Settings: 40xOil inverted objective (Nikon Eclipse TE300 Corp, Tokyo, Japan), Image size: 512×512 pixel, XY-pixel: 0.60 μm, Kalman filtration (n = 3).
For each sample, three replicates were analyzed. For each replicate, images were collected from 10 fields of view, chosen by arbitrary GDC-0941 molecular weight movements in the X-Y-direction. For each field of view, 3 images were collected at 4 μm intervals in the Z-direction. In total 90 images were collected per sample. The images were analyzed using ImageJ (version 1.44p, Wayne Rasband, Mizoribine clinical trial National Institute of Health, Bethesda, MD, USA, available at the public domain at http://rsb.info.nih.gov/ij/index.html). A threshold of 100 was applied to remove noise. Images were converted to binary images and image calculations using the AND and OR functions were applied as follows. Cells stained with both EUBmix and either one of ARC915 or
MX825 were removed from further analysis. The combined area of Archaea and Bacteria-positive cells, the total area with a signal, was calculated for all 90 images and all 3 probes. ARC915, although designed as a universal Archaea probe did not cover all 4SC-202 MX825 positive cells. The total area for Archaea was therefore counted as ARC915 positive cells plus MX825 positive cells not covered by ARC915. The relative abundance of Archaea was then calculated as the total area of Archaea divided by the combined area of Archaea and Bacteria. To analyze only images of flocs, and not dispersed cells, images with Montelukast Sodium a total area (both Bacteria and Archaea) lower than 1000 pixels were removed. Daime 1.1 [34] was used to generate images with all three probes used in the FISH analysis. Acknowledgements We thank the staff at Gryaab AB for assistance in obtaining samples and for providing data. We also thank The SWEGENE Göteborg Genomics Core Facility platform, which was funded by a grant
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