Team:BYU Provo/Results/Experimental

The enzymatic activity of α-Amylase was characterized to determine its capacity to inhibit biofilm formation by V. cholerae. Samples were prepared by adding 50 µL of V. cholerae culture to 1 mL of a high salt LB. The samples were then treated by the addition of purified α-Amylase in various concentrations and allowed to incubate at 30°C for 48 hours. After 48 hours the samples were examined and a distinct difference was seen in the amount of biofilm formed in the treated and untreated samples.

The samples were then transferred to eppendorf tubes and centrifuged at 1,300 × g for two minutes, the supernatant was discarded to remove the growth media, and the samples were resuspended in 200 µL ddH2O. The samples were then stained with 50 µL of a 0.03% CV solution and allowed to incubate for five minutes. The samples were again centrifuged at 1,300 × g for two minutes and the supernatant containing excess CV was discarded. The pelleted biofilm was then washed with 800 µL of 95% EtOH without resuspension, centrifuged for 30 seconds, and the EtOH was discarded. The EtOH wash was repeated twice more for a total of three washes. The samples were then resuspended in 200 µL EtOH, transferred to a 96-well plate, and incubated for five minutes. The plate was then shaken for ten seconds and absorbance readings were taken for each sample at 540 nm.

The above graph shows the average absorbance readings for samples treated with 0, 2.5, 12.5, and 25 µL of α-Amylase. There is a distinct reduction in the amount of biofilm growth between untreated and treated samples, with samples treated with 25 µL α-Amylase showing a 65.8% decrease in biofilm formation after 48 hours. While this clearly shows the ability of α-Amylase to inhibit biofilm formation by V. cholerae, further characterization is needed to determine the capacity of α-Amylase to degrade preexisting biofilms.