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 µl) 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.

  1. Using a capillary pipette, place a drop of the broth culture at the edge of the coverslip. Capillary action will draw the liquid under the coverslip. Wait 1-2 min. for the movement to stop and the cells to settle. Then with the low-power objective focus on the grid in the center of the slide; you should see a crosshatched area containing 25 squares each containing 16 squares:

  2. You will not be able to see many bacteria at this magnification so turn the high dry power lens (40X) into line. do not use the oil immersion lens with these chamber, as it just goomers the chamber and coverslip.

  3. Count the number of bacteria in 10-15 of the 1/400 mm2 squares and calculate an average cell number.

  4. To be statistically correct you should use a dilution such that there are no more than three cells in each small (1/400 mm2) square and the total number of cells counted is at least 100.

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.

For example:

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.

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