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1. Preparation of Bacterial Nanocellulose (BNC)
Note: All of the steps are performed under aseptic conditions, unless otherwise indicated.
- Prepare culture medium.
- Prepare 500 ml of liquid culture medium by combining 25 g of yeast extract, 15 g of peptone, 125.0 g of mannitol, and 500 ml of high purity water. Autoclave this mixture at 120 °C for 20 min and store at 4 °C.
- Prepare 100 ml of semisolid media by adding 15 g of agar to 5.0 g of yeast extract, 3.0 g of peptone, 25.0 g of mannitol, and 100 ml of high-purity water. Autoclave this mixture at 120 °C for 20 min. Once autoclaved, deposit 5 ml of the mixture in a 90 mm x 16 mm plastic Petri dish. Allow the solution to gel at 4 °C and store at this temperature until further use.
- Rehydrate Glucoacetobacter xylinus (G. xylinus) strain preserved in freeze-dried vials by adding 1 ml of liquid culture medium and pipetting up and down, as indicated by the manufacturer's instructions.
- Inoculate the Petri dishes containing semisolid media with small droplets of bacterial suspension using an inoculating loop. Make sure that the inoculum covers the entire Petri dish by moving the loop in a zig zag direction from the edge to the center of the dish.
- Incubate the Petri dishes at 26 °C for 72 hr in an incubator without CO2. Once the incubation period is complete, small white colonies are visible. If the colonies are not immediately used, store the Petri dishes at 4 °C by sealing the lid with Parafilm and placing the dishes upside down. The colonies can be stored in that way for up to 6 months.
- Transfer 2 ml of the liquid culture medium into each well of a 24-well tissue culture plate. Take two colonies with an inoculating needle from the inoculated Petri dishes and place them into the first well of the tissue culture plate. Repeat the same procedure for the remaining 23 wells.
- Incubate the tissue culture plate at 30 °C for 7 days. This will yield a total of 24 BNC pellicles with a diameter of 16 mm and a thickness of approximately 2-3 mm diameter, as depicted in Figure 1.Note: Do not disturb the bacterial culture at any point during the incubation period, for example, by shaking the plates. During the incubation period, G. xylinus extrudes glucopyranose sugar molecules to form a polymeric crystalline mesh in the air-liquid interface, which adopts the shape and size of the flask under static cultivation conditions. This polymeric matrix, known as bacterial nanocellulose (BNC), is conspicuous at the end of the incubation period.
- Collect the BNC pellicles from the growth media and sterilize them in 200 ml of 1% NaOH solution for 1 hr at 50 °C, in order to remove all traces of G. xylinus. Optionally, stir this solution at 300 rpm using a magnetic bar and a stirring plate. Discard the NaOH solution and add 200 ml of freshly prepared 1% NaOH solution. Repeat the same process once more or until the BNC pellicles in solution acquire a translucent appearance.
- Rinse the BNC pellicles with water three times and store them in high-purity water at RT. Make sure the BNC pellicles are completely submerged in the water and are not allowed to dry at any time.
- Autoclave the BNC pellicles at 121 °C for 20 min.Note: An in vivo subcutaneous study in the rat performed by Märtson and coworkers showed no degradation signs of the BNC after 60 weeks of implantation. Indeed, BNC is degradable in nature by microbial and fungal enzymes, which are absent in mammals. On the other hand, the biodegradability of the BNC can be the result of mechanical, chemical, and biological processes that weaken the microfibril network in vivo.
