Enumeration of soil bacteria with the green fluorescent nucleic acid dye Sytox green in the presence of soil particles Peter Klauth a, * , Ralf Wilhelm b , Erwin Klumpp a , Lothar Poschen c , Joost Groeneweg a a Agrosphere Institute (ICG-IV), Research Centre Juelich, Leo-Brandt Str., Juelich 52425, Germany b Biologische Bundesanstalt Braunschweig (BBA), Germany c Abteilung Sicherheit und Strahlenschutz (ASS), Research Centre Juelich, Juelich, Germany Received 1 June 2004; received in revised form 2 July 2004; accepted 2 July 2004 Available online 13 August 2004 Abstract Total counts in soils are usually determined using fluorescent dyes, such as DAPI or Sybr green, due to fluorescence enhancement if they are bound to nucleic acids. Unfortunately, these commonly used dyes stain soil particles as well. Therefore, besides fluorescence enhancement, sufficient spectral differentiation is also required. We present a new procedure that overcomes the problems of visualising bacteria on surfaces in soil and avoids the separation of soil particles to a large extent. Spectral differentiation between bacteria and soil matrix is achieved by using Sytox green and a suboptimal excitation wavelength. Bacteria exhibit a bright green fluorescence, while soil particles fluoresce blue or red. Slight homogenisation and sedimentation of the sand and coarse silt that were too big for microscopic investigations were the only separation steps required. We compared the proposed Sytox green staining with Sybr green staining. The recovery of Sybr green-stained cells amounted to 38%, whereas in samples stained by Sytox green 81% of the spiked cells were counted. Sytox green can also be combined with fluorescence in situ hybridisation (FISH) using deep red dyes such as Cy5. D 2004 Elsevier B.V. All rights reserved. Keywords: Sytox green; Total counts; Digital image analysis 1. Introduction Fluorescent dyes and epifluorescent microscopy have been used for more than 25 years for the rapid and sensitive enumeration of total counts of bacteria in soils and sediments (Hobbie et al., 1977; Porter and Feig, 1980; Zweifel and Hagstro ¨m, 1995). Fluorescent dyes optimally suited for total counts should stain all bacteria independent of their physiological state and metabolic activity. Therefore, fluorescent dyes should preferably stain cellular DNA to mark DNA-containing particles as total counts (Zweifel and Hagstro ¨ m, 1995). The presence of autofluorescent particles and debris stained by unspecific binding of the fluorescent dyes in samples from soils and sediments limits the use of the most common dyes such as DAPI and 0167-7012/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2004.07.004 * Corresponding author. Tel.: +49-2461-61-8663; fax: +49- 2461-61-2518. E-mail address: p.klauth@fz-juelich.de (P. Klauth). Journal of Microbiological Methods 59 (2004) 189 – 198 www.elsevier.com/locate/jmicmeth