Yesterday I tested whether pre-coating glass tubes with DNA helps competent cells form biofilms. The answer is clearly No, but the results are nevertheless interesting, in a "That's peculiar!" way.
I did pretty much what I had planned (see previous post): I added 2 ml of a DNA solution to new glass culture tubes (in high-salt, low-pH), left it for an hour at room temperature, removed the solution and let the tubes dry for several hours at 37 °C. I then rinsed these tubes and untreated tubes with the high-salt low-pH buffer. I then added 3 ml of either high-density (~2x10^8 cfu/ml) or low density (?~10^7 cfu/ml?) cultures of the five strains I wanted to test, and left them for either 4 hr (high density cultures) or 18 hr (low density cultures) at 37 °C, just sitting in a rack. When the time was up I dumped out the cells, rinsed the tubes once with phosphate-buffered saline (no vortexing), and added 2 ml of a 0.1% solution of crystal violet in water. (I tried putting the crystal violet in my high-salt low-pH buffer but it wouldn't dissolve). I let the tubes sit for 10 min, dumped out the crystal violet, rinsed them with water, and let them dry. Then I resuspended the dye in 1 ml of 95% ethanol and measured its absorbance. The amount of dye that stuck to the tubes should measure the amount of cells that were stuck to the tubes.
I was hoping that the DNA treatemnt would increase the number of cells sticking to the sides of the tubes, especially for the two hypercompetent mutants (sxy-hyp and murE-hyp), but not for the non-competent mutants (sxy- and pilA-).
The graph shows that the DNA treated tubes (light blue and light green) had LESS, not more, stain.
This could have just meant than my pre-treatment didn't work - that the DNA had washed off the tubes before I added the cells. But that wouldn't explain why the untreated tubes had more cells that didn't wash off, especially after the long incubation. Instead, the DNA treatment appears to have done something that actively decreased cell adhesion.
My first look at the 18 hr cultures had me expecting a very different result. In all of the cultures in the no-DNA tubes, most of the cells had sunk to the bottom of the tube, but most of the +DNA tubes were cloudy all the way up. This led me to think that they had thick biofilms, but now I think that the cells were just more evenly suspended in the medium. Unfortunately I didn't measure the cell densities or anything else, or do replicates, or even carefully check which of the +DNA tubes looked less cloudy than the others (I think it was the pilA mutant).
So I guess I should repeat the whole thing. But first I should put some thought into possible explanations... ...OK, I have no idea. There shouldn't have been much DNA in solution in the +DNA tubes, given that I did rinse them before adding the cells. But I could test this by having tubes where I just added 1 µg of DNA in solution to the culture. Might the soaking in DNA have modified the physical properties of the glass surface in some way? This time I should treat the -DNA control tubes exactly the same way I treat the +DNA tubes. I guess I also need to do replicates this time. I won't bother with the high-density 4-hr incubations, I'll just do overnight ones that start at low cell density.
I'll also count each culture's cfu/ml at the start of the incubations, and check the ODs at the end (with and without mixing up any settled cells. And I'll be more meticulous in my washing steps.
Better go pour a few plates and start preparing the tubes...
murE mutagenesis planning
9 hours ago in RRResearch