Fundamentals of Microbiology

2.5.7 Quantification of microorganisms / Measurement of cell numbers

Direct microscopic count (DMC)

The microbial load in a sample can be quantified by direct counting. Using a counting chamber is easy, quick and inexpensive. It also gives information about the size and morphology of microorganisms.

Petroff – Hausser counting chamber can be used for counting bacteria. Haemocytometer can be used for larger eukaryotic microorganisms. These are specially designed slides, that have chambers of known depth with an etched grid on the chamber bottom. The number of microorganisms in a sample can be calculated by taking into account the chamber’s volume and the dilution of the sample.

The bacteria in several of the central squares are counted (usually at 400x to 500x magnification). The average no. of bacteria in these squares is used to calculate the concentration of cells in the original sample.

There are 25 squares covering an area of 1 mm². The total number of bacteria in an area of 1 mm² is calculated / counted. The chamber is 0.02 mm deep and therefore,

Bacteria / mm³ = number / square x 25 squares x 50

The number of bacteria per cm² is 1000 times this value.

For eg. You have counted 20 bacteria in 1 square

20 x 25 x 50 = 25000 x 1000 = 2.5 x 10⁶ / ml

Disadvantages of this method

1. Very small volume is sampled and therefore the results are not accurate, unless the microbial population is large.
2. It is not possible to distinguish live and dead cells.