IADR Abstract Archives
In situ fluorescent labeling for rapid biofilm mass screening
Objectives: To develop a high-throughput in vitro screening assay using an MBECTM device and in situ fluorescence labeling to quantify biofilm mass as an alternative to directly measuring colony forming units or biofilm mass to evaluate the efficacy of oral biofilm treatments.
Methods: Streptococcus mutans biofilms were grown within MBECTM devices on polystyrene pegs by immersing the pegs into 96-well plates containing brain-heart infusion medium supplemented with a small molecular weight (3000kDa) Alexa Fluor® 488-labeled dextran. Confocal microscopy was used to verify that the fluorescently-labeled dextran was incorporated into the S. mutans biofilm matrix in situ. The biofilms were exposed to oral rinses containing chlorhexidine or cetylpyridinium chloride, or water as a negative control over a period of 24 hours. Upon completion of the treatment cycle, biofilms were dislodged from the MBECTM device by sonicating the pegs in a black, 96-well plate containing buffer. The relative biofilm mass was measured in the supernatant in each well by measuring the Alexa Fluor® emission at 525nm (490nm excitation) using a fluorescence plate reader. An improved method to detach biofilms from the polystyrene MBECTM substrate by enzymatically digesting the biofilm matrix will also be presented.
Results: Results: Treatment with oral rinses containing chlorhexidine or cetylpyridinium chloride caused a statistically significant decrease (p<0.05, n=8 for each treatment group) in the relative fluorescence of in situ-labeled biofilm compared to treatment with water using an unpaired two-tailed Student’s t-test. The mean fluorescence intensity values are summarized in the table below. The results indicate that treatment with these materials decreased the mass of treated biofilms compared to treatment with water.
Conclusions: In situ fluorescent labeling of S. mutans biofilms grown on MBECTM pegs provides a high-throughput quantitative technique to screen oral care products intended to kill, disrupt, or prevent biofilms.
Methods: Streptococcus mutans biofilms were grown within MBECTM devices on polystyrene pegs by immersing the pegs into 96-well plates containing brain-heart infusion medium supplemented with a small molecular weight (3000kDa) Alexa Fluor® 488-labeled dextran. Confocal microscopy was used to verify that the fluorescently-labeled dextran was incorporated into the S. mutans biofilm matrix in situ. The biofilms were exposed to oral rinses containing chlorhexidine or cetylpyridinium chloride, or water as a negative control over a period of 24 hours. Upon completion of the treatment cycle, biofilms were dislodged from the MBECTM device by sonicating the pegs in a black, 96-well plate containing buffer. The relative biofilm mass was measured in the supernatant in each well by measuring the Alexa Fluor® emission at 525nm (490nm excitation) using a fluorescence plate reader. An improved method to detach biofilms from the polystyrene MBECTM substrate by enzymatically digesting the biofilm matrix will also be presented.
Results: Results: Treatment with oral rinses containing chlorhexidine or cetylpyridinium chloride caused a statistically significant decrease (p<0.05, n=8 for each treatment group) in the relative fluorescence of in situ-labeled biofilm compared to treatment with water using an unpaired two-tailed Student’s t-test. The mean fluorescence intensity values are summarized in the table below. The results indicate that treatment with these materials decreased the mass of treated biofilms compared to treatment with water.
Conclusions: In situ fluorescent labeling of S. mutans biofilms grown on MBECTM pegs provides a high-throughput quantitative technique to screen oral care products intended to kill, disrupt, or prevent biofilms.
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