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
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. 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, ground the yeast to powder form. Add every 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 by aspiration.
       3. Score agglutination after 5 minutes on a scale of "–" to "+++", based on http://www.sciencedirect.com/science/article/pii/S0022283603005916 (Citation: 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 fluorescence.
Materials
       • EtBr stock solution: 225μl of (0.5 g/L ethidium bromide in water), 75mg of NaN3, and 15mL 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 λex 510 nm / λem 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 color at 23 degrees C (picture on the left) than at 37 degrees 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 23oC 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.