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Measuring OD600

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Measuring OD600

Postby Redinorun » Tue Dec 29, 2015 5:58 pm

I am measuring absorbance at 600 nm (with a spectrophotometer) to find the effect of certain substances on bacterial growth (Escherichia coli, Lactobacillus plantarum). I heard there is a difference between absorbance and optical density and after an internet search I'm now even more confused. Some say it's the same, some say it isn't.

(I've found this explanation but I'm still not 100% sure:
Optical density is the amount of attenuation -- or gradual intensity loss -- that occurs when light passes through an optical component, such as a neutral density filter. Absorbance considers only absorption within the optical component.)

Could anyone explain the difference? And if I'm measuring absorbance, can I still write 600nm as units or is this not the same?
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Re: Measuring OD600

Postby claudepa » Thu Dec 31, 2015 3:53 pm

I believe that all comes from the Beer Lambert law: log(Io/I)=(eps)lC
Io is the intensity of the light beam arriving on the sample
I is the intensity of the light beam after the sample
eps is a constant
l is the size of the cuvette
C is the concentration of the absorbing sample in the cuvette
For me (am I right?) log(Io/I) is either absorbance or optical density.
I was sometimes surprised to see that some people use sophisticated spectrophotometers without knowing this very basic (and very useful) law of physics.
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Postby Redinorun » Fri Jan 01, 2016 12:39 pm

Well, thanks for the reply, but I still didn't really understand that - your last part about "log(Io/I) is either absorbance or optical density" didn't make any sense. But it made me search for the answer in physics. And yes, the answer does lie in Beer-Lambert's law.

I think it's like that:

Absorbance: A = -log (I/I0); where:
I = intensity of light that passes through the sample;
I0 = initial light intensity

But also: A = aλ × b × c; where:
aλ = absorptivity coefficient which depends on wavelength
b = length of path
c = concentration of analyte

Optical density measured by the spectrophotometer: OD = A/L; where:
L = thickness of the sample (eg. the kivette thickness is 1cm) on which the optical density depends

The optical density is also directly proportional to the concentration.

Therefore optical density and absorbance are not the same thing.
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Re: Measuring OD600

Postby claudepa » Fri Jan 01, 2016 9:15 pm

Hi, I am pleased that your research on Beer Lambert law , that I did not use since a very long time, did bring you some more information. I think we are not very far since, if I also remember my maths from very far, -log(I/Io)=log(Io/I). Also as usually the thickness is 1cm, this means OD=A since L=b=1. when I used spectrophotometers I prefered double beams since the reference beam normally allows the mesurement of Io. I still believe that very person using a spectrophotometer should first learn the Beer Lambert law. Bravo for raising interesting points.
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Postby Redinorun » Sun Jan 03, 2016 5:10 pm

Thanks for the help. :) So in this case it doesn't matter if I say I measured optical density or absorbance to see the growth of microorganisms?
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Re: Measuring OD600

Postby claudepa » Tue Jan 05, 2016 11:53 am

Hi. Physics is one thing, application of physics to biology is an other one. I feel concerned since my initial background was essentially mathematics and physics before going to biology. In the world of cell and molecular biology I have known for a long time everybody is using optical density. I should not be surprised that many biologists do not know what is exactly absorbance. But they know very well how to follow the growth of a bacterial culture with OD. So, to my mind, better speaking of OD.
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