OD (Optical Density) vs absorbance in biochemistry & OD600 monitoring of bacterial growth
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- Опубликовано: 2 июл 2024
- OD stands for “Optical Density” and it’s a measure of how much light “gets lost” as. It passes through something. The higher the OD, the less of that light can pass through. It’s related to, but not identical to, “absorbance” - it’s basically “absorbance + light scattering”. Absorbance is where molecules specifically snatch up photons making up light of a specific wavelength (due to their chemical makeup) whereas light scattering is where light waves kinda bounce off of particles. If you look to see how much light is lost on its path through a sample, what you measure will depend on both of those things (plus the detector size, distance, etc. since if the light is scattered too far it won’t be captured). Thus, OD per se can’t distinguish between the two. BUT by measuring the OD at wavelengths at which either absorbance or scattering is minimal, we can use OD to determine each.
blog: bit.ly/odbiochemistry
So, for example, we commonly measure concentrations of proteins and nucleic acids (especially of purified, non-scattering solutions) based on their absorption in the ultraviolet range (typically at wavelengths of 280 and 260 nm, respectively). Here, with those pure solutions,, scattering is minimal. So the terms absorbance and OD are often used interchangeably. If we want to measure scattering (to assess turbidity) we measure at a longer wavelength. For example, we commonly assess bacterial growth by measuring “OD600” which is a measure of light of wavelength 600nm making it through. The more big stuff there is crammed into a solution, the more scattering there will be. So scattering increases as the “turbidity” increases. For example, as bacteria grow and divide in liquid media (food) such as LB (lysogeny broth), the solution becomes more turbid and the scattering increases. And this is reflected in a higher “OD600.” By tracking this value over time we can monitor bacterial growth and know when to do things like add IPTG to induce protein-making.
We typically induce at an OD of ~0.6-0.8 for LB, higher for TB (~1.5-1.8), which is a richer media so you can grow to higher density
Absorbance can be useful for measuring concentrations of various molecules because it’s more “picky” - rather than just have to bounce off, in order for light to be absorbed, the energy of that light’s photons has to be the “exact” energy to excite the molecule’s electrons just right. Much more on this in other posts but bottom line is that what wavelength a molecule or part of a molecule will absorb is a property of its chemical makeup. And the molecules we work with in biochemistry typically absorb high-energy (thus short wavelength) light.
Light scattering, however, depends less on that chemical makeup of the individual molecules and more on the size and distribution of particles.
Thus, although OD and absorbance are technically different, they are often used interchangeably (especially outside of a physics context).
In terms of what it means mathematically, I will leave the complex stuff to the physicists (and provide links) but it simplifies down to
OD = log10(Io/I)
Where “Io” is the incident light (the light that hits the sample) and I is the light that makes it through.
The higher the OD, the less light makes it through and the log10 part has the consequence that every 1 OD change represents a 10-fold change in light of the measured wavelength making it through. So, for example:
OD 1 = 10% light makes it through
OD 2 = 1% light makes it through
OD 3 = 0.1% light makes it through
In order to get an accurate OD reading you need to be sure to measure and subtract out the background (eg measure the OD of media that contains no bacteria).
And you need to make sure you’re in the linear range. For bacteria, if you’re measuring above 1, you’ll want to dilute your sample before measuring and take the dilution into account.
Also be aware that the actual value you measure may depend on the measurement device (how big the aperture to the detector is, etc.)
Why 600? 600 nm light is reddish. So greenish molecules absorb it. Bacteria aren’t greenish (at least normally) so we don’t need to worry about absorbance affecting the OD, simplifying things so that the OD is just measuring scattering. 600 nm light is also low energy so it won’t hurt the bacteria.
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Thank you so much! Your channel is a gem for me! I study biotechnologies, and my professor does not explain the topics in such great detail. I will share your content with my classmates.
Thanks so much! I'm so glad I could help!
It is important to mention that the O.D. is a relative quantity. In order to use it you first need to perform a time-consuming calibration step that requires taking volume samples, diluting said volumes to exact dilution factors, plating in petri dishes, incubating the petri dishes, counting the resulting colonies, repeating the process and taking averages.
All while making sure the bacterial population density is within 10 +/- 3 million cells per mL so the O.D. stays under 1.0 units for a 1 cm optical path.
Hello. I'm working on measurements of E Coli in LB media (200 ml) in an Erlenmeyer Flask. We are keeping it in the incubator without stirring at 37 °C. And then we do the measuremnt extracting 4 ml aprox cause' this what the spectrophotometer needs for measuring. I was wondering if it's important to mix the culture before taking the measurement. I read in one of the resources from the comments that advice. Also, I don't know if it's necessary stirring during the growth or if it's better to let the bacteria to growth by its own. I've search for explanation about stirring but I didn't find it, just some papers say they do and others not.
typically you grow the cells with shaking
@@thebumblingbiochemist Thanks :)
good resources:
IMPLEN, “OD600 (Cell Density, Bacterial Growth, Yeast Growth)“ www.implen.de/od600-diluphotometer/od600/
BiteSizeBio, “Is Your Bacterial Culture Still Growing? A Primer on OD600 Measurements” by Adrienne Huntress, July 28, 2021
bitesizebio.com/41100/is-your-bacterial-culture-still-growing/#:~:text=Optical%20density%20measures%20the%20degree,more%20the%20light%20is%20scattered
Eppendorf, “OD600 Measurements Using Different Photometers Why does the absorbance value of turbidity measurements vary using different photometers?’, September 2015 www.eppendorf.com/product-media/doc/en/148370/Detection_White-Paper_028_BioPhotometer-D30_BioSpectrometer-family_OD600-Measurements-Different-Photometers.pdf
more on recombinant protein expression in bacteria: bit.ly/bacterialproteinoverexpression ; longer video: ruclips.net/video/ePwVeXxU26w/видео.html ; just the gist version… ruclips.net/video/vcCApiKl80I/видео.html
more on absorbance: bit.ly/dnauvbeer ; RUclips: ruclips.net/video/cUxh2DCqYBo/видео.html
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
Can we count colonies of bacteria?
Yes