Experiments Using Multiple Media and Multiple Blank Wells

By Dr. Barry Hall

When using GrowthRates, it is essential to specify either a blank OD (-b option) or a blank well (-w option) on the command line so that all OD readings can be corrected for the absorbance of the medium. The -w option is much preferable because that allows for drift, precipitation or other effects that may alter the OD of the blank (uninoculated medium) over the course of the experiment.  GrowthRates can apply only a single blank well to the calculations, but what is to be done when several media, which have different ODs, are used in an experiment?

Scientist in lab holding a 96 well plate with samples for analysis

Consider an experiment in which the growth rates of three different strains are measured in four different media such as LB broth, Mueller-Hinton broth, SOC broth, and M9-glucose medium. The three-broth media are dark, are colored differently, and will have different ODs. M9-glucose medium is clear and will have still another OD. Let us assume that well B12 contains uninoculated LB broth, D12 contains uninoculated Mueller-Hinton broth, F12 contains uninoculated SOC broth and H12 contains uninoculated M9-glucose. Strains in A12–B11 are growing in LB broth, those in C1-D11 in Mueller-Hinton broth, etc.  How do we specify the blank wells for all four media?

The answer is to run GrowthRates four times on the same input file. For the first run the command line might be GrowthRates -i infile.txt -w 24. Well B12 is well number 24 and contains LB broth. From that run we get the growth rates for wells A1–B11, all of which are strains growing in LB.

The second run command line might be GrowthRates -i infile.txt -w 48 . Well D12 is well 48 and contains Mueller-Hinton broth. From that run we get the growth rates for wells C1–D11, all of which are strains growing in Mueller-Hinton broth.

You can see where this is going.  From one experiment, using the four GrowthRates runs to analyze the results we get correct growth rates for each of the media. Since each GrowthRates run takes only about 30 seconds, in less than five minutes we have all the necessary results. The only caution is that we need to rename the resulting output files from “infile.summary” and “infile.results” to something like “LB.summary” and “LB.results” so that each successive run does not overwrite the output of the previous run.

Categories: Best Practices

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