I coated some coverslips with poly-L-lysine, using the rub-until-dry method I was shown. I then assembled the coverslips into 'chambers' like those described here, and tested whether competent B. subtilis cells would stick to them.
I wanted to use chambers for these tests because that's what I'll be using with the optical tweezers. Another benefit turned out to be that I could compare how well cells stuck to the coated coverslip surfaces to how well they stuck to the untreated surfaces of the glass slides that form the bottoms of the chambers.
I let the cells sit in the chamber for 5-10 minutes at room temperature, and then washed them out by flowing about 10 volumes of medium through the chamber, introducing it in tiny drops from a pipette tip at one end and absorbing the flow-through with a square of blotting paper at the other end. Then I looked at the chamber under the microscope, comparing the cells on its upper and lower surfaces.
In most tests I saw little or no difference between the treated and untreated surfaces; quite a few cells were stuck on both. The largest volume of poly-L-lysine solution I used did give some patches where many cell stuck, but these were at the edges of the coverslip, where the rub-until-dry treatment hadn't reached well. Cell density (on both surfaces) was also generally higher at the edges of the chambers - I think this may just v=be because the washing is less effective at the edges.
I also tested my DNA-coated beads. They didn't stick any better than the cells did, which is good. But there were lots of clumps of beads - perhaps I didn't vortex them well.
In a previous experiment I had better results with coverslips that had been presoaked in acid alcohol, so I'll try this again.
I'll try higher concentrations of poly-L-lysine.
I'll try using more dilute cells.
I'll try H. influenzae cells as well as B. subtilis cells, because their surfaces have very different chemistries.
I'll try incubating the chambers+cells upside down before washing the cells out, so the cells will settle on the coverslip rather than the slide. Though, for the tweezers experiments, it doesn't matter which surface the cells are stuck on.
I'll try marking a reference spot on the coverslip, so I can track whether cells present after the first wash are removed by the second one.
I'll reread the papers that did tweezers studies of competent cells, and email their authors for advice.
Welcome to the 4th Reich part 1.
4 hours ago in Angry by Choice