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 approximately 100 cells, a minimum number of cells needed for a statistically significant count.
For large cells, you can simply count the cells inside the four large corner squares (Figure 3C-F) and the middle one (Figure 3B). For a dense suspension of small cells you may wish to count the cells in the four outer and middle squares of the central square (Figure 3B) or make a more dilute suspension.
Remember if a cell overlaps a ruling, count it as “in” if it overlaps the top or right ruling, and “out” if it overlaps the bottom or left ruling (Figure 3G).
The area of the middle (Figure 3B) and each corner square (Figure 3C-F) is 1 mm x 1 mm = 1 mm2: the depth of each square is 0.1 mm. The final volume of each square at that depth is 100nl.
Once you have obtained the total cell count, cell concentration can be calculated from the following formula: