the 25 smaller squares is 4.0 X 10 ml.
- Each of the 25 smaller squares is further divided into
16 squares, which are the smallest gradations on the
hemacytometer. The volume over these smallest squares is .25
X 1 ml.
- Given these volumes, the number of cells in a sample
can be determined by counting the number of cells in one or
more of the squares. Which square to use depends on the size
of the object to be counted. Whole cells would use the
larger squares, counted with 10X magnification. Isolated
mitochondria would be counted in the smallest squares with
at least 40X magnification.
For the yeast suspension, count the number of cells in 5 of
the intermediate, smaller squares of the hemacytometer. For
statistical validity, the count should be between 10 and 100
cells per square. If the count is higher, clean out the
hemacytometer and begin again with step 3, but use the next
dilution in the series.
Record the dilution used, and the five separate counts.
- Average your counts, multiply by the dilution factor,
and calculate the number of cells/ml in the yeast
suspension. Record this information in the space provided.
Area of each square = _______________
x 0.1 mm depth = volume of each square.
Volume of each square = _______________
Average number of cells per mm = _______________
Number of cells per cm
(1000 x above) = _______________
Note: Number of cells per cm
is also number per ml.
Number of cells per ml __________ x dilution factor (200) =
__________ cells per ml of whole blood.
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org