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Multiplex FISH analysis of a six-species bacterial biofilm.
- Source :
-
Journal of microbiological methods [J Microbiol Methods] 2004 Jan; Vol. 56 (1), pp. 37-47. - Publication Year :
- 2004
-
Abstract
- Established procedures use different and seemingly incompatible experimental protocols for fluorescent in situ hybridization (FISH) with Gram-negative and Gram-positive bacteria. The aim of this study was to develop a procedure, based on FISH and confocal laser scanning microscopy (CLSM), for the analysis of the spatial organization of in vitro biofilms containing both Gram-negative and Gram-positive oral bacteria. Biofilms composed of the six oral species Actinomyces naeslundii, Candida albicans, Fusobacterium nucleatum, Streptococcus oralis, Streptococcus sobrinus, and Veillonella dispar were grown anaerobically for 64.5 h at 37 degrees C on hydroxyapatite disks preconditioned with saliva. Conditions for the simultaneous in situ hybridization of both Gram-negative and Gram-positive bacteria were sought by systematic variation of fixation and exposure to lysozyme. After fixation and permeabilization biofilms were labeled by FISH with 16S rRNA-targeted oligonucleotide probes ANA103 (for the detection of A. naeslundii), EUK116 (C. albicans), FUS664 (F. nucleatum), MIT447 and MIT588 (S. oralis), SOB174 (S. sobrinus), and VEI217 (V. dispar). Probes were used as 6-FAM, Cy3 or Cy5 conjugates, resulting in green, orange-red or deep-red fluorescence of target cells, respectively. Thus, with two independent triple-hybridizations with three probes carrying different fluorescence-tags, all six species could be visualized. Results show that the simultaneous investigation by FISH of complex biofilms composed of multiple bacterial species with differential Gram-staining properties is possible. In combination with the optical sectioning properties of CLSM the technique holds great promise for the analysis of spatial alterations in biofilm composition in response to environmental challenges.
- Subjects :
- Carbocyanines chemistry
DNA Probes chemistry
DNA Probes genetics
DNA, Bacterial chemistry
DNA, Bacterial genetics
Dental Plaque microbiology
Fluoresceins chemistry
Fluorescent Dyes chemistry
Gram-Negative Bacteria genetics
Gram-Positive Bacteria genetics
Microscopy, Confocal methods
Specimen Handling methods
Biofilms growth & development
Gram-Negative Bacteria isolation & purification
Gram-Positive Bacteria isolation & purification
In Situ Hybridization, Fluorescence methods
Subjects
Details
- Language :
- English
- ISSN :
- 0167-7012
- Volume :
- 56
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Journal of microbiological methods
- Publication Type :
- Academic Journal
- Accession number :
- 14706749
- Full Text :
- https://doi.org/10.1016/j.mimet.2003.09.003