E-coli on glass. Frames taken by phase contrast microscopy utilizing 
fluorescent dye (white) to indicate microbial membrane permeability
resulting from bacterial death

  Surfacine treated glass (start)

 Control untreated glass (start)

 Surfacine treated glass (90 minutes)

 Control untreated glass(90 minutes)

Movie (side-by-side comparison)  Download AVI movie (1.7M)
How was it done?

Ten microliters of log phase culture of E. coli (in TSB) was placed onto either a Surfacine-coated 22 x 40mm glass coverslip or an untreated control coverslip. A disc of agarose (2mm thick X 10mm in diameter), infused with tryptic soy broth containing 0.84mM SYTO 9 dye (Molecular Probes), was placed atop the bacteria. SYTO 9 collects in dead bacteria after its cell walls degrade. A 15mm coverslip was placed atop the agarose, excess media removed by blotting and the entire prep - agarose between two coverslips - was sealed with melted paraffin and petroleum jelly.

The sealed prep was mounted on the stage of a Nikon Microphot maintained at 37 degrees C and viewed using a 100x phase contrast oil immersion lens. Video from an Optronics DEI 750 3-chip color CCD was captured as individual time-lapse frames at 3 second intervals on a Macintosh G3 computer. At every 100th frame (every 5 minutes), phase contrast illumination was blocked, and a fluorescence image captured. Fluorescence frames (shown in white) were morphed to show the relative time-course of bactericidal activity of Surfacine and superimposed on the phase contrast frames.

 Who did it?  
 Microscopy by Jim Sullivan,