Protocol

Use of Fluorescence In Situ Hybridization and the daime Image Analysis Program for the Cultivation-Independent Quantification of Microorganisms in Environmental and Medical Samples

Department of Microbial Ecology, Vienna Ecology Centre, University of Vienna, A-1090 Vienna, Austria

Corresponding author (daims{at}microbial-ecology.net)

INTRODUCTION

Conventional cultivation-based methods to measure microbial abundance are unsuitable for quantifying uncultured microorganisms that constitute the majority of microbial life in most environmental or medical samples. This problem is solved by the quantification approach described here, which combines fluorescence in situ hybridization (FISH) with rRNA-targeted probes and digital image analysis. By measuring the areas of probe-labeled biomass in randomly recorded image pairs, an unbiased estimate of the relative biovolume of the population of interest can be obtained. This approach expresses abundance as "biovolume fraction" (relative to the total biovolume of the whole microbial community). This value equals the share of biochemical reaction space occupied by the quantified population and thus can be more relevant ecologically than absolute cell numbers (e.g., a few large cells can contain the same biovolume as many small cells). Another advantage lies in the complete independence of this method from the morphology of the quantified organisms. Regardless of whether the target microbes occur as single cells in plankton samples, as filaments, or as dense aggregates in biofilms, this cultivation-independent method allows the composition of complex microbial communities to be determined.