Microbial populations have traditionally been studied in carefully controlled, laboratory-grown cultures. New metagenomic approaches are being developed to study these organisms in environmental or medical samples. The July issue of Cold Spring Harbor Protocols presents a method developed by Holger Daims from the University of Vienna for quantifying populations of microorganisms in a variety of naturally occurring conditions such as plankton samples or biofilms. Use of Fluorescence In Situ Hybridization and the daime Image Analysis Program for the Cultivation-Independent Quantification of Microorganisms in Environmental and Medical Samples combines fluorescent in situ hybridization using rRNA-targeted probes with digital image analysis. The results show an organism’s “biovolume fraction” in a given sample; this indicates the share of biochemical reaction space occupied by the quantified population and can be more relevant ecologically than absolute cell numbers. Like all of our featured articles, this protocol is freely available to subscribers and non-subscribers alike.