This article details a protocol for preparing bacterial biofilm colonies for morphological analysis using electron microscopy. The process involves fixation, dehydration, and careful mounting of the biofilm to preserve its structure.
Begin with bacterial biofilm colonies grown on a nutrient agar plate.
These biofilms consist of a hydrophobic extracellular matrix that protects the encapsulated bacteria from host immune responses.
Add a fixative and gently rotate the plate until the biofilm begins to float.
Seal the plate and incubate with gentle shaking to preserve biofilm structure.
Remove the fixative, add buffer, and incubate to stabilize the biofilm structure.
Using a Pasteur pipette, gently detach the colony from the agar.
Add increasing concentrations of ethanol to dehydrate the bacterial colonies.
Transfer a floating colony onto an ethanol-soaked filter paper and air-dry in a closed chamber.
Mount dried colonies on a carbon tape-coated stub and bridge with carbon tape to prevent electron beam-induced charging.
Next, coat the biofilm with a conductive metal layer to enhance imaging contrast.
The biofilm is now ready for morphological analysis by electron microscopy.
To begin sample fixation, first prepare enough fresh fixative solution for the desired number of biofilm colonies. Carefully add 5 milliliters of fixative to each petri dish, and avoid pipetting directly onto colonies. Colonies will begin to detach from the Agar and float.
Carefully seal the plates with parafilm, and incubate on a rotary shaker for two hours at room temperature. Transfer the plates to 4 degrees Celsius storage overnight.
Gently remove the fixative liquid with a Pasteur pipette, connected to a vacuum.
Add 10 milliliters of 100 nanomolar sodium cacodylate, 5 millimolar calcium chloride buffer to wash the biofilm, and incubate for five minutes. Carefully detach the center of the biofilm colony from the Agar plate with a Pasteur pipette.
To air-dry colonies, cut cellulose filter paper into quarters, and then submerge one section in a 100% ethanol.
Carefully transfer one floating biofilm colony onto the paper. Place the paper in a Petri dish lined with dry filter paper. Then, cover the dish, and leave it in a chemical hood to dry overnight.
In order to preserve the morphology of the biofilm colony, it is important to underlay the floating biofilm completely with the cellulose paper. Once on the paper, the biofilm cannot be readjusted.
Coat an electron microscopy stub with carbon tape, and then use tweezers to carefully transfer the biofilm colonies onto the stub.
After connecting each colony to the stub, by adding a thin bridge of carbon tape, store the stubs in a desiccator for 24 hours or until needed.
This step requires a steady and precise hand, because at this stage the biofilms are very fragile and easily fractured. The challenge is to mount a substantial part of the biofilm without cracks.
On the day of the examination, place the colonies into a gold-palladium sputter coater. Coat the samples for two minutes at a 60 degree angle. Repeat this step twice, rotating samples 120 degrees in between.
Finally, coat the samples once for three minutes from the top. Samples are now ready for imaging on the SEM.
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