Question

What is the Absorbance of a cell suspension? How does absorbance at 400 nm change with the number of cells in a cell suspension?

Answer 1

• Absorbance can also be called as Optical Density or OD. This refers to the quantity of light absorbed by any given solution at different wavelengths.
• In order to study the absorbance of a cell suspension, we make use of a UV-VIS spectrophotometer.
• By using light scattering, we are able to know the optical density of the given cell suspension. This reading gives an idea about the number of cells per unit volume in the cell suspension.
• In the method, we have to take the optical density reading as well as count the cells using the direct method.
• This method is not efficient in differentiating between live and dead cells.

UV-VIS SPECTROPHOTOMETER:

• UV- VIS or also called Ultraviolet-Visible Spectrophotometry, is a type of absorption spectroscopy
• It measures the light absorbance in the ultraviolet (180-390nm) as well as the visible regions of the light (390-780nm).
• It has various applications and detects several molecules.

Principle:

• UV- VIS spectroscopy follows the Beer-Lambert's Law.
• Beer Lambert's Law states that the quantity of light absorbed by a substance in the solution is directly proportional to the amount or concentration of that substance in the solution and the length of the light that traverses through the solution.

Instrumentation:

• Light sources:
  ---UV source- could be a mercury arc lamp, hydrogen or xenon discharge lamp
  ---Visible source- Tungsten Halogen Lamp
• Collimator- it is useful in making the dispersed light rays into a parallel line using silica or quartz lenses.
• Monochromator- used to separate the desired wavelength of light from the spectra like a prism.
• Filter- absorbs the unwanted wavelengths.
• Gratings- convert polychromatic light into monochromatic light.
• Sample holder- cuvettes to hold the desired sample.
• Detector- convert the light signals to electrical signals like a photovoltaic cell.
• Amplifier- amplifies the received signals for read-out.

The relationship between concentration and absorbance is clearly stated as Beer Lamber's Law. Usually, 600nm is the optimum wavelength for bacterial cell suspension. If we consider 400 nm, it is a strong yellow region, and also because the suspension is yellow in color, LB absorbs the light and tend to have high absorbance when compared to that of 600nm.