What you need to know about OD - Bitesize Bio
Optical density is useful and reasonably accurate in the exponential growth phase when the numbers of dead cells is minimal - and so is a good measure of . Optical density and absorbance both measure the amount of light "absorbed" when passing through an optical component, but there are. Absorbance comes from absorption which refers to the process of absorbing light physically. But absorbance measures the attenuation of the transmitted solar.
The speed of transmitted light is slower in the most optically dense materials than the less dense materials. Thus, with the increase in the index of refraction value, the optical density increases which decreases the speed of light in that material.
It is given by Beer-Lambert Law of Absorbance. This is especially used in quantitative spectroscopy. It shows a linear relationship between absorbance capacity and concentration of an absorber towards the electromagnetic radiation.Beer Lambert's Law, Absorbance & Transmittance - Spectrophotometry, Basic Introduction - Chemistry
The Beer-Lambert Law represents the Absorbance A in terms of absorptivity coefficient, the path length, and the concentration of analyte. In the absorption spectrometer, all the wavelengths of light pass through sample cell which contains solution of a substance and also through the reference cell.
Thus, the intensity of the light is measured which passes through the reference cell in terms of Io and I stands for the intensity of the light which passes through the sample cell. The optical density is directly proportional to the concentration. Optical density is measured by spectrophotometer. This is used in the biological research laboratory for measuring the concentration of the substance.
It is especially used to measure the concentration of bacteria in a biological suspension. The amount of scattering light is measured with the use of spectrophotometer.
The visible light passes through suspension of the cell and it scatters the light. The high-level of scattering indicates presence of more bacteria or other material which can be measured in a spectrophotometer. The optical density at a specific wavelength correlates with the various phases of bacterial growth. The measurement of concentration is done at maxima of the absorbance spectra because there is least change in absorbance with change in the wavelength due to zero value of slope of the absorbance spectra.
Measurement of Cell Concentration in Suspension by Optical Density
The measurement of optical density is a common method to quantify various important parameters like concentration of cell, production of biomass or alteration in the cell morphology.
The measurement is based on detecting the concentration of substances by using Beer-Lambert law as the absorbance is directly proportional to the concentration of the absorbing substance of the sample. Generally, small thickness cuvettes are used for good result approx 10mm thick sample. This value serves to determine the inoculum and the baseline to use in the test. The suspensions shall be used immediately. The first is that the technician is instructed to use an inoculum of about microorganisms per milliliter and then instructed to determine this by plate count.
Colony forming units CFU and cells are two different measures and this will inevitably lead to difficulties as the unfortunate lab worker cannot guarantee the number of cells in the suspension, only the number of CFU found.
However, we can accept the scientific inaccuracy as the numbers will generally work out. The more serious problem is the instruction to use the plate count CFU for determination of the inoculum for the test, and that the suspension shall be used immediately.
This quite frankly cannot be done. If you use the suspension immediately, the plate counts are unavailable, if you use the plate counts to set the inoculum, then the suspension is at least a day old.
Contrast these instructions with those in the USP 2 for the same exercise: The estimate of inoculum concentration may be performed by turbidimetric measurements for the challenge organisms. Refrigerate the suspension if it is not used within 2 hours]. Determine the number of cfu per mL in each suspension …to confirm the initial cfu per mL estimate.
This value serves to calibrate the size of the inoculum used in the test.
However, the turbidometric measure of the cells is also only an approximation of CFU. Thus the instruction to confirm the numbers after the test is underway with the plate count is an important control on the test.
Optical Density - Optical Density Formula & Optical Density of Spectrophotometer | BYJU'S
This article will explore the turbidometric approximation for cell numbers, and important controls on the process as well as potential pitfalls to the method. Theory Light scattering techniques to monitor the concentration of pure cultures have the enormous advantages of being rapid and nondestructive. However, they do not measure cell numbers nor do they measure CFU.