4-7 Using a counting chamber
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The direct cell counts will be demonstrated using Petroff-Hauser cytometers. These are glass slides with precisely machined chambers and coverslips, so that cells in very small volumes can be counted using the microscope.
The chamber is formed by laying the coverslip on the elevated borders of the central well area. See the diagram below:
Figure 4-12 Arrangement of the Petroff-Hauser counting chamber
The drawing shows the correct placement of the cover slip. Once the coverslip is placed on the slide, a small amount of culture (about 10 Âµl) is placed in the well on the left or right of the viewing area. Capillary action then brings the liquid onto the viewing area with the grid.
Figure 4-13 shows two images of a Petroff-Hauser counting chamber. Use this to see what it looks like and for performing a count of the microbes.
To determine the concentration of bacteria in the original culture use the following formula
Figure 4-14 Formula for the counting chamber
Use this formula for calculating the number of cells per ml from the count obtained using a counting chamber. Nc is the average number of cells counted per square and D is the dilution of the samples placed on the slide.
The 103 is there as a conversion factor from mm3 as measured by the chamber to cm3 (a.k.a. ml) as typically expressed for culture density. Here is a more detialed explanation of that conversion factor:
1 ml = 1 cm3 = 1 cm x 1 cm x 1 cm
1 cm = 10 mm
so 1 ml = 10 mm x 10 mm x 10 mm
or 1 ml = 103mm3
If you use one drop (without dilution) from a broth culture: and find an average of 2.31 squares per cell, your results would be:
Figure 4-15 Calculation of cell number from a counting chamber
If an average of 2.31 cells if found in a 10-1 dilution, the formula would appear as shown here with a result of 4.62 x 108 cells per ml of culture.