Expanding the microbiologist toolbox via new far-red-emitting dyes suitable for bacterial imaging.

IMPORTANCE: By harnessing the versatility of fluorescence microscopy and super-resolution imaging, bacteriologists explore critical aspects of bacterial physiology and resolve bacterial structures sized beyond the light diffraction limit. These techniques are based on fluorophores with profitable photochemical and tagging properties. The paucity of available far-red (FR)-emitting dyes for bacterial imaging strongly limits the multicolor choice of bacteriologists, hindering the possibility of labeling multiple structures in a single experiment. The set of FR fluorophores characterized in this study expands the palette of dyes useful for microbiologists, as they can be used for bacterial LIVE/DEAD staining and for tagging the membranes of viable Escherichia coli and Bacillus subtilis cells. The absence of toxicity makes these dyes suitable for live-cell imaging and allows monitoring of bacterial membrane biogenesis. Moreover, a newly synthesized FR-fluorophore can be employed for imaging bacterial membranes with stimulated emission depletion microscopy, a super-resolution technique capable of increasing the resolving power of conventional microscopes.

An image processing-based quantification of gram variability in Acinetobacter baumannii.

Gram staining differentiates bacteria as gram-positive and gram-negative, depending on their cell wall constituents, and coloring cells in violet and pink, respectively. Sometimes, a subpopulation of the same bacterial species assumes different colors, ranging from pink to violet, for reasons that are not completely understood yet. We analyze conventional brightfield images and use an automated pipeline to count pink and violet cells. The ImageJ-based processing algorithm quantifies the gram variability in Acinetobacter baumannii ACICU in the stationary phase of growth with a percentage of 66% pink cells. Different bacterial strains and cell growth stages have been considered. RESEARCH HIGHLIGHTS: Gram staining differentiates bacteria into gram-positive (violet) and gram-negative (pink). Gram variability represents an inhomogeneous distribution of pink and violet cells within the same species. We developed an ImageJ-based pipeline for the quantification of Gram variability in Acinetobacter baumannii.