Quantification of polysaccharides fixed to Gram stained slides using lactophenol cotton blue and digital image processing

Abstract

Indigo rings and circles emerged when I added the non-specific polysaccharide stain lactophenol cotton blue to Gram stained slides. I attribute the dark blue staining to the presence of capsular polysaccharides and bacterial slime associated with clumps of Gram-negative bacteria.  Since all bacterial cells are glycosylated and concentrate polysaccharides from the media, the majority of cells stain light blue. The contrast between dark and light staining is sufficient to enable a digital image processing thresholding technique to be quantitative with little background noise. Prior to the addition of lactophenol cotton blue, the Gram-stained slides appeared unremarkable, lacking ubiquitous clumps or stained polysaccharides.  Adding lactophenol cotton blue to Gram stained slides is a quick and inexpensive way to screen cell cultures for bacterial slime, clumps and biofilms that are invisible using the Gram stain alone.