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Team:Toronto/Project/Assays
From 2013.igem.org
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<p style = "font-size:18px;"><b><u>Cell density</u></b><br/> | <p style = "font-size:18px;"><b><u>Cell density</u></b><br/> | ||
Principle: OD 600 is used as a surrogate for number of cells. | Principle: OD 600 is used as a surrogate for number of cells. | ||
| + | <br> | ||
<br>Materials: | <br>Materials: | ||
<br> Plate reader | <br> Plate reader | ||
| Line 47: | Line 48: | ||
<br> | <br> | ||
Procedure: | Procedure: | ||
| - | + | <br> 1.Measure OD 600 for the entire plate of cultures in the micrometer plate | |
| - | + | reader. | |
| + | <br> | ||
| + | <br> | ||
| + | <p style = "font-size:18px;"><b><u>Aggregation</u></b><br/> | ||
| + | Principle: Measure cell density in culture below surface, aggregated cells will have | ||
| + | sunk to the bottom of the well. | ||
| + | <br> | ||
| + | <br> | ||
| + | Materials | ||
| + | <br> Plate reader | ||
| + | <br> 96-well plate with clear bottom | ||
| + | <br> | ||
| + | Procedure: | ||
| + | <br> 1. Remove 150 μL of culture volume from overnight culture plates.<br> | ||
| + | <br> 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.<br> | ||
| + | <br> 3.Measure OD 600<br> | ||
| + | <br> | ||
| + | <br> | ||
| + | <p style = "font-size:18px;"><b><u>Crystal violet</u></b><br/> | ||
| + | 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.<br> | ||
| + | <br> | ||
</p> | </p> | ||
</font> | </font> | ||
Revision as of 13:01, 27 September 2013
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.
