Microbiology & Immunology Publications

Surface properties of catheters, stents and bacteria associated with urinary tract infections

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International Biodeterioration and Biodegradation





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The use of fluorescence spectroscopy to evaluate Pseudomonas aeruginosa population in suspension and in young developing biofilms on a glass surface is described. The fluorescence emission at 460 nm for NADH/NAD(P)H when excited at 327 nm was used to monitor quantitative increase in cell numbers in suspension and during biofilm development over a 36-h period. NADH/ NAD(P)H fluorescence ratio, allowed for the discrimination of living cells from dead cells both in suspension and in biofilms. Living cells, yielded ratios of 0·38 ± 0·4 and dead cells yielded ratios of 0·23 ± 0·05. The overlap between the two population baseline studies was less than 5% (p < 0·05). The fluorescence spectra of living P. aeruginosa and dead cells at identical concentrations when excited at 334 nm has a fluorescence maxima at 450 nm which is mainly due to NADH/NAD(P)H and partly due to riboflavin. Viability of living and dead cells was confirmed by plating 0·1 alquots from suspensions onto agar and incubation at 37°C for 24 h. The living suspension yielded 10 CFU and no growth from the suspension of dead cells. A comparison of the determination of P. aeruginosa cell concentration by optical density, quantitative cell counts and fluorescence intensity (at 327 nm, 334 nm and 404 nm) are presented and show a linear relationship. These findings will allow the real time determination of the number of viable cells within a biofilm and in suspension and the determination of the ratio of living versus dead cells following exposure to antibiotic. This will lead to further understanding of the basic mechanisms of action of antibiotics on adherent biofilms. © 1992. 5

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