"
Team:Toronto/Project/Assays
From 2013.igem.org
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
Procedure:
1. Wash wells of overnight culture by removing well contents and pipetting in
350μl of double distilled water.
2. Reaspirate immediately by touching pipette to the side of the plate.
3. Add 350 μL of CV stock solution to well.
4. Incubate for 30 min. at room temperature.
5. Aspirate all of the liquid.
6. Repeat 350μl double distilled water wash 3 times. Make sure to empty the
well completely on the last wash.
7. Add 350 μL of ethanol/acetone.
8. Incubate for 15 min. while gently shaking.
9. Transfer the entire amount to an empty well on the microtiter plate.
10.Measure absorbance at 600 nm.
