title: Antimicrobial testing for surface-immobilized agents with a surface-separated live-dead staining method.
abstract: Modification of a traditional live-dead staining technique based on fluorescence microscopy has yielded an improved method capable of differentiating surface-immobilized antimicrobial agents from those agents acting via solution diffusion processes. By utilizing an inoculation chamber comprised of 50 µm polystyrene spheres as spacers between test substrate and coverslip control surfaces, three distinct bacterial cell populations can be probed by fluorescence microscopy for antimicrobial activity: (1) cells adhered to the coverslip, (2) cells adhered to the substrate, and (3) mobile cells in solution. Truly immobilized antimicrobial agents were found efficacious only at the substrate surface, while elutable agents were effective against all three populations. Glass surfaces derivatized with either quaternized poly dimethylaminoethylmethacrylate (pDMAEMA) or 3-(trimethoxysilyl) propyldimethyloctadecyl ammonium chloride (Si-QAC) were compared with bare glass control surfaces after contact and 4 h incubation with Staphylococcus aureus. pDMAEMA surfaces were both antimicrobial and immobilized, whereas the Si-QAC surfaces were only observed to be antimicrobial via active diffusion. In contrast to conventional thinking, Si-QAC surfaces showed no kill after removing all Si-QAC elutables via rinsing procedures. The semi-quantitative surface-separated live-dead staining (SSLDS) technique provides mechanistic insight and represents a significant improvement relative to current microbiological test methods for evaluating immobilized, antimicrobial agents.
authors.affiliation: Division of Technology Resources, Baxter Healthcare Corporation, WG3-2S, 25212 West Illinois Route 120, Round Lake, Illinois 60073, USA. john_bruce_green@baxter.com
authors.forename: John-Bruce D
authors.initials: JB
authors.lastname: Green
authors.forename: Susan
authors.initials: S
authors.lastname: Bickner
authors.forename: Phillip W
authors.initials: PW
authors.lastname: Carter
authors.forename: Timothy
authors.initials: T
authors.lastname: Fulghum
authors.forename: Michelle
authors.initials: M
authors.lastname: Luebke
authors.forename: Mark A
authors.initials: MA
authors.lastname: Nordhaus
authors.forename: Steve
authors.initials: S
authors.lastname: Strathmann
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001422:Bacterial Adhesion; D018914:Cells, Immobilized; D008826:Microbial Sensitivity Tests / Q000379:methods; D050296:Microbial Viability / Q000187:drug effects*; D008856:Microscopy, Fluorescence; D008863:Microspheres; D011137:Polystyrenes; D013194:Staining and Labeling / Q000379:methods*; D013211:Staphylococcus aureus / Q000187:drug effects*
chemical_list: D000900:Anti-Bacterial Agents; D011137:Polystyrenes