I've been working on the components of the optical tweezers experiments, with modest progress. The tweezers apparatus still had problems when I was there the other day (it wasn't aligned properly?), but I have lots of other parts to work on. Here's a list:
- The RA of the tweezers lab showed me a way to pre-clean coverslips, and I need to test the ones he prepared to see if they give more consistent results than the ones I've been using. (Coat his and mine with poly-L-lysine, make into chambers, test binding of competent cells.)
- I need to test whether washing treated coverslips with culture medium (BHI) or protein (BSA) blocks cell and bead binding, because once I have cells bound to the surface I don't want beads to stick to it.
- I need to make a big batch of competent cells and freeze them in 0.5 ml aliquots, because the cells I made before were contaminated. (The contaminants were flagellated cells that otherwise looked just like H. influenzae - they were the spinning wiggling cells I described in my last post.)
- I need to test whether cells that have stuck to a coverslip are still able to grow. To do this, my plan is to replace the medium in the chamber with medium containing low-melt agarose, and once this has set incubate the chamber at 37 °C for a few hours and then examine the cells under the microscope, looking for clusters of cells (microcolonies). As a control I'll first try this with cell-free chambers and agarose medium that already contains growing cells.
- For today I'm just going to work with H. influenzae, but I should also test these factors with B. subtilis cells.