This study investigates the interactions between Streptococcus mutans and Candida albicans in the formation of cross-kingdom biofilms. The methodology involves culturing these pathogens in a simulated oral environment to understand their synergistic behavior in oral pathogenesis.
Begin with cultures of Streptococcus mutans, an oral pathogenic bacterium, and Candida albicans, an opportunistic fungal pathogen.
Transfer isolated colonies into a nutrient-rich medium and incubate overnight to ensure active growth.
Centrifuge to pellet the cells. Remove the supernatant and resuspend the cells in sterile saline to a defined concentration.
Take a multiwell plate coated with human salivary solution to mimic the oral environment.
Add C. albicans and incubate to allow attachment to the salivary coating. Wash with sterile saline to remove non-adherent cells.
Add heat-inactivated serum and incubate to stimulate hyphal development. Wash off excess serum.
Introduce S. mutans, then sucrose-rich medium and incubate. S. mutans secretes glucosyltransferase, which binds to C. albicans mannans.
This enzyme catalyzes sucrose into glucans on the fungal surface, anchoring bacteria to hyphae and promoting proliferation and matrix formation.
This method yields cross-kingdom biofilms modeling microbial synergy in oral pathogenesis.
Grow S. mutans and C. albicans on blood agar plates at 37 degrees Celsius under aerobic conditions.
Then transfer single colonies of each organism to test tubes filled with 5 milliliters of brain heart infusion, and grow them for an additional 18 hours.
On the next day, centrifuge the cultures at 1,200 times g for five minutes and discard the supernatant. Resuspend the cells in physiological saline and adjust the OD550 to 0.5 for both C. albicans and S. mutans.
Then dilute the S. mutans suspension 1 to 10 to achieve equivalent concentrations. Pipette 50 microliters of sterile salivary solution into the wells of an optical bottom 96-well plate for microscopy.
Incubate the plate for 30 minutes at 37 degrees Celsius, then wash the plate three times with 100 microliters of sterile physiological saline, and empty the wells.
Add 100 microliters of C. albicans suspension to each well, incubate the plate at 37 degrees Celsius for 90 minutes, then wash the well three times with saline.
Next, add 100 microliters of heat-inactivated fetal bovine serum to each well. Incubate the plate for two hours, then wash it three times with saline.
Empty the wells but leave a 20 microliter reservoir to avoid excessive shear forces. Add 100 microliters of S. mutans suspension and 150 microliters of BHI with 5% sucrose to each well. Then incubate the plate at 37 degrees Celsius for 24 hours or longer.
When cultivating older biofilms, replace the medium daily. At the end of the cross-kingdom growth phase, wash the plate five times with sterile physiological saline.
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