Journal of Methods Microbiological Journal of Microbiological Methods 38 (1999) 177–182 www.elsevier.com / locate / jmicmeth An automated technique for most-probable-number (MPN) analysis of densities of phagotrophic protists with lux AB labelled bacteria as growth medium a, a a b * Flemming Ekelund , Søren Christensen , Regin Rønn , Eliza Buhl , c Carsten Suhr Jacobsen a Terrestrial Ecology, Zoological Institute, University of Copenhagen, Universitetsparken 15, DK-2100, Copenhagen Ø, Denmark b Department of Ecology, Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Denmark c Department of Geochemistry, Geological Survey of Denmark and Greenland, Thoravej 8, DK-2400 Copenhagen NV, Denmark Accepted 27 June 1999 Abstract An automated modification of the most-probable-number (MPN) technique has been developed for enumeration of phagotrophic protozoa. The method is based on detection of prey depletion in micro titre plates rather than on presence of protozoa. A transconjugant Pseudomonas fluorescens DR54 labelled with a luxAB gene cassette was constructed, and used as growth medium for the protozoa in the micro titre plates. The transconjugant produced high amounts of luciferase which was stable and allowed detection for at least 8 weeks. Dilution series of protozoan cultures and soil suspensions were inoculated into micro titre plates amended with a suspension of the transconjugant. After 45 days measurement of light emission allowed detection of individual wells in the titre plates, where protozoan grazing had removed the inoculated bacteria.  1999 Elsevier Science B.V. All rights reserved. Keywords: Heterotrophic flagellates; Lux AB labelled bacteria; Naked amoebae; Protozoa; Sediment; Soil 1. Introduction commonly used for enumeration of phagotrophic protozoa, direct counting procedures, eventually Protozoa constitute an essential component in both combined with staining, and the most-probable-num- terrestrial and aquatic ecosystems, as they regulate ber (MPN) technique. Both methods have weaknes- bacterial populations and stimulate mineralization ses and advantages compared to each other. In some processes (Ekelund and Rønn, 1994; Fenchel, 1987). systems they may supplement each other, whereas in Enumeration of protozoa is therefore essential in other systems only one of the methods will be useful ecological studies. Two different approaches are (Ekelund and Rønn, 1994; Stevik et al., 1998). In systems with only a minor fraction of solid material, e.g. the pelagic compartment of aquatic ecosystems, a direct counting procedure is often *Corresponding author. Tel.: 1 45-3532-1275; fax: 1 45-3532- preferred because it is considered more accurate and 1250. E-mail address: fekelund@zi.ku.dk (F. Ekelund) precise than the MPN-procedure (Fenchel, 1982; 0167-7012 / 99 / $ – see front matter  1999 Elsevier Science B.V. All rights reserved. PII: S0167-7012(99)00086-X