Growing Mycobacterial Biofilm as a Model to Study Antimicrobial Resistance

Our research focuses on bacterial metabolism, especially energy metabolism, which is crucial for growth and survival. Despite extensive biochemical and structural data, systems level understanding and adaptive mechanisms remain unclear. We aim to deepen this knowledge and to address antimicrobial resistance.

Our protocol enhances the physiological relevance of lab experiments by using biofilms, which mimics the natural bacterial growth better than the planktonic methods. Adapting mycobacteria biofilms will offer a great system to understand the genus'lifestyle, and it will also offer a screening system for antimicrobials. We are not the first ones to produce mycobacterial biofilms.

There are several groups using this as a model. Our motivation behind producing this protocol was to simplify the procedures as much as possible so many labs can adapt this system as a model. To begin, with a serological pipette, dispense two milliliters of autoclave lysogeny broth media into two sterile 14-milliliter test tubes inside a laminar hood.

Add 20 microliters of filter-sterilized 5%TWEEN 80 to the test tubes with a micropipette. Next, add the cryostock of mycobacterium smegmatis into a tube with an inoculation loop. Incubate the tubes for 48 hours in a shaker incubator at 300 rotations per minute and 37 degrees Celsius.

To prepare the secondary cultures, dispense two milliliters of autoclave lysogeny broth media into two sterile 14-milliliter test tubes. Then use a micropipette to add 20 microliters of filter-sterilized 5%TWEEN 80 to the test tubes. Next, with a micropipette, transfer 20 microliters of the primary culture to one of the tubes.

Incubate the cultures in a shaking incubator at 300 rotations per minute and 37 degrees Celsius until they reach an OD600 of 0.6 to 0.8. To begin, in a laminar hood, dispense six milliliters of Sauton's media, supplemented with 2%glucose into the wells of a six-well plate. Inoculate the wells with 120 microliters of the secondary cultures of mycobacterium smegmatis.

Keep one uninoculated well as a media control. Next, pipette the culture up and down with a one-milliliter micropipette for even mixing. Cover the lid, and carefully seal the plate with paraffin film.

Incubate the plate in a static incubator at 37 degrees Celsius for seven days. For a visual estimation of biofilm growth, place the plates in a gel documentation system under epi white light illumination. To measure dry weight, weigh a sheet of blotting paper.

Extract the biofilm from the six-well plate, and transfer it onto the paper with a spatula. Carefully collect most of the biofilm from the culture. Place the biofilm on the blotting paper.

Place the blotting paper in an incubator until the biofilm dries completely. Then measure the combined weight of the paper and biofilm. Biofilm pellicles were visible from day three onwards.

Reticulation of the cultures improved with the addition of 2%glucose to Sauton's media. Biofilm development was visible between days three to six. A film with slight reticulation was observed on day three.

This film matured by day five and disintegrated from day six.