Hi,
Most protocols I have seen involve preparing the media as usual and autoclaving. Then, the media is cooled to the pour temperature and a solution of aqueous Congo Red is supplemented by sterile filtering to the media with a final conc. from 0.05-0.1% wt/vol.
For example, if one was growing E. coli biofilms, one would prepare salt-free LB (10g tryptone, 5g yeast extract per L culture) and autoclave at 121°C for 30 min, including an autoclave-safe stir bar. While it cools, prepare 0.5g of Congo Red in a minimal volume of water (probably about 50 mL to dissolve it completely). Once the media is cool enough to pour (~50°C) add the Congo Red solution through a 0.22 µm syringe-driven filter to the media under sterile conditions and stir well on a magnetic stir plate until the Congo Red is fully incorporated. Pour the agar plates in Petri dishes as usual.
Dear Sumesh,
We have used Congo red plates in the past to assess cellulose expression by pseudomonads. This has been reasonably successful, though there are a couple of points well worth considering:
First, media and bacterial growth may effect the strength of the Congo red colour (i.e., the redness). Pseudomonads often produce siderophores, and the yellow fluorescence produced by our strains alters the Congo red plate colour at times (we can reduce this effect by adding FeCl3 to our media). It is better if you can use a media which does not have a very strong intrinsic colour (e.g., a minimal media to which you add sugars or amino acids).
Second, media with high NaCl levels will cause the Congo red to aggregate, so it is best to omit or use lower NaCl concentrations than normal. For example, we have used standard LB without NaCl. Always spin your Congo red stock solution down in a centrifuge (say, 1 min at max speed) to pellet any aggregations that form during storage.
Third, although you can easily 'see' the Congo red colour by eye, it is very difficult to take a digital image of this, as we see the red saturation in a different way to the camera. It is therefore really important that you pour Congo red plates so they all have the same volume / depth. Don't try adding Congo red to a poured plate before or after it sets, as it is impossible to get a uniform background colour. If you are taking pictures, try putting the plates in the fridge for a couple of hours or over-night before taking pictures, and use white and black backgrounds to get a better image.
Fourth, you probably need to try out a range of plates with different levels of Congo red, in order to find the concentration best for your work. We have used Congo red at 0.001% (w/v) for LB no NaCl plates we normally incubate for 2 - 3 days before assay.
Regards,
Andrew
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