(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 see more 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. For the verification of a possible cross hybridization of the specific FISH probe with non-target individuals the NonEUB338 probe was used standardly. This nonsense probe is reverse complementary to EUB338 probe and has no known 16S rRNA target. The test was conducted using a mixed culture of Methanosarcina barkeri (Archaea) and Propionibacterium acne (Bacteria) (Figure 5B). Whereas hybridization of M. barkeri / P. acne mixed culture

using the probe find more ARCH915 resulted in a high hybridization rate of about 80% of all cells, no fluorescence signal was determined with NonEUB338. This indicates that the chosen hybridization conditions did not promote any cross hybridization of archaeal FISH probe with bacterial cells in this culture. Furthermore, FISH without any probe was performed with the same sample to evaluate possible background fluorescence because it is well known that P. acne exposed a low red autofluorescence [33, 34]. As expected, in

this experiment the control sample of the mixed culture showed minor background fluorescence (Figure 5B). Another factor influencing the result of Flow-FISH is the choice of the fixative for the necessary cell fixation immediately after sampling. Because most environmental samples include both Gram-negative and Gram-positive prokaryotes, it is generally recommended to prepare both, formaldehyde- as well as ethanol-fixed samples. In this study, both fixation procedures were carried out with pure cultures of C. thermocellum, as a typical representative for Gram-positive prokaryotes in biogas reactors, as well as samples of UASS biogas reactor. In case of C. thermocellum, the fixation with 50% ethanol led to an increased G protein-coupled receptor kinase hybridization rate when using the bacteria universal probe EUB338 (Figure 5C). In contrast, in case of the UASS reactor sample, the fixation with 3.7% formaldehyde resulted in better hybridization rates than obtained after ethanol fixation regardless of which FISH probe was applied. The sum of archaea and bacteria cell counts in formaldehyde fixed samples achieved about 90% of total cell counts determined by flow cytometry (Figure 5C). Interestingly, the percentage of archaea, i.e. about 40% of total cell counts, is relatively high compared with previously published results [4, 23, 35, 36].