We present a protocol for structural and compositional analysis of natural oral biofilm from orthodontic appliances with in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Oral biofilm samples were collected from palatal expanders, scraping acrylic-resin flakes off their surface and referring them for molecular processing.
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.
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.
The authors have nothing to disclose.
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.
Oligonucleotides | Target organism | Fluorochrome |
---|---|---|
EUB338 – 338III | Eubacteria | Cy3 |
LGC354 A-C | Firmicutes | FITC |
Bac303 | Bacteroidetes | Cy5 |
Mut590 | Streptococcus mutans | FITC |
POGI | Porphyromonas gingivalis | FITC |
Table 3. Oligonucleotides (refer to probeBase13 for details).
Component | Company | Comments |
---|---|---|
4% paraformaldehyde | 4% solution in PBS | |
1 x PBS | 3 x solution prepared | |
ddH2O | Millipore | |
Ethanol (100%) | Merck | |
Lysozyme | Roth | 100 mg/ml stock solution |
Formamide | Roth | 99.5% solution |
5 M NaCl | Roth | |
1 M Tris-HCl | Roth | |
2% SDS | Roth | |
FISH probes (oligonucleotides) | Invitrogen, Sigma | |
ProLong Gold antifadent | Invitrogen | |
Nail polish | ||
Equipment | ||
Incubator | Thermo Scientific | |
Water bath | IKA Tez | |
50 ml tubes | Sarstedt | |
Polysine-coated slides | Thermo Scientific |
Table 4. Materials and equipment.