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

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I discovered indigo rings and circles in Escherichia coli ATCC® 25922™ cultures when I added the non-specific polysaccharide stain lactophenol cotton blue to Gram stained slides sampled from 96-well plates used to measure quantitative growth kinetics (QGK) in virtual colony count antimicrobial assays. I attribute the dark blue staining to the presence of capsular polysaccharides and bacterial slime associated with clumps of cells. Since all bacterial cells are glycosylated, 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 for circular or ring-shaped structures that imply the presence of slime fixed to the glass. These polysaccharides indicate a possible mechanism of resistance to antimicrobial peptides such as defensins, lectins with high affinity for polysaccharides and glycosylated proteins. Adding lactophenol cotton blue to Gram stained slides is a quick and inexpensive way to screen cell cultures for bacterial slime, clumps and biofilms, revealing details of polysaccharide secretion that are missed using the Gram stain alone. Combined with QGK threshold times, the lactophenol cotton blue Gram stain followed by digital image processing provides quantitative information useful for quality control, environmental monitoring and detection of clumping environmental factors.