I first did two controls. The first one was to compare H. influenzae cultures started in several different ways - from fresh and day-old colonies, and at various dilutions, and to see how close the growth of replicate cultures is. I used 10 replicate wells for each culture condition, and the results were excellent - all of the growth curves could be superimposed, with enough resolution to even see a tiny 'diauxic' pause in the growth, where the cells were switching from an exhausted nutrient to one they hadn't used yet.
The second control really should have been done first - a test for 'edge effects' and other inconsistencies in growth between the different wells in the culture plate. I was concerned that different wells might experience slightly different growth conditions (temperature, oxygen, whatever). In my previous test I had semi-randomized the arrangement of the different cultures in the wells, to average out any effects. For this test I put aliquots of the same culture in all the wells. The results were worse than I'd expected.
First, here's the superimposed growth curves from all 100 wells in this experiment. The X-axis is time and the Y-axis is OD. Although we don't see any growth for the first 300 minutes, the cells are growing, but their density is so low that they don't cause detectable changes in the OD readings. Again we see the little diauxic pause, here at OD = 1.05. The cultures peak with a tiny growth spurt at OD 1.4 and then the OD slowly declines.
A look at the innards of the Bioscreen suggests that these wells are probably a bit cooler than the others. The postdoc used his R awesomeness to make a heatmap showing the differences in growth rates for the time point indicated by the red line above, and I massaged it onto the schematic of the well layout, shown below. The wells on the right side aren't cooler, because the tray that holds the plates has space for a second tray on that side.
So now I'm doing 8 replicate runs for each of the competence mutants, and I'm using the wells that are orange or red in the heat map as control wells, putting plain medium with no cells into them. So far the results are very boring - all the mutants grow at the same rate.