180 Chapter 6 Fluorescence in situ hybridization (FISH) and PHA staining for confocal laser scanning microscopy To visualize “Ca. Methanoperedens” and the bacterial community, biomass was fixed at three salinities (0%,1.5%, and 3%). 2 ml of bioreactor samples were fixed with 3% (v/v) paraformaldehyde (PFA) to later perform Double Labeling of Oligonucleotide Probes (DOPE)-FISH for improved signal. Two “Ca. Methanoperedens” specific probes and the EUBmix probe set were employed (Supplementary Table 5) (Biolegio, Nijmegen, The Netherlands). Fixation was followed by sample stabilization using 1% of gelatin with 0.1g/l chromium sulfate and followed by an overnight hybridization at 46ºC with a 35% formamide hybridization buffer, washed (10 min at 48ºC) with a washing buffer, ice-cold water-dipped and air-dried. We checked for autofluorescence with controls that included no probes. Prior to microscopy, samples were embedded in Vectashield with DAPI (Vector Laboratories Inc., Burlingame, USA). Microscopy was performed in a Leica SP8-White Light Laser Confocal Microscope (Leica Microsystems, Wetzar, Germany). For visualizing PHA storage, the same fixed biomass hybridization protocol was combined with Nile Red (10 µl for 10 min) staining (Sigma-Aldrich, San Luis, USA) and DAPI-free Vectashield (Vector Laboratories Inc., Burlingame, USA). The FISH probe employed was single labelled with the Alexa350-fluorophore targeting “Ca. Methanoperedens” (NDAMOARCH_641) (Supplementary Table 5), a fluorophore that does not overlap with the Nile Red excitation/emission spectrum (Biolegio, Nijmegen, The Netherlands). We discarded autofluorescence with controls that included no probe or no Nile Red. Confocal microscopy images reported for this study are available at the Image Data Resource FigShare with reference: https://figshare.com/s/1f0f21a456665259077c Polyhydroxyalkanoate (PHA) quantification Our initial approach involved linking PHA production to “Ca. Methanoperedens” by employing Nile Red staining microscopy with “Ca. Methanoperedens”- directed FISH. Subsequently, we proceeded to evaluate PHA abundance in the “Ca. Methanoperedens” biomass, sampling at salinities 0%, 1.5%, and 3%. Here, PHAs were derivatized to polyhydroxy acids so that the methyl derivative could be analyzed by GC-MS. For that, the freeze-dried samples (10-20 mg) were first re-dried for 15 min at 100ºC in 12 ml-crystal vials (sealed with teflon-coated caps). Later,
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