Bio-Rad CHEF Genomic DNA Plug Kits - Manual (Page 11)

A. 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 below:
Average Cells per square
×
Dilution Factor
×
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.
For Example: 500 cells in 10 squares = average of
50 cells /square x 5 (dilution factor) x 10 4 = 2.5 x 10 6 cells per
ml. For 1 ml of plugs you need 1 ml x (5 x 10 7 ) cells final
concentration divided by 2.5 x 10 6 actual cells concentration =
20 ml of cell suspension is required to make 1 ml of agarose
plugs.
B. 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
5 x 10 7 cells desired
actual cell concentration
× ml of plugs to be made = ml of cell suspension
to use.
Cells Counted
Number of Center Squares
= Average Cells per Square
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9.3 Hemocytometer Usage
A hemocytometer is usually divided into nine large
squares (Figure 1). Each large square is 1 x 10 -4 cm 2 or 0.1
mm 3 , two such squares are shown the figure with darkened
borders (A&B). 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 is subdivided into 25
smaller squares. The smaller circle in the center square (C)
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. 1. Hemocytometer grid.
A
B
C
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LIT510E 2/11/99 3:47 PM Page 16