Measurement of Bacterial Cell Growth

Direct Microscopic Count

The number of bacterial cells in a medium can be measured by direct microscopic count using Petroff-Hausser counting chamber.

These are specially designed slides that have square on the grid has definite depth and volume.

The direct microscopic method is easy, inexpensive and relatively quick to count bacterial cell number. However, using this method dead cells are not distinguished from living cells.

Coulter Counter

Coulter counter is an electronic device used to count number of bacteria and other microorganisms such as protozoa, microalgae and yeasts.

This device is provided with a tiny orifice 1×10⁻¹² m in diameter. This orifice connects the two compartments of the counter which contain an electrically conductive solution (electrodes).

In this method, the sample of bacterial cells is forced through the small orifice. On the both sides of the orifice, electrodes are present to measure the electric resistance or conductivity when electric current is passed through the orifice.

Every time a bacterial cell passes through the orifice, electrical resistance between the two compartments (electrodes) increases momentarily or the conductivity drops. The generates an electrical signal which is automatically counted.

Each electrical signal represents the counting of one bacterial cell.

Membrane-Filter Technique

Microbial cell numbers are frequently determined using special membrane filters possessing millipores small enough to trap bacteria.

In this technique, a water sample containing microbial cells is passed through the filter. The filter is then placed on solid agar medium or on a pad soaked with nutrient broth and incubated until each cell develops into a separate colony.

A colony count gives the number of microorganisms in the filtered sample, and specific media can be used to select for specific microorganisms.

Dry Weight Technique

This is one of the simple indirect methods to determine bacterial cell numbers or growth Portions of a culture can be taken at particular intervals and centrifuged at high speed to sediment bacterial cells to the bottom of a vessel.

The sedimented cells (called a cell pellet) are then washed to remove contaminating salt with distilled water, and dried in an oven at 100-105°C to remove all water, leaving only the mass of components that make up the population of cells.

The drying process is continuing till a constant weight is achieved.

Dry weight indicates cell growth in terms of inorganic material they utilized from medium. Increase in dry weight correlated with number increment of bacterial cells.

Turbidometric Estimation (Turbidometry)

Rapid cell mass determination is possible using turbidometry method. Turbidometry is based on the fact that microbial cells scatter light striking them.

Since the microbial cells in a population are of roughly constant size, the amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number.

Further increase in concentration results in greater turbidity. When a beam of light is passed through a turbid culture, the amount of light transmitted is measured Reduction in transmittance is correlated with the number of cells present

Flow Cytometer

When a bacterial cell suspension run through the cytometer, sheath fluid (for flow support) is used to hydrodynamically focus the cell suspension through a small nozzle.

When the cells pass through this narrow passage, a laser beam falls over them, and scatter when diverted by the cells.

A detector in front of the light beam measures scattering . Number of scatterings is directly indicating the number of cells pass through passage, and give the data in digital form of number of cells.

MTT Assay

This is an in-vitro method for only VIABLE bacterial cell growth measurement (cell number measurement).

This method is based on the response of bacterial cell to external factors. This assay is based on the principle of the reduction of tetrazolium salts to form coloured product, and the intensity of colour is measures and correlated with bacterial cell proliferation.

The yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) is reduced by metabolically active cells (Live cells), by the action of dehydrogenase enzymes and produce Formazan (a purple colour product) The intensity of the formazan is measured at 500-600 nanometers using a spectrophotometer. The darker the solution, the greater the number of viable, metabolically active cells.