Abstract
Dark blue rings and circles emerged when the non-specific polysaccharide stain lactophenol cotton blue was added to Gram stained slides. The dark blue staining is attributable 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. The presence of cell clumping provides a possible explanation of the presence of persisters and paradoxical points observed in Virtual Colony Count antimicrobial assays, and suggests a phenotypic resistance mechanism to antimicrobial peptides involving capsular polysaccharides.
Highlights
The virtual colony count (VCC) microbiological assay has been used for over a decade to measure the effect of antimicrobial peptides such as defensins and LL-37 against a variety of bacteria. (Ericksen et al, 2005; Zhao et al, 2013)
Blue Gram stain Glass slides were scrubbed with PCMX hand soap using a pipe cleaner. 10 μL of cells sampled from broth cultures in 96-well plates after VCC assays using twice-concentrated Mueller Hinton Broth (MHB) in the outgrowth step were added to the slides and equilibrated to ambient humidity overnight
Clumps were observed in E. coli cultures and in open cuvettes Macroscopic clumps were observed in 25 mL TSB batch cultures of E. coli ATCC® 25922TM grown at 37°C in early exponential phase to an expected optical density at 650 nm (OD ) of approximately
Summary
The virtual colony count (VCC) microbiological assay has been used for over a decade to measure the effect of antimicrobial peptides such as defensins and LL-37 against a variety of bacteria. (Ericksen et al, 2005; Zhao et al, 2013). The application of lactophenol cotton blue, ordinarily used to visualize fungi by staining cell wall polysaccharides such as chitin, revealed circles and rings consistent with the caramelized residue of polysaccharides, which presumably included capsular polysaccharides and slime secreted concomitantly with clump and biofilm formation. These dark blue circles and rings could be consistent either with a heterogeneous subpopulation of E. coli or with slight contamination with a second strain
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