Establishment of Flow-FISH protocol. The average percentage of cells hybridized with AlexaFluor488 labeled oligonucleotide probes for bacteria (EUB338), archaea (ARCH915), and the nonsense probe NonEUB338 was determined by flow cytometry at 488 nm excitation: (A) Effect of dehydration on FISH hybridization rate using pure cultures of E. coli and Pseudomonas fluorescens; +D = with dehydration steps before hybridization, -D = without dehydration steps before hybridization. (B) Proof of possible cross hybridization effects using mixed culture of Methanosarcina barkeri (archaea) and Propionibacterium acne (bacteria). (C) Influence of fixative on FISH hybridization rate using a pure culture of Clostridium thermocellum and two independent samples of a mesophilic UASS biogas reactor (UASS-1 and UASS-2); F = sample was fixed with 3.7% formaldehyde, E = sample was fixed with 50.0% ethanol. For all experiments a control sample without any FISH probe was applied to detect possible cell autofluorescence. All samples were pretreated with purification procedure 1-C2-S2-H1-F2. Error bars resulted from three different measurements.