Plasmid-mediated conjugation is an important mechanism through which bacteria establish new genetic traits in the environment. Here we used a combination of direct viable counting (DVC), fluorescence in situ hybridization (FISH), and green fluorescent protein (GFP) gene expression to estimate plasmid transfer frequencies (TFs) on nutrient-limited media at the single cell level. Conjugation experiments on nutrient-rich and -limited media were carried out using Pseudomonas fluorescens as a donor of a broad-host-range plasmid (RK2) tagged with the GFP gene and P. putida as the recipient. FISH-GFP and DVC-FISH-GFP allowed for the accurate detection of donor, recipient and transconjugants by fluorescence microscopy. The TFs obtained by these culture-independent approaches were 1 to more than 3 orders of magnitude higher than those determined by selective plate-counting. Lower and variable TFs obtained by selective plate-counting emphasize the importance of culture-independent approaches in plasmid transfer studies. The application of DVC before FISH-GFP on nutrient-limited medium produced elongated and/or fattened cells and resulted in a better fluorescence signal in target bacteria cells and better accumulation, maturation and fluorescence-emission of GFP within transconjugant cells, facilitating the detection and identification of cells compared with FISH-GFP alone. The DVC-FISH-GFP could be useful for other conjugation studies using bacteria with low metabolic activity under oligotrophic/stressful conditions or studies which require a precise determination of plasmid transfer events during short-term conjugation experiments.