A comparison of enumeration methods for culturable Pseudomonas fluorescenscells marked with green fluorescent protein

The detection of bacteria in environmental samples using genetic markers is valuable in microbial ecology. The green fluorescent protein (GFP) reporte r gene was studied under nutrient starvation conditions at 4 degrees C, 23 degrees C and 30 degrees C in Pseudomonas fluorescens R2fG1 cells tagged wi th a red-shifted gfp. Fluorescence intensity was not significantly differen t in cells maintained in a buffer for at least 48 days at all the tested te mperatures. gfp-Tagged R2fG1 cells were introduced into bulk soil microcosm s and soil microcosms with wheat seedlings. GFP-marked cells were enumerate d immediately after inoculation into soil and again in soil and root sample s after 10 days. Counts of culturable colonies were obtained from drop plat es using 5-mu l aliquots of serial dilutions viewed with an epifluorescent microscope. Traditional spread plates (using 100-mu l aliquots) and the mos t-probable-number (MPN) method using a spectrofluorometer were also used to enumerate the GFP-marked Pseudomonas cells in soil, rhizosphere and rhizop lane samples. Microcolonies were visualized on root surfaces under the epif luorescent microscope after immobilizing in agar and incubation for 24 h. C ounts from traditional spread plates were significantly higher (P < 0.05) t han the population estimates of the MPN method for all treatments at any sa mpling time. Counts using the drop plate method, however, were not signific antly different (P < 0.05) except in one treatment, and provided similar es timates in half the time of spread plates and at an estimated third of the cost. (C) 2000 Elsevier Science B.V. All rights reserved.