1Department of Orthodontics and Maxillofacial Orthopedics, Medical University of Graz, 2Institute of Hygiene, Microbiology and Environmental Medicine, Medical University of Graz, 3Department of Prosthodontics, Restorative Dentistry, Periodontology and Implantology, Medical University of Graz, 4Institute of Plant Sciences, Karl-Franzens-University Graz
Klug, B., Rodler, C., Koller, M., Wimmer, G., Kessler, H. H., Grube, M., et al. Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy. J. Vis. Exp. (56), e2967, doi:10.3791/2967 (2011).
Confocal laser scanning microscopy (CLSM) of natural heterogeneous biofilm is today facilitated by a comprehensive range of staining techniques, one of them being fluorescence in situ hybridization (FISH).1,2 We performed a pilot study in which oral biofilm samples collected from fixed orthodontic appliances (palatal expanders) were stained by FISH, the objective being to assess the three-dimensional organization of natural biofilm and plaque accumulation.3,4 FISH creates an opportunity to stain cells in their native biofilm environment by the use of fluorescently labeled 16S rRNA-targeting probes.4-7,19 Compared to alternative techniques like immunofluorescent labeling, this is an inexpensive, precise and straightforward labeling technique to investigate different bacterial groups in mixed biofilm consortia.18,20 General probes were used that bind to Eubacteria (EUB338 + EUB338II + EUB338III; hereafter EUBmix),8-10 Firmicutes (LGC354 A-C; hereafter LGCmix),9,10 and Bacteroidetes (Bac303).11 In addition, specific probes binding to Streptococcus mutans (MUT590)12,13 and Porphyromonas gingivalis (POGI)13,14 were used. The extreme hardness of the surface materials involved (stainless steel and acrylic resin) compelled us to find new ways of preparing the biofilm. As these surface materials could not be readily cut with a cryotome, various sampling methods were explored to obtain intact oral biofilm. The most workable of these approaches is presented in this communication. Small flakes of the biofilm-carrying acrylic resin were scraped off with a sterile scalpel, taking care not to damage the biofilm structure. Forceps were used to collect biofilm from the steel surfaces. Once collected, the samples were fixed and placed directly on polysine coated glass slides. FISH was performed directly on these slides with the probes mentioned above. Various FISH protocols were combined and modified to create a new protocol that was easy to handle.5,10,14,15 Subsequently the samples were analyzed by confocal laser scanning microscopy. Well-known configurations3,4,16,17 could be visualized, including mushroom-style formations and clusters of coccoid bacteria pervaded by channels. In addition, the bacterial composition of these typical biofilm structures were analyzed and 2D and 3D images created.
1. Specimen collection
2. Biofilm fixation
3. Dehydration of fixed Samples
4. In-situ hybridization
6. Representative Results:
Scraping biofilm off fixed orthodontic appliances (Figure 5) yields suitable flakes (Figure 6) that can be hybridized directly onto coated glass slides for microscopy. In this way, different groups of orobiome bacteria can be identified in their natural three-dimensional environment by tagging bacterial rRNA with differently labeled specific probes (Figures 7 and 8). In Figure 7, biofilm was stained with EUBmix (green, all bacteria) and LGCmix (yellow, Firmicutes). Firmicutes appear in green, as they were stained with yellow and blue. In Figure 8, biofilm was stained with EUBmix (red, all bacteria), Bac303 (blue, Bacteroidetes) and POGI (yellow, Porphyromonas gingivalis). Porphyromonas gingivalis is shown in yellowish white, as all three probes bind to its DNA and the overlap of colors results in a white signal. Morphological differences between groups of bacteria can also be identified (Figures 9 and 10). Large clusters of coccoid bacteria are shown in Figure 9, where staining was performed with EUBmix (green, all bacteria). Different shapes of oral bacteria were visualized in Figure 10, where coccoid and filamentous bacteria can be distinguished by staining with EUBmix (red). Also, a typical mushroom-like structure of the biofilm can be processed via 3D modeling of the CLSM data (Figures 11 and 12) Click here to watch a movie of the 3D modeling.
Figure 1. Fixed palatal expander in situ.
Figure 2. Nola Dry Field System.
Figure 3. Sterilized pliers, gloves and tray.
Figure 4. Removed expander.
Figure 5. Scraping off biofilm with a sterile scalpel.
Figure 6. Resin flakes directly hybridized onto coated glass slides.
Figure 7. CLSM image: differentiation of a specific bacterial group. 2D overlay of 3D CLSM stack data. Biofilm stained with EUBmix (green, all bacteria) and LGCmix (yellow, Firmicutes).
Figure 8. CLSM image: differentiation of a specific bacterial group. 2D overlay of 3D CLSM stack data. Biofilm stained with EUBmix (red, all bacteria), Bac303 (blue, Bacteroidetes) and POGI (yellow, Porphyromonas gingivalis).
Figure 9. CLSM image: differentiation of specific morphologies. 2D overlay of 3D CLSM stack data. Biofilm stained with EUBmix (green, all bacteria). Large clusters of coccoid bacteria (arrows).
Figure 10. CLSM images: differentiation of specific morphologies. 2D overlay of 3D CLSM stack data. Biofilm stained with EUBmix (red, all bacteria). Coccoid (arrow below) and filamentous bacteria (arrow above) can be distinguished.
Figure 11. Mushroom structure, 3D views from below. Stacks of biofilm flakes (scraped off the surface of an orthodontic appliance), stained with EUBmix and processed with IMARIS (CLSM image).
Figure 12. Mushroom structure, 3D side view. Stacks of biofilm flakes (scraped off the surface of an orthodontic appliance) processed with IMARIS (CLSM image).
|Hybridization buffer (200 μl)|
|5 M NaCl||36||36||36|
|1 M Tris-HCl||4||4||4|
|Any FISH probe||2||2||2|
Table 1. Constituents of hybridization buffer (concentrations in μl).
|Washing buffer (50 ml)|
|5 M NaCl||4500||2150||300|
|1 M Tris-HCl||1000||1000||1000|
|0.5 M EDTA||0||500||500|
|ddH2O||44 500||46 350||48 200|
Table 2. Constituents of washing buffer (concentrations in μl).
Movie 1. Click here to watch the movie.
The protocol herein described is a highly workable approach to staining biofilms collected from hard materials. Sampling and hybridization are the most critical steps. During sampling, care must be taken that slices of adequate thickness are collected to ensure that the biofilm structure remains intact. During hybridization, it is essential to avoid excessive fluctuations in temperature, thus avoiding non-specific binding, or loss of binding, of the fluorescently labeled probes. This FISH protocol is an elegant method to stain normal heterogeneous biofilm directly on glass slides without disrupting its composition. The slides can immediately be used for microscopy without a need to move the sample once again. In this way, an improvement over staining in tubes is achieved by less shear force being applied to the biofilm. Slices > 50 μm thick can be prepared and investigated in this fashion.
No conflicts of interest declared.
This work was supported by the Hygiene Fonds of the Medical University Graz. All subjects gave their informed consent. Institutional approval of the study protocol was obtained from the Medical University Graz.