Fluorescence imaging of microbial communities

Cells were fixed in growth medium with the addition of 2.5% formaldehyde for 2 h at room temperature. After this period, they were washed three times in 1× phosphate-buffered saline and then stored at −20 °C in a mixture of absolute ethanol and 1× phosphate-buffered saline (1:1). The oligonucleotide probes used in this study were the general bacteria-targeting EUB338 (ref. ^{59 (link)}), the Methanomicrobiales-targeting MG1200 (ref. ^{60 (link)}) and the Lokiarchaea-specific DSAG-Gr2-1142 (ref. ^{7 (link)}) (Supplementary Table 5). The DNA-hybridization chain reaction procedure was performed as described previously^{61 (link)} and the samples were imaged on the Eclipse Ni-U epifluorescence microscope (Nikon) using Gryphax (v.1.1.8.153). Images were processed using ImageJ^{62 (link)}.