Quantifying Enzyme-Induced Biofilm Suppression in Pathogenic Bacteria

Abstract

Source: Tay, S. B., et al. Anti-virulent Disruption of Pathogenic Biofilms using Engineered Quorum-quenching Lactonases. J. Vis. Exp. (2016)

The video demonstrates a method to quantify biofilm biomass in a pathogenic bacterium, comparing untreated controls with samples treated with a quorum-quenching enzyme. The enzyme disrupts bacterial signaling during early incubation, reducing cell adhesion. After washing and further incubation, control wells form dense biofilms, while treated wells show reduced formation. A cationic dye is used to stain the biofilm, and absorbance is measured to assess biomass.

Protocol

1. Crystal Violet Quantitation of Biofilm Formation in A. baumannii S1

1. Grow a 5 mL culture of Acinetobacter baumannii S1(A. baumannii) in Lysogeny broth (LB) (tryptone 10 g/L, yeast extract 5 g/L) at 30 °C in a shaking incubator (220 rpm) for 16 hr.
2. Adjust the culture of A. baumannii S1 to a desired optical density at 600 nm (OD₆₀₀) of 0.8. Using a 96-well plate, inoculate the bacteria culture (1:100 dilution) into fresh LB containing 10 µl of purified GKL enzyme (40 mg/ml); the new culture's final volume is 100 µl.
3. Prepare a control culture as well; this will not contain any enzyme. Repeat similar conditions to yield the desired number of replicates.
4. Cover the plate with a lid and place it into a sealed 10 L plastic container. Incubate the plate at 30 °C for 3 hr before gently removing the media.
5. Add another 100 µl of fresh LB medium to the well and incubate the plate for 21 hr at 30 °C.
6. After the second period of incubation, gently remove all the media. Wash the planktonic bacteria cell with 200 µl sterile water. Ensure that there is only minimal disturbance to the cells during washing.
7. Add 100 µl of 1% crystal violet solution to each well and incubate for 15 min at room temperature. Remove the crystal violet solution by washing the well with 200 µl sterile water. Repeat the wash two more times.
8. Add 100 µl of 33% acetic acid to each well and incubate for 15 min with gentle shaking; this will dissolve the dye.
9. Quantitate the amount of biofilm formed by measuring the absorbance of crystal violet at 600 nm. The amount of crystal violet is proportional to the amount of biofilm formed.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tryptone	BD	211705
Yeast Extract	BD	212750
96-well plate	Costar	3596
Crystal Violet	Sigma-Aldrich	C6158
Acetic Acid	Lab-Scan	PLA00654X	Caution: Flammable
Synergy HT Microplate Reader	BioTek