Visualizing acidophilic microorganisms in biofilm communities using acid stable fluorescence dyes

Bacteria in acidophilic biofilm communities, i.e. acid streamers and snottites, obtained from a subsurface mine in Königstein were visualized by fluorescence microscopy using four new fluorescent dyes (DY-601XL, V07-04118, V07-04146, DY-613). The pH of the bulk solution in which these bacteria thrive was pH 2.6 to 2.9. The new fluorescent dyes were all able to clearly stain and microscopically visualize in-situ the bacteria within the biofilm community without changing pH or background ion concentration. The commonly used fluorescent dyes DAPI and SYTO 59 were also applied for comparison. Both dyes, however, were not able to visualize any bacteria in-situ, since they were not stable under the very acid conditions. In addition, dye V07-04118 and dye DY-613 also possess the ability to stain larger cells which were presumably eukaryotic origin and may be attributed to yeast cells or amoeba-like cells. PCR analyses have shown that the dominant bacterial species in these acidophilic biofilm communities was a gram negative bacterium of the species Ferrovum myxofaciens. The presented four new dyes are ideal for in-situ investigations of microorganisms occurring in very acid conditions, e.g. in acidophilic biofilm communities when in parallel information on pH sensitive incorporated fluorescent heavy metals should be acquired.