I need to carry out MIC on some plant extract against some bacteria using spectrophotometer to check the optical density of the dilution culture
Hi Ewanisha
600nm is an easy to obtain wavelenght of the optical light, you avoid possible strong UV absorbances of dissolved molecules and the defined wavelenght helps to standard your measurings. 600nm is most common, sometimes you have other wavelengths like 550nm in literature
The measurement is also reliable under "imperfect" optical conditions in MTPs with only one straight cuvette wall and the quality of your photometer does not need to be high-end.
Sometimes you have turbidity measuring systems like orbisphere that work with the full optically visible spectra, but these measurings mostly require strict calibrations and are not so easy to handle like a single filtered wavelength.
good luck
https://www.researchgate.net/post/Why_is_bacterial_density_measured_at_OD_600/1
https://www.researchgate.net/post/Bacterial_density_at_other_wavelengths_besides_OD6001
read these discussions
Remember for bacterial cell density you are measuring light scattering (i.e., not Absorbance) - most bacteria are colorless; therefore, the amount of light they absorb is negligible and most of reduction in transmitted light is due to light scattering by the cells. However, if the growth media has a color then it will absorb a portion of the light and interferes with your readings.
Therefore, you can use a wave length anywhere between 400nm and 700nm to measure ODs. But remember that the amount light scattered is proportional to the ratio of the cell (particle) size to the wavelength of the incident light. In other words, the same number of cells/ml will have a higher scattering (OD value) at 400nm than at 600nm.
The reason most microbiologists use 600nm (red range of the spectra) for measuring ODs of the culture is:
- Bacteriological media are yellow in color and will have high absorbance at 400nm but very low Absorbance at 600nm. If you are using a minimal salt media (i.e., no peptone, yeast extract...) and the growth will be spare, you can use 400nm and that will give higher ODs.
Remember, the relationship between OD and Dry weight of cells/ml is linear only at low cell densities (you need to determine the linear range for your organism). Therefore, after the culture reaches an OD where linearity is no longer exists, you need to dilute the culture before taking the OD readings then multiply the diluted reading by the dilution factor to get the correct OD.
Good Luck,
OD600 is preferable to UV spectroscopy when measuring the growth of a cell population because:
· At λ=600 nm, the cells will not be killed.
· UV light cause small to medium mutations in bacteria, potentially altering or destroying genes of interest, but the wavelengths (600 nm) is not harmful for both culture and cells.
· This wavelength (λ=600 nm) is not absorbed by yellow medias like NB, TSB and LB. Therefore, by monitoring the rate of change in optical density at 600 nm, it is possible to identify the lag, log, and stationary phases of a culture.
In case of bacterial cell density , we basically measure the light scattering by the bacterial cell and not the absorbance, as bacteria's are colorless and they absorb only negligible amount of light and most of it is scattered, and also the media that we mostly use are yellow to orange color giving absorbance maximum from 500-600nm. that's why this wavelength is preferred.
Most of the bacteria are colourless and they only scatter light so it is always better to use a higher wavelength (less energy) i.e. 600 nm to check the cell density because
1. High wavelength has less energy so less harmful for a particular bacterium.
2. Light of a shorter wavelength gets scattered more easily than the higher wavelength and using a shorter wavelength may interfere with your OD value.
3. Most of the culture media in microbiology are red, orange, yellow sometimes green so it's better to use one wavelength near the wavelength of a respective colour to get error-free data.
The method is referred to as turbidimetric and spectrophotometric technique. These techniques offer a straightforward, quick, and practical way to estimate total biomass. They are often carried out at a certain wavelength that each bacterium prefers. The light scattered by a suspension of cells during turbidimetric measurements is proportional to the cell concentration. As an alternative, spectroscopy may be used with a cell suspension's absorbance or transmittance.