This is a lot faster than Wolfe-Simon et al. reported for GFAJ-1 cells in their liquid medium (about two doublings per day was the fastest), and it's a hell of a lot faster than my GFAJ-1 cells are growing in the liquid version of the same medium. I think the cells in liquid medium are still alive, but the numbers from my crude counts haven't really changed at all in the past two days, and have hardly increased from when I inoculated them five days ago. It's not for lack of phosphate; if anything, the cells in medium with little or no added phosphate are doing better than the cells in medium with the full 1.5 mM phosphate that's also in the agar medium.
Do the cells just prefer growing on an agar surface to growing in a liquid? Does the agar contain some trace nutrient they need? Or chelate away some inhibitory trace component of the liquid medium? Is it something about being in a petri dish? Or being sealed? (The petri dishes were wrapped with parafilm so they wouldn't dry out during long incubations, but the tubes of liquid can breathe through their loose caps.) I've made a fresh batch of agar plates, which should let me test these ideas.
- Compare growth on plate from previous batch to growth on new batch.
- Spot 10 µl of each liquid culture onto plates (I did this three days ago too, so I can compare colony counts).
- Compare growth with the two vitamins to growth without pantothenic acid, with and without thiamine.
- Compare growth with and without parafilm wrapping.
- Test whether cells will grow in liquid medium if it's overlaid on agar medium.
- Test whether cells will grow in liquid medium if it's in a petri dish.
And I've found a Petroff-Hausser counting chamber, so maybe I can improve my microscope counts. I've also had advice from several colleagues who count cells by flow cytometry or microscopically - I'm still digesting this.
This thing isn't known to prefer low-oxygen, is it? (I'm not a microbiologist so pardon if it's a dumb question)
ReplyDeleteIt's relatively common for things to prefer growing on plates - they do scavenge from the agar. Which type are you using? I use Oxoid No. 1 and then wash it with ether, ethanol and adiculated water to get rid of soluble organics and metals. If you're being really particular, why not try agarose plates? Decadent, I know, but so much cleaner.
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@xenobiologist: the paper didn't say anything about its oxygen preferences, and it doesn't seem to care whether its plates are sealed with parafilm (upcoming post). In general Halomonas species are pretty versatile (thought they like high salt).
ReplyDelete@Rich: The goal is to get them to grow in liquid, not to stop them growing on agar.
Interesting, if his halomonas simply loves to grow on agar, that the thing we are also challenging with other bacteria.
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