Preparation of Bacterial Biofilm Colonies for Morphological Analysis

Abstract

Source: Bucher, T., et. al., Methodologies for Studying B. subtilis Biofilms as a Model for Characterizing Small Molecule Biofilm Inhibitors. J. Vis. Exp.(2016)

This video demonstrates the preparation of mature bacterial biofilm colonies for high-resolution imaging. It covers fixation, dehydration, mounting, and metal coating steps to preserve and visualize biofilm morphology.

Protocol

Biofilm Colony Sample Preparation for Scanning Electron Microscopy

1. Grow bacterial biofilm colonies.
2. Prepare a fresh batch of 2% (v/v) glutaraldehyde, 3% (v/v) paraformaldehyde solution in 100 mM sodium cacodylate, 5 mM calcium chloride buffer, pH 7.3. Prepare 5 ml of fixative for every Petri dish of 8.5 cm diameter.
   CAUTION: Glutaraldehyde and paraformaldehyde are hazardous. Handle them with safety equipment inside a chemical hood. Discard the solutions and the contaminated materials in the hazardous waste.
3. Carefully add the fixative to the biofilm colonies, without dispensing directly on top of the biofilms.
   NOTE: Due to the hydrophobic character of the biofilm colony, the colonies slowly detach from the agar and start to float.
4. Carefully seal the plates with a strip of Parafilm. Incubate on a rotary shaker for 2 hr at room temperature and subsequently transfer the plates to 4 °C overnight.
5. The next day, carefully remove the liquid with a Pasteur pipette connected to a vacuum pump.
6. Carefully add 10 ml 100 mM sodium cacodylate, 5 mM calcium chloride buffer to wash the biofilm and incubate for 5 min. Gently remove the liquid with the Pasteur glass pipette from the corner of the plate to avoid damaging the biofilm, and add fresh washing solution by gentle pipetting. Repeat this step once.
7. For the dehydration of the biofilm colonies, proceed with the following steps: 2x 5 min in ddH₂O; 2x 20 min in 30% ethanol; 2x 20 min in 50% ethanol; 2x 20 min in 70% ethanol; 2x 20 min in 96% ethanol; 2x 30 min in 100% ethanol.
   1. Add 15 ml of liquid for every Petri dish of 8.5 cm diameter in each step and remove the liquid carefully after each incubation.
8. Use one of two different methods for drying the samples from ethanol.
   1. For air-drying from ethanol:
      1. Cut a cellulose filter paper (diameter of 9 cm) into quarters. Briefly submerge one quarter in 100% ethanol, and then carefully transfer one floating biofilm colony onto it. Put the wet filter paper in a Petri dish lined with filter paper. Cover the Petri dish and let the biofilm colonies dry overnight in a chemical hood.
9. Coat an electron microscopy stub with carbon tape. With the help of tweezers, carefully transfer the biofilm colonies onto the stub. Connect each colony to the stub by adding a thin bridge from carbon tape, which is crucial for charge elimination under the electron beam. At this stage, handle the biofilm colonies with care as they are very fragile. Store the samples in a desiccator for at least 24 hr or until examination.
10. The day of the examination, with the scanning electron microscope (SEM), sputter-coat the biofilm colonies for 2 min at a 60° angle in a gold-palladium sputter-coater. Repeat this step twice and rotate the samples by 120° in between. At the end, sputter-coat the samples once for 3 min from the top. The 20 nm thin layer of gold-palladium improves the conductivity and enhances the contrast of the sample for imaging in scanning electron microscopy.
11. Store the samples in a desiccator to avoid the rehydration of the sample until imaging with a scanning electron microscope.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Glycerol anhydrous	Bio-Lab Itd	712022300
Ethanol anhydrous	Gadot	830000054
Razor blade	Eddison	NA
Circular cellulose filter papers	Whatman, 90 mm	1001-090
Glutaraldehyde	EMS (Electron Micoscopy Science), 25% in water	16220
Paraformaldehyde	EMS, 16% in water	15710
Sodium cacodylate	Merck, 214.05 g/mol	8.2067
Calcium chloride 2-hydrate	Merck, 147.02 g/mol	1172113
Stub-aluminium mount	EMS, sloted head	75230
Carbon adhesive tape	EMS	77825-12
Shaker 37 °C	New Brunswick Scientific Innowa42	NA
Incubator 30 °C	Binder	NA
Incubator 23 °C	Binder	NA
Rotary Shaker - Orbitron Rotatory II	Boekel	NA
S150 Sputter Coater	Edwards	NA
Environmental Scanning Electron Microscope	XL30 ESEM FEG Philips (FEI)	NA