Bio-Rad CHEF-DR II System - Manual (Page 19)

3.8 Hemocytometer Usage
A hemocytometer is usually divided into nine large squares (Figure 3.1). Each large square
is 1 x 10 -4 cm 2 or 0.1 mm 3 , one such square (A) is shown in the figure with darkened borders.
The large circle around the center square (B) represents your field of view at 100x power
(10x objective lens, 10x eye piece). The center square (C) is subdivided into 25 smaller
squares. The smaller circle in the center square represents your field of view at 400x power
(40x objective lens, 10x eye piece). These 25 center squares are further divided into 16 squares.
Fig. 3.1. Hemocytometer grid.
A. Mammalian or Tissue culture cells:
Because of the large size, tissue culture cells are able to be counted at 100x power. Count
10 of the large squares, five on each side of the hemocytometer. Determine the average cells
per square using the equations below:
Hemocytometer Equations:
For Example: 230 cells in 10 squares = average of 23 cells /square x 5 (dilution factor)
x 10 4 = 1.2 x 10 8 cells per ml. So for 5 ml of plugs you need 5 ml x 5 x 10 7 cells final con-
centration divided by 1.2 x 10 8 cells/ml concentration = 0.41 ml of cell suspension is required
to make 5 ml of agarose plugs.
16
A
B
C
Cells Counted
= Average Cells per Square
Number of Squares
Desired Cell Concentration
(ml of plugs to be made) = ml of cell suspension needed
Actual Cell Concentration
(Average Cells per Square)(Dilution Factor)(10 4 ) = Cells per ml.