Home › Cell Biology › Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy
Cell Biology JoVE (Open Access) Citable · DOI

Oral Biofilm Analysis of Palatal Expanders by Fluorescence In-Situ Hybridization and Confocal Laser Scanning Microscopy

DOI: 10.3791/2967-v
What you'll learn
  • ✓ Collect and prepare oral biofilm samples from orthodontic appliances
  • ✓ Apply FISH labeling to identify microbial taxa in biofilm
  • ✓ Acquire and interpret confocal microscopy images of biofilm structure
Protocol

Biopharma Insights 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.

Difficulty
advanced
Total time
~4–6 hours per sample (collection ~30 min; fixation overnight; dehydration ~2 hrs; FISH + imaging ~2–3 hrs)
Biosafety
BSL-1

Steps

1
Collect biofilm and fix samples

Scrape acrylic-resin flakes bearing oral biofilm from palatal expanders and fix samples in appropriate fixative solution to preserve biofilm structure and microbial morphology.

▶ 01:51
2
Dehydrate fixed biofilm samples

Process fixed samples through graded ethanol or acetone series to remove water and prepare specimens for in-situ hybridization and microscopy imaging.

▶ 03:38
3
Perform FISH and acquire confocal images

Apply fluorescently labeled oligonucleotide probes to dehydrated samples and acquire three-dimensional images using confocal laser scanning microscopy to visualize biofilm composition and spatial organization.

▶ 04:34
4
Interpret biofilm structural and compositional results

Analyze confocal image stacks to identify microbial taxa, assess biofilm architecture, and evaluate spatial distribution of different bacterial populations within the biofilm matrix.

▶ 07:21
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