Quick note from the Bibel lab 3 - PVKA phosphate solubilization assay

Yep - these agar plates are teal!!!! And I finally broke in my lab coat with proof my scienceing is real! The teal color isn’t mold, instead it’s a dye called bromocresol green. And although it looks cool, it’s actually not the coolest part of this media. Instead, the really cool part, the super special part is the calcium phosphate. Basically it’s an inorganic form of phosphate. Phosphorus is super duper important to life as we know it. It gives you the P in ATP among other things! But, in calcium phosphate, it’s all tied up in a way that organisms can’t easily use. In fact, a LOT of the phosphorus that’s in soil is tied up in an accessible form like this. Which is a big problem. It’s one of the reasons why so much phosphate has to be put in fertilizer, because it gets all tied up bound to calcium, etc. when it’s added to soil.
What’s really cool though is that some microbes, like some bacteria, have a superpower which allows them to solubilize that phosphate. They can do this by doing things like secreting organic acids that can kind of take the phosphate away from the calcium and other metals. This makes it more usable both for themselves as well as for things around them like plants. So they can promote plant growth (so generous of them right?)
These plates are way that you can test for phosphate-solubilizing superpowers. The base of it is called Pikovskaya agar (PVKA). By itself, if you do not have the dye, it’s milky white. It’s all cloudy. You can still see the cloudiness with the dye. Because that cloudiness comes from the calcium phosphate. If that gets solubilized, what’s gonna happen is the media is gonna clear up. So you can plate bacteria, then look to see if the media around the bacteria clears up. This will let you see if the bacteria were able to soluble the phosphate. If so, you would see a spot of bacteria and then around that you’ll see a clear halo.
But the halo itself might not be clear to see! Unless you have a pH indicator! In our case, bromocresol green, though other dyes like bromothymol blue are also sometimes used. The reason why you add this is that the way that the bacteria are able to make the phosphate soluble is by secreting acidic stuff. So the media around them would acidic around the bacteria. And that would cause the dye to protonate, which would cause it to change color (to more of a yellow). So, then you’d have a halo that is different color and that’s gonna make it easier to see.
Unfortunately, when syringe filtering the stock solution of the dye, I managed to squirt some dye on my coat… (Those syringe filters without Luer locks are accidents waiting to happen…) Despite that, we got it filtered. Then Rory made some of these plates, spotted the bacteria on it and we’ll see what happens. Hopefully one of these at least one of these strains is able to solubilize the phosphorus. Which would increase its bioremediation potential!
We are using the plates for the screening stage, but they’re mainly qualitative. You can compare the halo diameters but that’s it. If you want to get more number-y and exact, there are broth-based methods. As well as some alternative media formulas that may be better for better purposes.
Here’s the paper we’re going off of.
Joe, M. M., Deivaraj, S., Benson, A., Henry, A. J., & Narendrakumar, G. (2018). Soil extract calcium phosphate media for screening of phosphate-solubilizing bacteria. Agriculture and Natural Resources, 52(3), 305-308. doi.org/10.1016/j.anres.2018....
Unfortunately, they don’t tell you the final concentration of dye, so I had to look elsewhere. I couldn’t find anyone give one for the PVKA, but found a different media composition that used the dye at 0.00022% w/v (g/100 mL), so we went with that, making a 0.22% stock solution (thus 1000x) in ethanol, adjusting the pH to ~7.2 with 0.1M NaOH, then adding that to the PVKA before autoclaving and pouring.
Fingers crossed!
more about bacterial media & agar plates: bit.ly/bacterialmedia
more about all sorts of things: #365DaysOfScience All (with topics listed) 👉 bit.ly/2OllAB0 or search blog: thebumblingbiochemist.com
#scicomm #biochemistry #molecularbiology #biology #sciencelife #science #realtimechem

[@MrBarryyoung]

Sounds like fun. Good Luck!

[@thebumblingbiochemist]

Thanks!

[@brandoncrooks1681]

if you were a phosphate, would you be bound cAMP or ATP or ADP or NADPH or cGMP or some different molecule? I'm a cAMP kind of guy.

[@thebumblingbiochemist]

ooh - maybe creatine?

[@saunglaseije176]

Hope you can find that bacteria ❤

[@saunglaseije176]

Also,that an exciting-phenomenon! Another step forward 🎉