Explain the Working Principle of Spectrophotometer?

The working principle of spectrophotometer is graphically presented in Figure.

Figure: Working principle of spectrophotometer

Spectrophotometer measures either the amount of light transmitted (% transmission) or the amount of light absorbed (A) by a sample, as you may have noticed from Figure. Here monochromatic light (beam of light at a single wavelength) is passed through a liquid sample. Depending upon the cells suspended in the culture, light is scattered. The scattering of light is directly proportional to the cell mass or indirectly to the cell number. More the number of cells, more would be scattering of light. The unscattered light is measured by a photoelectric cell and is recorded as 0% to 100% transmission (T).

However, in practice, cell density is represented in form of optical density or absorbance because itis directly proportional to the cell concentration while transmission is inversely related to cell density. Population growth can be easily measured spectrophotometrically as long as the population is high enough to give detectable turbidity. At very high concentration of cells, light scattered by one cell may be rescattered by another cell and it may appear as unscattered. This results in the loss of linearity between cell number and turbidity at very high cell concentration. Commonly used wavelengths for bacterial turbidity measurements are 540 nm (green), 600 (orange) or 660 nm (red). Estimation of cell number is based on turbidity measurement. Turbidity increases as the cell number increases.