FIG 6.

Prelabeling pneumococcal cells with fluorescent vancomycin or fluorescent bocillin but not fluorescent cephalosporin prevents localized labeling with the FDAA HADA. Strain IU1945 (D39 Δcps) cells were grown exponentially in BHI broth, pretreated with the indicated antibiotics (pseudocolored red), washed, and labeled with HADA for 5 min (pseudocolored green), washed, and imaged by epifluorescence microscopy as described in Materials and Methods. There was minimal variation in labeling patterns for >90 individual cells in diplococci or short chains for each treatment examined in microscopic fields in two biological replicates, and representative cells are shown. Fluorescent cephalosporin is specific to a limited number of PBPs (47), whereas fluorescent bocillin and fluorescent vancomycin label all PBPs and PG pentapeptides, respectively (see references 33 and 48). See the text for additional details. Scale bar = 1. P, phase-contrast image; H, HADA labeling; C, V, and B, fluorescent-cephalosporin, vancomycin, and bocillin, respectively; O, overlay of green and red fluorescence images.