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Measuring and Counting with a Light Microscope

Author(s): David R. Caprette, PhD

Using a Hemacytometer

One entire grid with Neubauer rulings can be seen at 40x.The main divisions separate the grid into 9 large squares (like a tic-tac-toe grid). Each square has a surface area of one square mm, and the depth of the chamber is 0.1 mm. Thus, the entire counting grid lies under a volume of 0.9 mm-cubed. The center grid is finely divided so that one can use the same chamber for counting dilute or very concentrated cells of variable diameters.

To perform the count, determine the magnification needed to recognize the desired cell type and systematically count the cells in selected squares so that the total count is 100 cells or so (number of cells needed for a statistically significant count). For large cells, this may mean counting the four large corner squares and the middle one. For a dense suspension of small cells, you may wish to count the cells in the four 1/25 sq. mm corners plus the middle square in the central square. To avoid overestimating cell counts, overlapping cells should be accepted or rejected according to a predetermined scheme, as described earlier in the presentation.

Here is how to determine a cell count. First, divide the total count by 0.1 (chamber depth) then divide the result by the total surface area counted. For example, if you counted a total of 125 cells in the four large corner squares plus the middle, divide 125 by 0.1, and then divide the result by 5 mm-squared, which is the total area counted (each large square is 1 mm-squared). 125/ 0.1 = 1250. 1250/5 = 250 cells/mm-cubed. There are 1,000 cubic mm per ml, so you calculate 250,000 cells/ml. Sometimes you will need to dilute a cell suspension to get the cell density low enough for counting. In such cases, you will need to multiply your final count by the dilution factor. For example, suppose you had to dilute a suspension of Chlamydomonas 10 fold. Suppose you obtained a final count of 250,000 cells/ml, as above. In this case, the count in the original (undiluted) suspension would be 10 x 250,000 which is 2,500,000 cells/ml.