I am expected to track cell growth by measuring the incubating culture's cell concentration every 30 minutes or so. So my questions are: Why do I need to do serial dilution (assuming that I do)? Why can't I just fill up a cuvette with the cuvette's volume's worth of incubating culture and measure that against the blank every 30 minutes? And if I do need to dilute, how far should I dilute (1:100, 1:1000, etc)? This is for a culture filled with E. coli.
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4 Answers
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One is not normally required to serially dilute E. coli cultures for spectrophotometric measurements, at least in the experiments where the OD value is important.
For most protein expression work, the consensus is to start the induction at OD 0.6, and for most chemically competent cells, the optimal OD ranges from 0.3 to 0.8 depending on the competency protocol. At no point in time do you need to perform dilutions, as they are all within the linear range of cell count:OD ratio.
The only time you need to dilute the culture is when you want to find the OD of a very concentrated culture (for example after 24 hours of vigorously shaken incubation).
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Reading the optical density of a log phase microbial culture at a fixed wavelength approximates the Beer-Lambert law where A = ecl. This relationship is not linear as the Absorbance approaches 1.0 so at higher cell densities you will eventually underestimate the cell density of the culture. Likewise the law is not accurate below an Absorbance below ~ 0.05, so a 10-fold dilution should be fine. Eventually the culture's growth will slow down.
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You should dilute (not necessarily serially dilute) if:
- You are exceeding the concentration range detectable by the machine
- Scattering starts overwhelming absorption (at high cell densities scattering will overwhelm absorbance). In such cases you can use a turbidimeter.
As such there is no issue of the law not holding true at certain limits of absorbance.
So a general thumb rule for dilution of cells is that the suspension should not look very turbid (You should be able to see your thumb from the other side of the test tube ;-) ).
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It depends on your purpose, but you could seed cells into multiwell plate and let microplate reader monitor the growth. Some microplate readers can incubate cells at a temperature you set, shake the plate, and monitor OD.