Submitted by Lauren French (download pdf)
 
  • Load the hematocrit with a small volume of the cell suspension (or a diluted volume).
  • With a 10x objective in the phase optics of a microscope, count the cells.  There are 9 large 1mm x 1 mm squares.  The total cells in one of these 9 squares are counted and multiplied by 104 to obtain the number of cells/ml.
  • In practice, count one row of the 5 small squares that comprise the center large square on the 3 x 3 grid of squares.  Average the numbers from each of the 5 small squares and multiply by 5 to get the number of cells x 104.

 

Example: In the 5 smallest squares, the average number of cells counted was 131 cells.  131 cells x 5 = 655 cells, which is 655 x 104 cells/ml in the cell suspension (which contains 5 mls less the volume to count which is inconsequential).  You may want to dilute the initial 5 ml volume by 10-fold to make counting cells a bit easier, however since the cells are fairly clumpy, good resuspension will be key to accurate counting.  By viewing the entire 3 x 3 grid of squares, it will be apparent how representative the center square is for all of the squares.

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