1
Table S1. Concentration of NaCl in the washing buffer corresponding to the amount of
formamide in the hybridization buffer. Concentrations are determined for stringent washing at 37°C after hybridization at 35°C.
% formamide in hybridization buffer mM NaCl in washing buffer
20 135 25 95 30 64 35 42 40 27 45 16 50 9 55 3 60 0
Fig. S1. Epifluorescence micrographs of bacteria in the mucus of an aquarium-reared L. pertusa
polyp. The image is an overlay of DAPI signals (blue) targeting DNA of coral cell nuclei and bacteria, and hybridization signals of probes EUB338 I-III with Cy3 TSA (red). Arrows mark mucus secretions with large amounts of bacteria. Scale bar, 100 µm.
2
Pixel brightness / arbitrary units
80 100 120 140 160
Relative pixel abundance / percent 0,0 0,5 1,0 1,5 2,0 2,5 3,0
3,5 LGC0355
LGC0355b
Fig. S2. Specificity cross check for probes LGC0355 and LGC0355b. Histograms of signal
brightness (expressed as pixel brightness values of digital images) for hybridization of the
firmicute Bacillus subtilis with probes LGC0355 and LGC0355b under the same conditions (35%
formamide in hybridization buffer). Arrows denote the main fluorescence signals of both probes.
3
a c e
f d
b
g
k h
j
Fig. S3. Epifluorescence micrographs of bacteria hybridized with probe EUB338 I. a through f)
Overlay images of DAPI (blue) and Cy3 signals (red). a, b) Alphaproteobacteria, environmental isolate. c, d) Gammaproteobacteria, Escherichia coli. e, f) Firmicutes, Bacillus subtilis. g
through k) Thin sections in the tentacle region of L. pertusa polyps. g, h) Red coral color variety,
4
station 1. j, k) Red coral color variety, station 2. Bacteria had been permeabilized with lysozyme (a, c, e, g, j) or not permeabilized (b, d, f, h, k) prior to hybridization. Scale bars, 20 µm (a through k).
a b
Fig. S4. Epifluorescence micrographs of bacteria on thin sections of the gastral cavity of L. pertusa polyps (red coral color variety, station 1). Thin sections were hybridized with probe EUB338 I, followed by Cy3 TSA. Bacteria had been permeabilized with lysozyme (a) or not permeabilized (b) prior to hybridization. Scale bars, 20 µm.
