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# Question about measuring bacterial OD

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### #1 funnymicrobe1

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Posted 06 July 2011 - 01:59 PM

Hi,
we usually measure our E.coli OD at 600nm in a volume of 1ml, (occasionally the colony forming units are also determined by serial dilution). Could someone please let me know how to measure bacterial OD600 using a microplate-reader. We do have a bioscreen machine where we use 200ul of 1:100 culture in replicates to obtain a growth curve. But, if I use 200ul of my 5ml growing culture in a 96 well plate to measure its OD at 600nm and use that as a reflection of bacterial concentration, how accurate is that? Also how does that compare with an OD measurement using 1ml of the culture. Is OD600=0.5 in 200ul culture same as OD600=0.5 in 1ml culture? Thanks.

### #2 bob1

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Posted 06 July 2011 - 07:32 PM

I don't know how the Beer-Lambert law applies to bacterial cultures, but the general equation for absorbance of a particular compound is:

Light Absorbance (A) = log (I0 / I)= KCL

Where:
Light Intensity entering a sample is "I0 "
• Light Intensity exiting a sample is "I"
• The concentration of analyte in sample is "C"
• The length of the light path in glass sample cuvette is "L" .
• "K" is a constant for a particular solution and wave length

### #3 pito

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Posted 07 July 2011 - 01:23 AM

it should be the same.
Its pure physics.
You should check the physics behind it like bob1 said.

Of course: when taking samples to measure the OD, make sure they are mixed well, if you work with smaller amounts you need to make sure you dont pipette, by accident, more cells for example.

PS.: test it for yourself: take a sample 1 ml , measure it, take from that 1ml sample 200µl, and measure that again. It should be the same...

Edited by pito, 07 July 2011 - 01:24 AM.

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### #4 funnymicrobe1

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Posted 07 July 2011 - 02:43 PM

Thanks for the response.
I agree, they should be the same and I tried to test it already. Did it in 200ul using the microplate reader and then did that in 1ml using a regular spec, there was a difference which I thought due to the difference between 2 readers. But if I add 100ul, 200ul and 300ul of the same growing culture into the wells of a 96 well plate, OD600 was coming very different, if 100ul shows 0.2, 200 ul is showing 0.39 and 300ul is showing 0.7 or something like that using a plate reader. This made me think of the media/culture column length, but then if I use 100ul, 200ul and 300ul of clean media blank, their OD600 are nearly identical. So, the difference must be coming from the number of bugs in the volume present. I guess, I need to adjust something on my settings but don't know what. (If it's of any use, I am using a Biotek synergy HT plate reader). Could you please comment, thanks for the help.

pito, on 07 July 2011 - 01:23 AM, said:

it should be the same.
Its pure physics.
You should check the physics behind it like bob1 said.

Of course: when taking samples to measure the OD, make sure they are mixed well, if you work with smaller amounts you need to make sure you dont pipette, by accident, more cells for example.

PS.: test it for yourself: take a sample 1 ml , measure it, take from that 1ml sample 200µl, and measure that again. It should be the same...

### #5 pito

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Posted 08 July 2011 - 05:44 AM

funnymicrobe1, on 07 July 2011 - 02:43 PM, said:

Thanks for the response.
I agree, they should be the same and I tried to test it already. Did it in 200ul using the microplate reader and then did that in 1ml using a regular spec, there was a difference which I thought due to the difference between 2 readers. But if I add 100ul, 200ul and 300ul of the same growing culture into the wells of a 96 well plate, OD600 was coming very different, if 100ul shows 0.2, 200 ul is showing 0.39 and 300ul is showing 0.7 or something like that using a plate reader. This made me think of the media/culture column length, but then if I use 100ul, 200ul and 300ul of clean media blank, their OD600 are nearly identical. So, the difference must be coming from the number of bugs in the volume present. I guess, I need to adjust something on my settings but don't know what. (If it's of any use, I am using a Biotek synergy HT plate reader). Could you please comment, thanks for the help.

and they were from the same sample? you measured it at the same moment?

Its possible you get bad readings because 100µl is not enough , the meniscus might be too different..
(http://www.nature.co...l/3700054a.html)

But I dont think this is the problem. I think you are doing something wrong.
(you said it yourself: the blanks all come out the same...)

Did you measure a few wells? Or did you just fill one well with 100µl, 1 with 200µl and 1 with 300µl?
Try to fill up an entire multiwell with a few lanes filled with 200, a few with 100 and a few with 300 and see what that gives.

Anyway: when I used a multiwell plate to measure OD, we always worked with the same volumes.. just to rule out small erros (like meniscus differences).

But normally it shouldnt matter (altough its possible there is a minimum volume you need to use due to technical problems.. not sure how that is in the late reader you use)

Maybe you stick your larger pipette tips too deep in the culture?

Use 1 pipette tip for the 3 measurements: a 0-500µl pipette (tip)
and see what that gives.
(if you have those: use a multipipette, pipette with 8 tips on it.. so you can easly fill one multiwell)

Edited by pito, 08 July 2011 - 05:52 AM.

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### #6 funnymicrobe1

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Posted 08 July 2011 - 08:13 AM

Yes, all the samples were from the same 4hr e.coli culture (so they were all homogeneous as far as I can tell) and yes I did 4 replicates for each volumes, all replicates gave similar values. I didn't use multichannel but did use a single tip to fill them up. I will look into the Biotek HT manual for the recommended volume per well, usually we always use 200ul with that plate reader for any kind of measurement.
I was looking at the Nature press article you have send, don't know much about the technicalities of optics, do you think the meniscus issue can affect scattering of light by the bacteria present in a solution, even though the blank media is not affected, can the size of the particle present matter? Thanks.

pito, on 08 July 2011 - 05:44 AM, said:

funnymicrobe1, on 07 July 2011 - 02:43 PM, said:

Thanks for the response.
I agree, they should be the same and I tried to test it already. Did it in 200ul using the microplate reader and then did that in 1ml using a regular spec, there was a difference which I thought due to the difference between 2 readers. But if I add 100ul, 200ul and 300ul of the same growing culture into the wells of a 96 well plate, OD600 was coming very different, if 100ul shows 0.2, 200 ul is showing 0.39 and 300ul is showing 0.7 or something like that using a plate reader. This made me think of the media/culture column length, but then if I use 100ul, 200ul and 300ul of clean media blank, their OD600 are nearly identical. So, the difference must be coming from the number of bugs in the volume present. I guess, I need to adjust something on my settings but don't know what. (If it's of any use, I am using a Biotek synergy HT plate reader). Could you please comment, thanks for the help.

and they were from the same sample? you measured it at the same moment?

Its possible you get bad readings because 100µl is not enough , the meniscus might be too different..
(http://www.nature.co...l/3700054a.html)

But I dont think this is the problem. I think you are doing something wrong.
(you said it yourself: the blanks all come out the same...)

Did you measure a few wells? Or did you just fill one well with 100µl, 1 with 200µl and 1 with 300µl?
Try to fill up an entire multiwell with a few lanes filled with 200, a few with 100 and a few with 300 and see what that gives.

Anyway: when I used a multiwell plate to measure OD, we always worked with the same volumes.. just to rule out small erros (like meniscus differences).

But normally it shouldnt matter (altough its possible there is a minimum volume you need to use due to technical problems.. not sure how that is in the late reader you use)

Maybe you stick your larger pipette tips too deep in the culture?

Use 1 pipette tip for the 3 measurements: a 0-500µl pipette (tip)
and see what that gives.
(if you have those: use a multipipette, pipette with 8 tips on it.. so you can easly fill one multiwell)

### #7 pito

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Posted 08 July 2011 - 10:19 AM

funnymicrobe1, on 08 July 2011 - 08:13 AM, said:

Yes, all the samples were from the same 4hr e.coli culture (so they were all homogeneous as far as I can tell) and yes I did 4 replicates for each volumes, all replicates gave similar values. I didn't use multichannel but did use a single tip to fill them up. I will look into the Biotek HT manual for the recommended volume per well, usually we always use 200ul with that plate reader for any kind of measurement.
I was looking at the Nature press article you have send, don't know much about the technicalities of optics, do you think the meniscus issue can affect scattering of light by the bacteria present in a solution, even though the blank media is not affected, can the size of the particle present matter? Thanks.

The meniscus does influence the reading yes.

But still, normally you shouldnt get that kind of differences unless maybe 100µl is just a too small volume for a good read out.. but the 700µl should not be so much different from the 200µl.

I dont know what the problem might be, maybe you pipetted wronly? It should not that you get different results.

Are you see you used the same blank as a reference each time? And you didnt touch the multiwellplate with your hands (the top and bottom) so that couldnt interfear?
You measured each time with the plate open and not with the lid on it?
...

Another thing: if you pipetted the culture did you see big differences? I mean: cells in 1 sample and not in the other perhaps?
And when pipetting, did you leave the well for a longer time when you measured the 700µl for exammple (precipitation of cells that could occur and give other results when measuring)
If you don't know it, then ask it! Better to ask and look foolish to some then not ask and stay stupid.

### #8 funnymicrobe1

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Posted 08 July 2011 - 04:27 PM

I did 100, 200 and 300ul, normally we only do 200ul. Just by eye, they look the same. The OD was measured with no lid on and the answer in no for the other concerns. I haven't checked the volume recommendations
for the plate reader yet (couldn't find the manual), but it could be that 100ul is too little and 300ul is too close or something. I will post after I find it, there must be something, thanks for helping me.

pito, on 08 July 2011 - 10:19 AM, said:

funnymicrobe1, on 08 July 2011 - 08:13 AM, said:

Yes, all the samples were from the same 4hr e.coli culture (so they were all homogeneous as far as I can tell) and yes I did 4 replicates for each volumes, all replicates gave similar values. I didn't use multichannel but did use a single tip to fill them up. I will look into the Biotek HT manual for the recommended volume per well, usually we always use 200ul with that plate reader for any kind of measurement.
I was looking at the Nature press article you have send, don't know much about the technicalities of optics, do you think the meniscus issue can affect scattering of light by the bacteria present in a solution, even though the blank media is not affected, can the size of the particle present matter? Thanks.

The meniscus does influence the reading yes.

But still, normally you shouldnt get that kind of differences unless maybe 100µl is just a too small volume for a good read out.. but the 700µl should not be so much different from the 200µl.

I dont know what the problem might be, maybe you pipetted wronly? It should not that you get different results.

Are you see you used the same blank as a reference each time? And you didnt touch the multiwellplate with your hands (the top and bottom) so that couldnt interfear?
You measured each time with the plate open and not with the lid on it?
...

Another thing: if you pipetted the culture did you see big differences? I mean: cells in 1 sample and not in the other perhaps?
And when pipetting, did you leave the well for a longer time when you measured the 700µl for exammple (precipitation of cells that could occur and give other results when measuring)

### #9 bob1

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Posted 08 July 2011 - 10:35 PM

Bubbles on the top of the liquid in the wells will affect absorbance readings!  Briefly flame the top of the wells to pop the bubbles.

### #10 alexh

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Posted 15 May 2012 - 07:46 AM

funnymicrobe1, on 07 July 2011 - 02:43 PM, said:

Thanks for the response.
I agree, they should be the same and I tried to test it already. Did it in 200ul using the microplate reader and then did that in 1ml using a regular spec, there was a difference which I thought due to the difference between 2 readers. But if I add 100ul, 200ul and 300ul of the same growing culture into the wells of a 96 well plate, OD600 was coming very different, if 100ul shows 0.2, 200 ul is showing 0.39 and 300ul is showing 0.7 or something like that using a plate reader. This made me think of the media/culture column length, but then if I use 100ul, 200ul and 300ul of clean media blank, their OD600 are nearly identical. So, the difference must be coming from the number of bugs in the volume present. I guess, I need to adjust something on my settings but don't know what. (If it's of any use, I am using a Biotek synergy HT plate reader). Could you please comment, thanks for the help.

pito, on 07 July 2011 - 01:23 AM, said:

it should be the same.
Its pure physics.
You should check the physics behind it like bob1 said.

Of course: when taking samples to measure the OD, make sure they are mixed well, if you work with smaller amounts you need to make sure you dont pipette, by accident, more cells for example.

PS.: test it for yourself: take a sample 1 ml , measure it, take from that 1ml sample 200µl, and measure that again. It should be the same...

Hi,

I believe that the reason you see a difference when you pipette 100 uL vs. 200 or 300 uL is due to the change in the path length through the liquid column.  As the volume of sample in the well increases, the column height/path length parameter of Beer's Law increases.  Thus, when the light is passing through the sample of greater path length, there is a greater number of cells to absorb the light.  It is helpful to report culture densities as optical density units per unit volume (e.x. ODU/mL) because ODU alone does not take into account the apparatus used to take a measurement.

Based on the rest of what you say, your technique sounds fine.  You should be able to work out this issue by doing some calculations differently.

Good luck!

### #11 leelee

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Posted 16 May 2012 - 12:51 AM

When you say you usually measure 1ml- is that in a cuvette in a spectro? If yes, then you aren't actually measuring the whole 1ml are you? Just the absorbance of light that passes through that small bit of the cuvette in the middle somewhere. Volume is irrelevant- its all about path length.

My guess is that the micro plate reader has been set up in such a way as to calculate the OD600 accurately for a 200ul volume. So when you add a different volume, you stuff the readings up. Again, as for spectro the volume is almost irrelevant- the 200ul is used so that the path length is known, and that is all.