Home › Microbiology › A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy
Microbiology JoVE (Open Access) Citable · DOI

A New Method for Qualitative Multi-scale Analysis of Bacterial Biofilms on Filamentous Fungal Colonies Using Confocal and Electron Microscopy

DOI: 10.3791/54771-v
What you'll learn
  • ✓ Grow bacterial biofilms on fungal hyphae in vitro
  • ✓ Prepare samples for confocal and electron microscopy analysis
  • ✓ Perform multi-scale qualitative analysis of biofilm structure
  • ✓ Interpret meso- and micro-scale biofilm morphology
Protocol

Biopharma Insights This protocol describes a new method to grow and qualitatively analyze bacterial biofilms on fungal hyphae by confocal and electron microscopy.

Difficulty
advanced
Total time
~3–5 days (culture growth + sample preparation + imaging)
Model organism
Pseudomonas fluorescens BBc6, Laccaria bicolor
Biosafety
BSL-1

Steps

1
Prepare bacterial and fungal cultures

Culture Pseudomonas fluorescens and Laccaria bicolor separately under appropriate conditions. Ensure cultures are viable and standardized before biofilm co-culture initiation.

▶ 00:50
2
Establish in vitro bacterial biofilm on fungal colony

Inoculate bacterial cells onto growing fungal hyphae to initiate biofilm formation. Incubate under controlled conditions to allow biofilm development on the fungal surface.

▶ 02:11
3
Prepare samples for confocal microscopy imaging

Fix and stain biofilm-colonized fungal samples using appropriate fluorescent probes or dyes. Mount prepared samples on slides for confocal microscopy visualization.

▶ 03:41
4
Acquire confocal and electron microscopy images

Image samples using confocal microscopy for 3D fluorescence analysis and electron microscopy for ultrastructural detail. Capture multi-scale data of biofilm organization on fungal hyphae.

▶ 05:35
5
Analyze meso- and micro-scale biofilm structures

Qualitatively evaluate confocal and electron microscopy data to characterize biofilm architecture, distribution, and cellular interactions at multiple spatial scales.

▶ 07:07
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