# What you need to know about OD600

If you use a spec to measure cell density, you may be making a very common mistake and taking inaccurate measurements as a result.

Specs are often used for measuring the density of suspension cultures, but the mistake that many people make is to record the OD given by the spec as an absolute value.

The OD value represents the amount of light that is absorbed by your sample. But that value is affected by the intensity of the light beam in the spec, and the spec design. This means that similar samples will give completely different OD values in different specs due to the specs having different bulbs, or even in the same spec over time, as the beam intensity reduces with the age of the bulb.

So recording an OD value in your lab book does not really mean anything as this number is as much dependent on your spec as the density of your culture.

## What you really want to know

What you really want to know from an OD600 reading is the density of the cells e.g. in cells/mL. And to get this you need a standard curve. In other words, like any other spec-based experiment you will ever perform,  you need to calibrate the absorbance value against the number you actually want to know.

For some reason, people don’t seem to remember this for OD600. I can’t think of any other experiment where people record the absorbance value as an absolute number like they do for OD600, but maybe you can think of some – tell me in the comments :).

Constructing a standard curve for OD600 is a bit tedious, but it is a good exercise to go through every 6 months or so (and for each spec you use).

Start by making a suspension culture of the cells you are interested in, then diluting to obtain a series of samples with ODs of =2, 1, 0.8, 0.6, 0.4, 0.2 and 0.1 on your spec. (NB: don’t make serial dilutions as these are very inaccurate).

For each OD you then want to know the cell density in cells/mL. So for each OD, make dilutions of 1 in 1×10^7, 1×10^6 and 1×10^5, then plate 1mL of each onto suitable plates and grow them up. Then count the number of colonies formed on the dilution that gives the most appropriate number and multiply up by the dilution factor to obtain the number of cells/mL in the original sample. These values can then be used to construct a calibration curve of OD vs cells/ML.

The conversion factor for your spec (the number of cells/mL represented by 1 OD unit) will be equal to the gradient of the linear portion of the curve (normally up to about OD=1).You can now use this factor to convert your OD reading to cells/mL and as long as you calibrate whatever spec you are using in the future, this number will be absolute and comparable between experiments, specs and years.

## Just when you thought it all made sense…

A word of warning though, since the OD of a sample is dependent on the size and shape of the particles in it, different cell lines can have completely different relationships between OD and cells/mL. This means that a separate calibration will be needed for each cell type you use, which is tedious, but better than recording meaningless and arbitrary numbers in your lab book.

1. Kamal Sadeghi on January 18, 2020 at 6:07 am

Great point 👌

2. Banday Mohd sadeeq on August 25, 2019 at 7:47 am

I am a neive to microbiology ,plz let me make clear about the relationship between o.d and cell number as I am doing work with yeast ,so what should be the correct od of the overnight culture in order to get good number of colonies on a plate ,my second question is what should be od of the culture if we are supposed to do transformation ,plz ellaborate this

3. Mustafa Thega on September 13, 2017 at 12:46 pm

Hello

Well done, that is much informative and clear for my.

Many thanks

4. Jess on February 20, 2016 at 9:24 am

oh great!
So making competent cells, which several protocols mention, should not be grown beyond OD600 = 0.4 is quite a tricky job.

5. wensan0000 on December 10, 2012 at 4:28 pm

Very impressive article. I am new hand in microbiology and would like to ask the calibration for OD600 measurement. Will the results of calibration vary between different stages of growth? ( eg. Will ythe value of CFU/ml in lag or log phase be different when OD600 value is adjust to the same?) Thank you.

• JackBean on October 24, 2013 at 10:18 am

yes, it will be different, because in the stationary phase some of the cells will be dead already, as is written in the article. Thus the OD600 will be higher, but CFU will be lower.

6. DK on December 28, 2008 at 10:19 pm

This means that similar samples will give completely different OD values in different specs due to the specs having different bulbs, or even in the same spec over time, as the beam intensity reduces with the age of the bulb.

OD600 is certainly a crap value that shouldn’t be used/published the way it is but your explanation as to why it is so is completely incorrect. It has nothing to do with the bulb/monochromator/etc – for that, there is blank value that should be taken and an internal calibration that ensures linear response with respect to the incoming light. Once these two are done, all spectrophotometers will give the same reading for *absorbing* samples. But what we measure with OD600 is not absorbance, it is scattering!

So here is a correct explanation: OD600 as a measure of cell concentration relies on scattering of the light – some light is “lost” because scattered light now travels in all directions. So what gets to the photomultiplier has two components: A1+A2 where A1 is the amount of light that was not scattered (function of cell concentration) and A2 is some proportion of scattered light that still made it in (scattered light coming from a slit in the cuvette holder is roughly a cone of light). It is A2 that is variable from spec to spec since it depends on geometry of the spec: sizes of the slit in the cuvette holder and photomultiplier, and also distance from the cuvette to the photomultiplier (the closer it is, the more scattered light will be mistakenly recorded as non-scattered thus lowering OD600 value).

But yes, of course, when it matters, one should take calibration curve – not only OD600 is not a linear function of cell concentration but different cells scatter differently. (For example, cells with inclusion bodies seem to scatter considerably more; different strains/species may scatter differently, etc).

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