Shine On You Deadly Bacteria

As I mentioned in a previous post, I've been working with a small research team on the development of an electrochemical sensor to detect pathogenic E. coli bacteria. I've already discussed the difficulty that I had in obtaining an accurate cell count to quantify the concentration of the E. coli cells in the soy broth suspension that they live in. Well, I've overcome the problems that I was having with cell counting and have moved on to fluorescent imaging of the cells. More specifically, I've been charged with labeling the E. coli with a fluorescent dye and exposing the labeled cells to our electrochemical devices functionalized with IgG antibodies that selectively bind to E. coli. Then, rather than taking electrochemical measurements, I want to take fluorescent images to correlate our findings from the sensor data to actual images of the cells bound to the device.

I've been working on fluorescently labeling the E. coli cells with fluorescein isothiocyanate (FITC) for a couple of months now, with essentially no success. FITC is an amine-reactive fluorophore, and so my plan was to label the anti-E. coli antibody with FITC, then allow the labeled antibodies to coat the surface of the cells. However, that didn't work out so well. After several attempts using this procedure, I ended up with zero observable or measurable fluorescence from the E. coli cells. I did learn some things along the way, though. Most notably, I learned that trying to label viable live cells is a fool's errand unless absolutely necessary, so I started heat killing the cells before the labeling procedure.

After these failed attempts, I decided to simplify things a bit. Why not try to label the E. coli with FITC directly, rather than trying to use the antibody as an intermediate? So that's what I did. I introduced the killed cells to a large concentration of FITC, allowed the binding reaction to take place for an hour or so, and then purified the labeled cells from the remaining free dye. The resulting solution of labeled E. coli showed strong fluorescence when analyzed using fluorescence spectroscopy, so I dropped the cell suspension on a microscope slide and took some fluorescent images. And...well...see for yourself:


Those small elongated dots are the brightly labeled rod-shaped bacteria. I should mention that, despite being stored in the dark, the fluorescent emission of the cells is dropping surprisingly quickly. I would guess that this may be an indication that rather than the expected covalent attachment of the FITC to surface proteins on the E. coli, the FITC diffused into the cell and is slowly diffusing back out again over time. Either way, the cells were initially nice and bright, which allowed me to take some good images of the labeled cells bound to our electrochemical sensor device, which is exactly what I needed to do.