When the coverslip is positioned correctly, the volume of liquid over each square (subgrid) is a known constant, 0.1mm 3 or 1.0 x 10-4mL. If cells are counted in each of the corner squares and in the central squares of each of the two chamber grids, a total volume of 1 x 10 -3 mL (10 x 0.1 mm 3 = 1.0 mm 3) will have been examined, and the cells per milliliter (con- centration) of the sample added to the hemocytometer can be determined by multiplying the count by 1000. It should be noted that when cells are diluted in stain or buffer during preparation, then multiplication by the appropriate dilution factor is also required to obtain the concentration of the sample. Determination of viability with the hemocytometer method involves the dye exclusion test. This test relies on the ability of living cells to exclude certain stains from crossing the cell membrane. Dead cells are permeable and will take up the stain.