1) All kinds of species! I'm working with random soil isolates, but mainly Pseudomonads. However, the stain fails both in coculture and in monoculture - so it doesn't seem that the other isolates are to fault.

2) Yes, that is exactly what I'm trying to do. I'm screening multiple isolates to see if any of them will make Bsub produce "more biofilm" (i.e. more cells or more matrix). ConA seemed like a perfect staining candidate in that it should be able to bind to the terminal mannose and glucose molecules that Bsub EPS is believed to contain.

Lovely insight, Harsh, I've had the exact same thoughts.

Currently, I'm using a 3610 expressing constitutive mKate2 and EGFP controlled by the EPS promoter. I have the same setup for the tasA promoter. I even have an mCherry-labelled tasA fusion for visualizing tasA, but currently no multi-labelled reporter strains with fluorophore-fusions to multiple matrix components (though that would be radical!)

The data looks spot on for the 3610 mKate/Peps-GFP, actually, but I've had the feeling that an "increase in biofilm" is more than just the number of Bsub cells and their EPS-expression. I though maybe I could visualize the entire biofilm (both Bsub and its partnering isolate) by staining the EPS, but now that I've actually done most of the screening with the Peps-reporter (almost two months later), I'm no longer sure it's necessary for our purpose, exactly like you also point out.

I also agree with the inability of the stain to reach the pellicle. I guess I just hoped that it would perhaps be able to bind to the EPS from the underside of the pellicle.

Thanks a lot for you answer!

The pellicles sometime are so fragile, that it's just not feasible to drain the medium, add stain and then remove the stain again (especially not in the small wells in a 96-well plate). Optimally, I would like to wash the pellicles also, but that just isn't possible. Perhaps it would be, if the pellicles were grown in pellicle induction medium (LBGM or MSgg), but I want to see subtle increases and decreases, and pellicles in LBGM are already too thick to image through with a confocal microscope.

I guess I wouldn't be able to tell apart the producer of the EPS, but I'm also looking at division of labor, so any kind of matrix promotion would be interesting. Perhaps I also erroneously assumed that the EPS in the matrix that could be stained with conA would be produced by Bacillus - probably not correct.

I've also already looked into flow cytometry, but I've had a lot of trouble with quickly and easily breaking apart the pellicles, so instead they just clog the machine. I'm screening upwards of a thousand isolates, so the method really needs to be high-throughput and currently, we only have a single probe sonicator, such that every single sample takes >60 s just to sonicate. That's a lot of time for a 96-well plate (and then just imagine the replicates!)

I also just tried to measure the fluorescence intensity with a fluorescent plate reader, but it's not sensitive enough to detect subtle differences in the pellicle. Possibly because the pellicle absorbs much of the light at the top? I'm not really sure - in any case, I was unable to show a difference with my positive control samples on a plate reader.

Thanks for the vote of confidence! I hope I can show some interesting stuff, once I'm through with the screening.