Bio-Rad CHEF-DR® III Variable Angle System - Manual (Page 17)

Fig. 3.1. Hemocytometer grid.
Mammalian or tissue culture cells
Because of the large size, tissue culture cells can 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:
Cells Counted
= Average Cells per Square
Number of Center Squares
Average Cells per Square x Dilution Factor x 10 4 = Cells per ml
Use the following ratio to determine how many ml of cell suspension to use to achieve the
desired cell concentration for the plugs.
5 x 10 7 Cells Desired
x ml of plugs to be made = ml of cell suspension to use
Actual Cell Concentration
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 actual cells concentration = 2.1 ml of cell suspension is required
to make 5 ml of agarose plugs.
Bacteria and yeast cells
Count five to ten of the 25 center squares, at 400x power, to get a representative sample
of your cell suspension. You should have approximately 25 to 75 cells per square. The cells
should be relatively free of clumps. Bacteria which naturally chain or grow in clusters are
relatively easy to count and do not have to be dispersed by chemical or enzymatic methods.
The Grams Crystal Violet aids in the visualization of bacteria.
Use the equations below to determine the cell concentration:
Cells Counted
= Average Cells per Square
Number of Squares
Average Cells per Square x 25 Squares x Dilution Factor x 10 4 = Cells per ml
Use the following ratio to determine how many ml of cell suspension to use to achieve the
desired cell concentration for the plugs.
Desired Cell Concentration
x ml of plugs to be made = ml of cell suspension to use
Actual Cell Concentration
A
B
C
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