title: Gram's Stain Does Not Cross the Bacterial Cytoplasmic Membrane.
abstract: For well over a century, Hans Christian Gram's famous staining protocol has been the standard go-to diagnostic for characterizing unknown bacteria. Despite continuous and ubiquitous use, we now demonstrate that the current understanding of the molecular mechanism for this differential stain is largely incorrect. Using the fully complementary time-resolved methods: second-harmonic light-scattering and bright-field transmission microscopy, we present a real-time and membrane specific quantitative characterization of the bacterial uptake of crystal-violet (CV), the dye used in Gram's protocol. Our observations contradict the currently accepted mechanism which depicts that, for both Gram-negative and Gram-positive bacteria, CV readily traverses the peptidoglycan mesh (PM) and cytoplasmic membrane (CM) before equilibrating within the cytosol. We find that not only is CV unable to traverse the CM but, on the time-scale of the Gram-stain procedure, CV is kinetically trapped within the PM. Our results indicate that CV, rather than dyes which rapidly traverse the PM, is uniquely suited as the Gram stain.
authors: †Department of Chemistry, Temple University, 1901 N. 13th Street, Philadelphia, Pennsylvania 19122, United States.
authors: Michael J
authors: MJ
authors: Wilhelm
authors: ‡Department of Biology, Temple University, 1900 N. 12th Street, Philadelphia, Pennsylvania 19122, United States.
authors: Joel B
authors: JB
authors: Sheffield
authors: †Department of Chemistry, Temple University, 1901 N. 13th Street, Philadelphia, Pennsylvania 19122, United States.
authors: Mohammad
authors: M
authors: Sharifian Gh
authors: †Department of Chemistry, Temple University, 1901 N. 13th Street, Philadelphia, Pennsylvania 19122, United States.
authors: Yajing
authors: Y
authors: Wu
authors: †Department of Chemistry, Temple University, 1901 N. 13th Street, Philadelphia, Pennsylvania 19122, United States.
authors: Christian
authors: C
authors: Spahr
authors: †Department of Chemistry, Temple University, 1901 N. 13th Street, Philadelphia, Pennsylvania 19122, United States.
authors: Grazia
authors: G
authors: Gonella
authors: †Department of Chemistry, Temple University, 1901 N. 13th Street, Philadelphia, Pennsylvania 19122, United States.
authors: Bolei
authors: B
authors: Xu
authors: †Department of Chemistry, Temple University, 1901 N. 13th Street, Philadelphia, Pennsylvania 19122, United States.
authors: Hai-Lung
authors: HL
authors: Dai
mesh_terms: D004926:Escherichia coli / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D005840:Gentian Violet / Q000032:analysis / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D010539:Permeability; D010619:Phenazines / Q000032:analysis / Q000378:metabolism*; D013194:Staining and Labeling
chemical_list: C035863:Gram's stain; D010619:Phenazines; D005840:Gentian Violet