A DUAL FLUORESCENCE TECHNIQUE FOR VISUALIZATION OF STAPHYLOCOCCUS-EPIDERMIDIS BIOFILM USING SCANNING CONFOCAL LASER MICROSCOPY

A new dual fluorescence technique is described which, when combined wi th scanning confocal laser microscopy (SCLM), can be used to visualize the components of biofilm produced by Staphylococcus epidermidis. Che mostat cultures of RP62A (a well-characterized slime-producing strain of S. epidermidis) were used to produce mature biofilm on polyvinylchl oride (PVC) disks immobilized in a modified Robbins device using a 'se ed' and 'feed' model system. Serial horizontal and vertical optical th in sections, as well as three-dimensional computer reconstructions, we re obtained on in situ biofilm using the dual fluorescence procedure. Bacteria were visualized by green autofluorescence excited at 488 nm w ith an Argon laser, Cell-associated and exocellular matrix material (s lime) was visualized by red fluorescence excited at 568 nm with a Kryp ton laser after interaction of the biofilm with Texas Red-labeled whea t germ agglutinin which is a slime-specific lectin marker. Structural analysis revealed that the cocci grew in slime-embedded cell clusters forming distinct conical-shaped microcolonies. Interspersed open chann els served to connect the bulk liquid with the deepest layers of the m ature, hydrated biofilm which increased overall surface area and likel y facilitated the exchange of nutrients and waste products throughout the biofilm. The combined dual fluorescence technique and SCLM is pote ntially useful as a specific noninvasive tool for studying the effect of antimicrobial agents on the process of biofilm formation and for th e characterization of the architecture of S. epidermidis biofilm forme d in vivo and in vitro on medical grade virgin or modified inert polym er surfaces.