I am working on a project where I am testing the resistance of a certain bacteria to copper and was testing it by adding copper at varying concentrations to r2a agar. However, despite sitting in a cold room for a week the agar at 0.08 M Cu 2+ is completely liquified and 0.00321 M Cu2+ is like a slushy. The other agar concentrations solidified. Is this happening because of the copper concentration and is there a way around this?
An interesting observation but I doubt about the such role of copper...
The copper will always be accompanied by a counter ion. Did you try another copper salt to confirm that copper is the culprit?
I actually had to look it up, because I know that bacteria normally don't like copper but this has been checked on Agar plates.
Looking around a few articles, it looks like your concentration is quite high.
Ma et al. [1] used a concentration of ~0.015 M (1,000 mg per L) and regarded it as very high.
But generally should not be a problem with solidifying. I would try to make a simpler medium like LB, as R2A uses a very low concentration of Agar, or supplement to ~10% agar and see if that works.
And it could always be a foolish problem like: missed calculation, bad ingredients etc. Try making it again with fresh reagents.
Good luck!
[1] Ma, Y., Rajkumar, M., & Freitas, H. (2009). Inoculation of plant growth promoting bacterium Achromobacter xylosoxidans strain Ax10 for the improvement of copper phytoextraction by Brassica juncea. Journal of Environmental Management, 90(2), 831-837.
@ C.A. (Kees) Kan I only have the one copper salt and I am working out of a high school lab so I was not able to check a diffrent salt (the copper I am using cupric nitrate Cu(NO3)2). I think the issue might be the dramatic change in pH caused by the addition of copper is what is preventing solidification (it goes from 7.4 to about 4.6)
Amir Nakar Thank you very much for your suggestions. I am using r2a because the bacteria I am using (Bacillus Arenosi) requires the low nutrient solution, and the reason my concentrations are so high is because I am attempting to make sure that the bacteria does not initially grow in those concentrations as the goal of the experiment is to build the resistance of the bacteria through artificial selection. The 0.08M concentration is actually the concentration of the most polluted soil on earth currently.
Christina Hermansen yes you are right. Its the pH that is affecting the setting of the agar. R2A contains Agar does not set in acidic conditions.
https://www.researchgate.net/post/How_would_you_make_agar_plates_at_pH_30
you can refer to this link to some suggestions on getting agar to set at acidic conditions. However what they suggested of doubling the agar concentration has its drawbacks. I would suggest you put in a buffer solution so that it will still fulfill your experiment requirement and yet make it feasible. Also note on the solubility of the salt in different pH. This needs to be closely monitored as well. Hope it helps cheers.
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