The Ct value, a product of free chlorine concentration and contact time, is the most significant factor in the disinfection of pathogenic microorganisms. Pulse introduction of a tracer at the reactor inlet and a collection of probes at the outlet are often used to determine contact time t from the residence time distribution (RTD) curve. Introduction of inert soluble tracers such as chloride, fluoride, and fluorescent dyes has long been considered sufficient to determine the flow-through path in chlorination reactors. It was generally agreed that the microorganisms are following the path of the soluble tracers. The current study compares RID curves of soluble tracers with curves of viruses and bacteria under a hypothesis that residence time can be influenced by the size of the solute. Tracer studies were performed in a bench-scale chlorination contact tank with fluorescent dyes rhodamine B (RB) and acridine orange (AO), viruses MS2 and T4, and bacterium Escherichia coli. The viruses and bacterium were stained with RB and AO, respectively. An inverse relation between residence time and size of solute has been observed. Mean residence times of 10.8, 7.8, 7.1, and 6.1 min were found for rhodamine B, MS2, T4, and E. coli, respectively. The RTD curves of labeled and native microorganisms closely resemble each other, and were significantly different from the RTD curves of soluble dyes. Fluorescence intensity of the dyes was found to be sensitive to free chlorine concentration C. A linear correlation over a free chlorine concentration between 0 and 3 mg/L was found. Determination of contact time in chlorination reactors by fluorescent labeling of microorganisms is a novel, simple, fast, and convenient procedure. The staining can provide precise microorganism-specific residence time in a chlorination reactor; in that sense the new tool has the potential to replace the inert soluble tracers used so far in RID experiments. (C) 2010 Elsevier By. All rights reserved.