# 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.