Team:Toronto/Project/Assays

Assay Protocols

This page describes the assay protocols for each genotype and stimulus. These assays were chosen because we wanted to assess the biofilm phenotype thoroughly as well as evaluate the components of the biofilm that are most critical towards the transition to the sedentary lifestyle. Our battery of assays contains assays to assess overall biofilm development: Crystal violet, Aggregation and Adhesion assays. However we also examine curli expression with the Congo Red assay, cellulose expression with the Congo Red and Calcofluor assays, Exopolysaccharide expression with the Colanic Acid assay and fimbriae expression with the Yeast Agglutination assay.
Input into these assays are a series of microtiter wells with a culture volume of 300 μμL and phosphate buffered LB as the reference medium.
In terms of a qualitative biofilm assay, our petri dish-based Colony Morphology assay assesses the appearance, colour and texture of E. Coli colonies subject to varying stimuli.

Cell density
Principle: OD⁶⁰⁰ is used as a surrogate for number of cells.
Materials:
       •Plate reader
       •96-well plate with clear bottom
Procedure:
       1. Measure OD⁶⁰⁰ 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 OD⁶⁰⁰

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 mobilised 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.
       •1400 μl 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 350 μl of double distilled water.
       2. Aspirate 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 350 μl double distilled water wash 3 times. Make sure to empty the well completely on the last wash.
       7. Add 350 μL of 80:20 ethanol:acetone solution.
       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.

Agglutination
Principle:Fimbriae bind mannose. Yeast cells display mannose molecules on their cell surface and can be induced to visibly clump together with E. coli cells that express functional fimbriae under the specific culture conditions.
Materials:
       • Mortar and pestle
       • Prepare the stock solution (yeast slurry) fresh on the day of measurement:
       Add half a teaspoon of Fleischmann's yeast granules.
       Using a mortar, grind the yeast to powder form. Add 500mg of yeast powder in 5mL of 10% PBS.
       • 96-well plate with clear bottom
Procedure:
       1. Add 60 μL of the yeast slurry with 300 μL of overnight culture into well.
       2. Mix briefly using a micropipette.
       3. Score agglutination after 5 minutes on a scale of "–" to "+++", based on http://www.sciencedirect.com/science/article/pii/S0022283603005916

Nishiyama M., Vetsch M., Puorger C., Jelesarov I., Glockshuber R. 2003. Identification and characterization of the chaperone-subunit complex-binding domain from the type 1 pilus assembly platform FimD. J. Mol. Biol. 330, 513–525.

Adhesion
Principle:To measure the number of adherent cells by washing microtiter wells and determining remaining cells through increase of ethidium bromide (EtBr) fluorescence.
Materials
       • EtBr stock solution: 225 μL of (0.5 g/L ethidium bromide in water), 75mg of NaN₃, and 15 mL of 1X TBE. Store in cool and dark place till usage.
       • 1:1000 dilution of Sigma Antifoam A
       • 96-well plate with clear bottom
Procedure:
       1. Aspirate overnight culture volumes from wells.
       2. Add 100 μl of EtBr stock solution.
       3. Measure fluorescence with λ^ex 510 nm / λ^em 595 nm.

Colanic acid [http://bio.huji.ac.il/upload/94(1).pdf]
Citation: Ionescu, M., Belkin, S. (2009). Simple quantification of bacterial envelope-associated extracellular materials, J. Microbiol.Methods, doi:10.1016/j.mimet.2009.06.020
Principle: When evaluated against OD⁶⁰⁰ or cell count, measuring the height of each PCV tube provides fast and simple evaluation of small differences in extracellular content.
Materials:
       • TPP easy read cell counter tubes (PCV tubes) from http://www.sigmaaldrich.com/catalog/product/sigma/z761001?lang=en&region=CA
       • Microcentrifuge
Procedure:
       1. Add 455 μL of cell culture into a clean PCV tube. Parafilm the opening.
       2. Spin at 4,000 RCF for 2 min.
       3. Note length of the packed cell column according to the graduations of the capillary.
       4. Packed cell volume is expressed as a value per OD⁶⁰⁰.

Colony Morphology
Reasoning:
The dyes in the agar plates will differentially stain cells based on whether or not they produce curli and cellulose (Nakhamchik 2008). Bacteria that exclusively express curli will bind to the congo red and will form red colonies, whereas bacteria that express only cellulose will form blue colonies. If both are expressed, purple colonies will be formed. Rough colonies are indicative of curli formation. In the below photograph, there is more variation in colour at 23°C (picture on the left) than at 37°C, reflecting the protein expression phenotypes that are accessible at the lower temperature. This is the reason the former temperature was chosen to incubate colony morphology plates.

Nakhamchik A., Wilde C, Dean A. Rowe-Magnus (2008). Curli Fibers Are Highly Conserved between Salmonella typhimurium and Escherichia coli with Respect to Operon Structure and Regulation.J. Bacteriol,vol. 180 no. 3,722-731.

Materials
       • LB Agar with 40 µg/mL Congo Red dye and 20 µg/mL Brilliant Blue dye
       • Petri dishes
       • Antibiotic
       • Inoculation loop for streaking
Protocol
       1. Prepare phosphate-buffered LB Agar containing 40 µg/mL Congo Red dye and 20 µg/mL Brilliant Blue dye along with appropriate antibiotic.
       2. Prepare overnight cultures of E. coli in phosphate buffered media.
       3. To save plates, divide each plate into 4 quarters using a lab marker or use a whole plate. Streak out bacteria from overnight culture using an inoculation loop.
       4. Incubate in darkness at 23°C for 48 h.
       5. Assess colony morphology.

Congo Red
Reasoning:
CR binds to curli and cellulose. The difference between the blank CR absorbance and absorbance of the supernatant taken after centrifugation will be proportional to the amount of CR by our substrates of interest.

Nakhamchik A., Wilde C, Dean A. Rowe-Magnus (2008). Curli Fibers Are Highly Conserved between Salmonella typhimurium and Escherichia coli with Respect to Operon Structure and Regulation.J. Bacteriol,vol. 180 no. 3,722-731.
W. E. Klunk, R. F. Jacob, and R. P. Mason, “Quantifying amyloid by congo red spectral shift assay,” Methods in Enzymology, vol. 309, pp. 285–305, 1999.

Materials for Congo Red:
       • 0.5 mM CR solution in ^ddH²O
       • Antifoam A concentration
       • 96-well plate with clear bottom
       • Microcentrifuge tubes
       • Centrifuge
       • Absorbance plate reader
Protocol
       1. To a 0.5 mM Congo red (CR) in ^ddH²O, add 15 μL of Antifoam A concentrate per 100 mL (final concentration 10ppm per well on 96-well plate) to prevent bubbles while pipetting.
       2. Pipette 200 μL of 1:100 dilution of overnight culture grown in phosphate buffered LB into 96-well plate well.
       3. Add 100 μL of phosphate buffered LB.
       4. Incubate for 48 h at 23°C in darkness.
       5. Add 20 μL of the 0.5 mM Congo red solution to well containing bacteria grown for 48 h.
       6. Pipette 150 μL of CR into another well for a blank measurement (the “blank CR” well).
       7. Incubate for 60 min.
       8. Transfer 200 μL into microcentrifuge tube.
       9. Centrifuge at 14,000 x g for 30 seconds.
       10.Transfer 150 μL to an empty well on the microtiter plate.
       11.Measure absorbance at 490 nm.
       12.Subtract absorbance value from absorbance value of the“blank CR”. This will give you the amount of bound CR.

Calcofluor
Reasoning:
CF binds to cellulose. The difference between the blank CF fluorescence and fluorescence of the supernatant taken after centrifugation will be proportional to the amount of CF bound by our substrates of interest.

Nakhamchik A., Wilde C, Dean A. Rowe-Magnus (2008). Curli Fibers Are Highly Conserved between Salmonella typhimurium and Escherichia coli with Respect to Operon Structure and Regulation.J. Bacteriol,vol. 180 no. 3,722-731.

Materials for Calcofluor:
       • 0.02 g/L CF solution in ^ddH²O
       • Antifoam A concentration
       • Black 96-well plate
       • Microcentrifuge tubes
       • Centrifuge
       • Fluorescence plate reader
Protocol
       1. The original calcofluor (CF) solution contained Evan's blue 0.5 g/L, CF 1.0 g/L in water (as purchased from Sigma). To a working solution of 0.02 g/L CF of original solution, add 15 μL of Antifoam A concentrate per 100 mL (final concentration 10ppm in each well of 96-well plate).
       2. Pipette 200 μL of 1:100 dilution of overnight culture grown in phosphate buffered LB into 96-well plate well.
       3. Add 100 μL of phosphate buffered LB.
       4. Incubate for 48 h at 23 degrees C in darkness.
       5. Add 20 μL of 0.02 g/L CF stock solution to first well.
       6. Pipette 150 μL of CF into another well for a blank measurement (the “blank CF” well).
       7. Incubate for 60 min.
       8. Transfer 200 μL of first well contents into microcentrifuge tube.
       9. Centrifuge at 14,000 x g for 30 seconds.
       10.Transfer 150 μL to an empty well on the microtiter plate.
       11.Measure fluorescence with λ^ex 352nm / λ^em 450 nm.
       12.Subtract fluorescence value from fluorescence of “blank CF” well to get amount of bound CF.