No staining method exists to enable simultaneous, high-resolution labeling of the gram-negative and gram-positive cell envelope. This project proposes the development of a cellular staining protocol, intended to visualize a “Tricolor” model in pathogenic bacterial species, selected for their relevance in infection and laboratory utility. The “Tricolor” concept has not been realized in gram-negative bacteria and few examples have been engineered in gram-positive models. The development of a comprehensive staining protocol will prove beneficial to cell biology research, offering precision labeling and visualization. A novel benzoboroxole-based dye, OM-488, was engineered for high-affinity complexation with carbohydrate-rich components of the outer membrane and cell wall. OM-488 was applied as a staining reagent to non-capsular species: E. coli, P. aeruginosa, Methicillin-resistant S. aureus (MRSA), and Methicillin-susceptible S. aureus (MSSA). Parameters of the staining protocol were refined. Serial dilution and colony counting were used to investigate OM-488 toxicity. Samples were imaged with fluorescent microscopy. Results between control and variable plates in serial dilutions revealed no significant differences in cell viability when treated with OM-488, indicating non-toxicity. Fluorescent probes exhibited optimal glycan targeting when prepared under neutral (7.4 pH) conditions and exact treatment durations. Subsequent imaging depicted precise selection and labeling of the outer membrane and cell wall. Future steps will incorporate HADA, a fluorescent D-amino acid, for peptidoglycan staining in addition to OM-488 to produce a dual color model. Continued trials will aim to prove the selective binding to cellular targets and eliminate spectra overlap and bleed-through (crosstalk) between fluorophores of HADA and OM-488.
