FISH was performed as previously described (43 ). Optimal formamide concentration for FISH probes was determined after performing hybridization at different formamide concentrations in the range of 0 to 70% (with 5% increments). The intensity of at least 50 cells was measured using ImageJ (64 (link)) software. Optimal hybridization conditions are described in Table S1 in the supplemental material. EUBmix (65 (link), 66 (link)) was used to target all bacteria, and NON-EUB (67 (link)) was used as a negative control for sequence-independent probe binding. Quantitative FISH (qFISH) biovolume fractions of individual genera were calculated as a percentage area of the total biovolume, hybridizing with both EUBmix probes and a specific probe. qFISH analyses, performed using the Daime image analysis software (68 (link)), were based on 30 fields of view taken at ×63 magnification. Microscopic analysis was performed with an Axioskop epifluorescence microscope (Carl Zeiss, Germany) equipped with a Leica DFC7000 T charge-coupled device (CCD) camera or a white light laser confocal microscope (TCS SP8 X; Leica). Multicolor FISH was performed as described by Lukumbuzya et al. (69 (link)). Raman microspectroscopy combined with FISH was used to detect intracellular storage polymers (polyP, PHA, and glycogen) in probe-defined species and was performed as previously described (70 (link)).
Free full text: Click here