Phylogenetic relationship of isolates F2001-L and F2001-O. The 16S rDNA-based tree is based on the results of a maximum likelihood analysis. Only sequences which share common residues in at least 50% of all available sequences from gram-positive organisms with low GC content were included for the calculation of this tree. The tree topology was evaluated and corrected according to the results of distance and maximum parsimony analyses. The bar indicates 10% estimated base changes.

FISH of fungal culture P5c7 with group-specific 16S rRNA oligonucleotide probes: (A) no probe, (B) EUB338mix-FITC, specific for the domain bacteria, (C) LGC354mix-Cy3, specific for Firmicutes. Round bacteria measuring less than 0.5 μm wide were detected with the probes EUB338mix-FITC and LGC354mix-Cy3, indicating that the bacteria belong to the Firmicutes. The superimposition of the images (D) shows an exact colocalization of the signals obtained. No autofluorescence of the bacteria was observed without probe (A).

FISH of fungal culture P5c7 without probe in Cy5, with the species-specific oligonucleotide probe BIF216-Cy3 (probe specific for the Paenibacillus isolates from the fermentor), and the Firmicutes-specific probe LGC354mix-FITC. The superimposition of the images results in a yellow color for the bacteria, indicating colabeling by LGC354mix-FITC (green) and BIF216-Cy3 (red).

FISH of fungal culture P5c7 without probe in Cy5, with nonEUB338-FITC, antisense probe, and with the Firmicutes-specific probe LGC354mix-FITC. After superimposing the images, the bacteria fluoresce red, which corresponds to hybridization of the LGC354mix-Cy3 probe, whereas they would have fluoresced yellow (green plus red) in the case of nonspecific hybridization with nonEUB338-FITC (green).

Selection of a gallery of CLSM pictures corresponding to FISH of fungal culture P5c7. Optical sections were realized every micrometer (one picture presented every 2 μm) in a sample hybridized with nonEUB338-FITC (control for the specificity of the hybridization) and LGC354mix-Cy3 (Firmicutes). Bacteria are expected to appear as red dots. The fungal cell wall fluoresces turquoise nonspecifically, delimiting the fungal cell compartment. Thick arrowheads indicate plain fungal cell wall when the optical section is above or beneath the hypha, and thin arrowheads indicate fungal cell wall flanking the fungal cytoplasm when the optical section occurs inside the hypha. Bacteria appear inside the fungal cell concomitantly with their appearance in the cytoplasm (sections 2 to 8 μm), between the two turquoise lines corresponding to the fungal cell wall.