Preparation of Bacterial Nanocellulose Pellicles from Cellulose-Producing Bacteria

Take a microplate filled with a nutrient medium containing mannitol.

Inoculate each well with a cellulose-producing bacterium and incubate under static conditions.

During incubation, the bacteria take up mannitol and metabolise it into glucose.

This glucose is transported to cellulose synthase complexes embedded in the bacterial membrane and polymerizes into glucan chains.

These chains extrude into the extracellular space, where they crystallize into microfibrils.

The microfibrils bundle into ribbon-like structures, forming a three-dimensional nanocellulose matrix.

As more nanocellulose is produced, it accumulates at the air-liquid interface, thickening into gelatinous pellicles moulded into the well's shape.

Harvest the pellicles, and incubate them in an alkaline solution to lyse the bacterial cells, then remove residual medium.

Rinse the pellicles repeatedly with water to eliminate any remaining alkaline solution. Store the pellicles in water to prevent dehydration. Then, steam-sterilize them to ensure sterility for further applications.

Transfer two milliliters of the liquid culture medium into each well of a 24 well tissue culture plate. Take two colonies with an inoculating needle from