A rapid fluorescent staining method using a tetrazolium dye and propidium iodide for the in-use assessment of disinfection of Pseudomonas aeruginosa biofilms on soft contact lenses showed that 11 to 13% of cells on lenses remained actively respiring and recoverable by culture methods after 30 min of exposure to 3% hydrogen peroxide.Suspension tests not only are inappropriate for the assessment of contact lens disinfectant efficacy but may produce results which give the disinfectant manufacturer and the lens wearer a false sense of security (3). Image analysis and the fluorescent probes 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and propidium iodide (PI) were used to investigate the disinfection of biofilms of Pseudomonas aeruginosa, an important eye pathogen, on hydrophilic contact lenses using 3% H 2 O 2 (the most widely used lens disinfectant). This novel approach offers a rapid and reliable assay representing in-use conditions. P. aeruginosa (NCIMB 6750) was grown in 10 ml of Trypticase soy broth (TSB), harvested by centrifugation (2,400 ϫ g), and washed three times with phosphate-buffered saline (PBS), and counts were adjusted following spectrophotometry at 660 nm. Eighteen sterile contact lenses (polyhydroxyethyl-methacrylate; water content, 38%) were placed, concavity downwards, in separate bottles containing 10 ml of TSB, inoculated with 0.1 ml of a 37°C overnight TSB culture of P. aeruginosa, and incubated for 4 h at 37°C with gentle agitation.Lenses were immersed in 10 ml of 3% H 2 O 2 for up to 30 min, with six samplings at 5-min intervals, rinsed in 10 ml of PBS, and placed in 5 ml of TSB containing 6,000 U of catalase ml Ϫ1 for 1 min (to neutralize the H 2 O 2 ). They were then cut radially to allow them to lie flat on microscope slides and stained with 5 g of PI (Sigma, St. Louis, Mo.) ml Ϫ1 and 10 mM CTC (Polysciences, Inc., Warrington, Pa.) in PBS. After 1 min of incubation with PI at 20°C and 1 h of staining with CTC in the dark at 20°C, the lenses were rinsed in PBS for 5 s and then examined by epifluorescence microscopy with excitation at 510 to 560 nm (using a 580-nm dichroic mirror and a 590-nm barrier filter) in a Nikon Optiphot microscope. Cells that took up PI and fluoresced bright red were deemed to possess compromised cell membranes, while respiring cells reduced CTC to give bright red, fluorescent formazan crystals within the cell. When these stains were used together, there were no significant differences (P Ͼ 0.05) between replicate experiments. Stained cells were distinguished from nonspecific reactions by overlaying the fluorescence and phase-contrast images.The image analysis system comprised a Sony charge-coupled device camera and a Seescan Solitaire image analyzer (Seescan Ltd., Cambridge, United Kingdom) with archiving to hard disk. Random numbers were generated to give coordinates of the sampling sites on the contact lenses. Counts were made from 20 random microscopic fields (each of an area of 33 by 40 m) in triplicate experiments. Twenty fields of view were chosen as the ...
