A non-invasive fluorescent staining procedure allows Confocal Laser Scanning Microscopy based imaging of Mycobacterium in multispecies biofilms colonizing and degrading polycyclic aromatic hydrocarbons

To study the micro scale interactions of Mycobacterium with bacteria belonging to other genera by means of Confocal Laser Scanning Microscopy (CLSM), a procedure was developed to non-invasively and fluorescently stain Mycobacterium without compromising the signal produced by commonly used fluorescent reporter genes. The procedure makes use of the commercial non-specific nucleic acid stain Syto62 and was optimized to efficiently stain Mycobacterium cells in suspensions and biofilms. The staining procedure was found non-invasive towards overall cell viability, biofilm architecture and fluorescence signals emitted by other organisms expressing the fluorescent reporter genes gfp and dsRed. The procedure was successfully applied to visualize the comportment of the PAH-degrading Mycobacterium sp. VM552 in triple species biofilms containing, in addition to strain VM552, the GFP labeled PAH-degrading Sphingomonas sp. LH128-GFP and DsRed-labeled Pseudomonas putida OUS82(RF), and colonizing a glass substrate coated with phenanthrene crystals in flow chambers. CLSM imaging and subsequent appropriate image processing of the biofilms show that the comportment of strain Mycobacterium sp. VM552 was largely affected by the presence of the other organisms. The data support the value of the staining procedure to study ecological questions about micro scale behavior and niche occupation of Mycobacterium in multi-species systems.