Candida Virulence & COVID-19: Enzymes & Biofilms
Okay, here’s a breakdown of the biofilm formation assay described in the text, summarizing the key steps. This is essentially a quantitative assay to measure how well Candida strains adhere to and form biofilms in 96-well plates.
Biofilm Formation Assay – Summary
1. Culture Preparation:
* Candida strains are initially grown on Sabouraud Dextrose Agar (SDA) at 35°C for 48 hours.
* Colonies are then transferred to Sabouraud dextrose broth and incubated overnight at 30°C with shaking (100 rpm).
* Cells are collected by centrifugation, washed with PBS, and resuspended in RPMI-1640 medium (pH 7.0, buffered with MOPS) to a concentration of 1 x 107 cells/mL.
2. Initial Adhesion (1.5 hours, 37°C, 75 rpm):
* 100 µL of the yeast suspension is added to each well of a 96-well plate.
* Negative control wells receive no yeast.
* Incubation allows initial attachment of cells to the well surface.
* Unattached cells are removed by washing with PBS.
3. Biofilm Development (66 hours,37°C,75 rpm):
* Fresh RPMI-1640 medium is added to each well.
* Plates are covered and sealed with parafilm to prevent evaporation.
* Extended incubation allows biofilm maturation.
* Non-adherent cells are removed by washing with PBS.
4.staining and Quantification:
* Wells are stained with 0.3% crystal violet solution for 30 minutes at 25°C (crystal violet binds to biofilm components).
* Excess stain is removed by multiple washes with distilled water.
* Wells are de-stained with 95% methanol.
* The absorbance (optical density – OD) of each well is measured at 570 nm using a microplate reader.
5. Determining Biofilm Formation:
* The OD cut-off point (ODc) is calculated as three standard deviations above the mean absorbance of the negative control wells.
* Wells with an OD value greater than the ODc are considered biofilm-positive.
Key Points & References:
* The protocol is based on previously published methods (references 23 and 21).
* RPMI-1640 medium with MOPS buffering is used to maintain a stable pH.
* Crystal violet staining is a common method for visualizing and quantifying biofilm biomass.
* The ODc is used to differentiate between importent biofilm formation and background noise.
Let me know if you’d like me to elaborate on any specific step or aspect of this assay!