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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.
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 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
Even after these remediation measures, uncovered 1 mL samples placed in the detector for 2 hours formed a macroscopic clump at the base of the cuvette accompanied by a decrease in optical density, suggesting that at least one clumping environmental factor (CEF) was concentrated by the fan and filter within detector acting as a dust trap
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 originally published plate configuration included a ring of 36 wells containing uninoculated Mueller Hinton Broth (MHB) capable of detecting crosscontamination (Ericksen, 2014). 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 indigo 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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