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Appl. Environ. Microbiol. doi:10.1128/AEM.01806-07
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

In situ activity measurement of suspended and immobilized microbial communities by Fluorescence Lifetime Imaging (FLIM)

Department of River Ecology, Helmholtz Centre for Environmental Research - UFZ, Magdeburg, Germany; Hochschule Magdeburg-Stendal (FH), Magdeburg, Germany

^* To whom correspondence should be addressed. Email: thomas.neu{at}ufz.de.

	Abstract

In this study the feasibility of Fluorescence Lifetime IMaging (FLIM) for measurement of RNA:DNA ratios in microorganisms was assessed. The fluorescence lifetime of a nucleic acid specific probe (SYTO 13) was used to directly measure the RNA:DNA ratio inside living bacterial cells. In vitro, SYTO 13 showed lower fluorescence lifetimes in DNA solutions compared with RNA solutions. Growth experiments with bacterial monocultures were performed in liquid media. The results demonstrated the suitability of SYTO 13 to measure the growth phase dependent RNA:DNA ratio in E. coli cells. The fluorescence lifetime of SYTO 13 reflected the known changes of the RNA:DNA ratio in microbial cells during different growth phases. As a result, the growth rate of E. coli cells strongly correlated with the fluorescence lifetime. Finally, the fluorescence lifetime of SYTO 13 in slow and fast growing biofilms was compared. For this purpose biofilms developed from activated sludge were grown as autotrophic and heterotrophic communities. The FLIM data clearly showed a higher fluorescence lifetime for the fast growing heterotrophic biofilms and a lower fluorescence lifetime for the slow growing autotrophic biofilms. Furthermore, starved biofilms showed lower lifetimes in comparison with biofilms supplied with glucose indicating lower RNA:DNA ratio in starved biofilms. It is suggested that FLIM in combination with SYTO 13 represents a useful tool for the in situ differentiation of active and inactive bacteria. The technique does not require radioactive chemicals and may be applied to a broad range of sample types including suspended and immobilized microorganisms.