Microscopic count of Bacteria

Enumeration of microorganisms is especially important in dairy microbiology, food microbiology, and water microbiology.

Direct Microscopic count/ Total cell count
Direct microscopic counts are possible using special slides known as counting chambers, consisting of a ruled slide and a cover slip. It is constructed in such a manner that the cover slip, slide, and ruled lines delimit a known volume. The number of bacteria in a small known volume is directly counted microscopically and the number of bacteria in the larger original sample is determined by extrapolation. Dead cells cannot be distinguished from living ones. Only dense suspensions can be counted.

Bacteria can be counted easily and accurately with the Petroff-Hausser counting chamber. This is a special slide accurately ruled into squares that are 1/400 mm² in area; a glass cover slip rests 1/50 mm above the slide, so that the volume over a square is 1/20,000 mm³i.e.
1/20, 000, 000 cm³. If for example, an average of five bacteria is present in each ruled square, there is 5 x 20,000,000 or 10⁸, bacteria per milliliter. A suspension of unstained bacteria can be counted in the chamber, using a phase-contrast microscope.

 The formula used for the direct microscopic count is:
The number of bacteria per cc = The average numbers of bacteria per large double-lined square X The dilution factors of the large square (1,250,000) X The dilution factor of any dilutions made prior to placing the sample in the counting chamber, e.g., mixing the bacteria with dye

Advantage of Direct Microscopic count

1. Rapid, Simple and easy method requiring minimum equipment.
2. Morphology of the bacteria can be observed as they counted.
3. Very dense suspensions can be counted if they are diluted appropriately.

Limitations of Direct Microscopic count

1. Dead cells are not distinguished from living cells.
2. Small cells are difficult to see under the microscope, and some cells are probably missed.
3.  Precision is difficult to achieve
4. A phase contrast microscope is required when the sample is not stained.
5. The method is not usually suitable for cell suspensions of low density i.e. < 10⁷ Cells per ml, but samples can be concentrated by centrifugation or filtration to increase sensitivity.