Team:Toronto/Project/Assays

Assay Protocols

Cell density
Principle: OD 600 is used as a surrogate for number of cells.
Materials:
       Plate reader
       96-well plate with clear bottom
Procedure:
       1.Measure OD 600 for the entire plate of cultures in the micrometer plate reader.

Aggregation
Principle: Measure cell density in culture below surface, aggregated cells will have sunk to the bottom of the well.
Materials
       Plate reader
       96-well plate with clear bottom
Procedure:
       1. Remove 150 μL of culture volume from overnight culture plates.
       2.. Place it into the empty microtiter well, at the edge of the plate. To prevent bubble formation, make sure to stop pipetting at the first stop.
       3.Measure OD 600

Crystal violet
Principle: Crystal violet binds to polysaccharides of the biofilm matrix. By measuring the binding to adhering biofilm, the amount of biofilm produced can be quantified. A stock solution of crystal violet is incubated with the culture, the supernatant is removed, bound crystal violet is mobilized with an ethanol/acetone solution and the absorption is determined.
Materials:
       Crystal Violet stock solution: To make 1L, add 0.3 g of crystal violet to a bottle and fill to 1L with double distilled water. Stir well. Store at room temperature till usage.
       1400μl double distilled water
       350 μL 80:20 ethanol: acetone stock solution
       • 96-well plate with clear bottom