LEVEL I

Figure 1.8 Improved Neubauer hemacytometer

**Materials**

- Microscope
- Hemacytometer and coverslip
- Suspension of yeast 13

**Procedure**

- 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.
14
- 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 squares marked 1-4, the area of each is 1
mm, and
the volume is .1 mm.
Since .1 mm
equals 10
ml, the number of cells/ml = Average # of cells per 1
mm times
10 times
any sample dilution.
- For the 25 smaller squares in the center of the grid
marked 5, each small square is 0.2 x 0.2 mm,
and the volume is thus 0.004 mm.
For small cells, or organelles, the particles/ml equals the
Average # of particles per small square times 25 X
10
times any sample dilution.
- Grids 1-5 are all 1 mm. Grids 1-4 are divided into 16 smaller squares (0.25 mm on each side), grid 5 is divided into 25 smaller squares (0.2 mm on each side). Grid 5 is further subdivided into 16 of the smallest squares found on the hemacytometer.

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.
Square # Cell Count 1 2 3 4 5 Area of each square = _______________ mm x 0.1 mm depth = volume of each square.

Volume of each square = _______________ mm

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. - The coverslip is 0.1 mm above the grid, and the lines etched on the grid are at preset dimensions.

Cell Biology Laboratory Manual

Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,

St. Peter, MN 56082 -- cellab@gac.edu