Preparation of Bacterial Biofilm Colonies for Morphological Analysis

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 t