By Dr. Barry Hall

When I measured microbial growth before the age of microtitre plate readers, I used a spectrophotometer. I knew that at a wavelength of 600 nm an OD (optical density) of 0.1 meant that there were 10^{8} cells per ml, and I knew that the range over which there was a linear relationship between OD and cells per ml was OD = 0.05 to OD = 0.6. I knew these things because I had calibrated my spectrophotometer. I used serial dilutions of a suspension of cells in buffer to determine the linear range of the instrument, and I plated samples of those serial dilutions for single colonies to determine the relationship between culture density and OD.

You can easily calibrate your plate reader in the same way.

# Linear Range of the Instrument

To understand the linear range of the instrument it is important to know what your plate reader actually measures. It functions as a spectrophotometer, shining light through the culture in a well and measuring the fraction of incident light that is detected by the detector above the well. That fraction of light detected is the Transmittance; the darker or cloudier the culture the lower is T, the Transmittance.

OD = -log_{10}T. If 0.1 of the incident light is detected, the OD is 1.0.

Two factors determine the OD: *absorbance* by the culture medium, and *light scattering* by the cells. Since we are interested in using OD to estimate the culture density it is necessary to **subtract** from the OD reading the OD that is due to absorbance. We determine that determining the OD of a blank well that contains the same volume of the same medium as the experimental wells.

Having corrected OD for absorbance, we now want to know the range over which OD is proportional to culture density. To determine that we grow an overnight culture, wash the cells by centrifuging the culture and resuspending it in ½ volume of buffer containing no growth nutrients. We make a series of two-fold dilutions in that same buffer and put identical volumes of the diluted cultures into a series of wells. The last well, which serves as a blank, should contain plain buffer.

We use the plate reader to take a single reading, then we plot the OD vs dilution. Over some OD range, the points will fall onto a straight line. As the OD increases further the points will curve over. Although we know that the cell densities are increasing by a factor of two, the OD is not increasing at that rate.

What is going on? OD is the result of some light being scattered off axis from the detector. The more cell the more light is scattered off axis and the higher is the OD. At a certain point there are so many cells that light that was scattered away from the detector by one cell is likely to be scattered back toward the detector by another cell. As long as the culture density is low enough that multiple scattering is rare the culture density is proportional to the number of cells, above that threshold the proportionality falls off.

We now know the range over which OD is a reliable measure of population density.

# Inferring Cell Number from OD

To infer the cell number corresponding to OD values, we plate cells from each dilution for single colonies. Since these were serial dilutions of the same resuspended culture, we will find that the number of cells (colony forming units) indeed does change by a factor of two at each dilution. But if you plot cell number vs OD, we find that at ODs above the linear rage of the instrument the ratio of cells to OD increases. You can use the curve you have just determined to estimate the culture density from OD even about the linear range of the instrument.

It is often the case that the exponential growth phase persists longer than is indicated by the OD, but the growth rate that is determined from the apparent exponential phase is accurate.

# Growth Rates

Growth rates are determined from the exponential phase of culture growth. During exponential phase there is a linear relationship between ln OD and time. Eventually the growth rate decreases (deceleration phase) and culture density shows little or no further increase, as shown by the maximum OD. It is sometimes useful to know the maximum density that can be obtained under the given conditions. But as we have seen, maximum OD does not directly represent the maximum population density. A well-done calibration curve can permit accurate interpretation of the maximum OD.