Because the GFAJ-1 cells have been growing well on plates (visible colonies in less than 48 hr), I've started using colony counts to follow their growth (or lack of it). Two little experiments highlight the problem.
First, I tested the growth of cells in several tiny colonies that had grown up in 48 hr on agar plates with the complete ML60 medium. I sucked each colony up into a micropipette tip and resuspended it in 100 µl of ML60. Then I spotted 1 µl, 10 µl and the remaining 89 µl onto a new agar plate and incubated it for 48 hr. The 1 µl spots each produced more than a thousand new colonies, which means that the original colonies each had more than 10^5 cells. This means that, on agar plates, the cells are doubling in less than 3 hours, much faster than their fastest doubling rate seen in Fig. 1 B of the Wolfe-Simon et al. paper (~12 hr).
Second, I tested the growth of cells in liquid ML60 medium. I had a stock of cells that had been grown in a layer of liquid ML60 overlying the surface of a ML60 agar plate. I serially diluted these cells in liquid ML60 (10^-1, 10^-2 ... 10^-6), and spotted 10 µl of each dilution onto agar plates. After 48 hr the 10^-5 dilution had produced about 50 colonies, indicating that the original stock had about 5 x 10^8 viable cells per ml. While the agar plates were incubating I also incubated the dilutions I'd made, and after 48 hr I plated them again. Now the 10^-5 dilution spots produced only ~1 colony, and the 10^-4 spots produced ~15. This means that 95% of the cells had died in the same medium that, when solidified with agar, they grew very well on!
Today I'm going to several more tests:
First, I'm going to make a completely new batch of the ML60 medium base, and test whether cells grow better in it.
I've already tested whether cells grow better sealed in screw-cap glass tubes (used by Wolfe-Simon et al.) rather than in loosely capped glass tubes - they don't. using the sealed tubes lets me test whether they grow better when gently agitated. I can't fit a roller wheel into my little 28°C incubator but I can fit a rocker platform. But I first needed to test whether the rocker motor produced too much heat, as this would raise the temperature of the incubator above 28°C. The temperature seemed OK last night, so they cells in sealed tubes have been rocking overnight.
Finally, I'm going to test whether GFAJ-1 cells use agar as a carbon source, by plating them on agar-solidified ML60 with no glucose, and by adding agar rather than glucose to the liquid medium. Maybe I'll also test whether they grow on ML60 solidified with agarose (much more pure than agar). I don't think that we have any gellan (an agar substitute), but I know that a lab upstairs does, so I might test that too.
The big question is, should we still try to test whether GFAJ-1 put arsenic in their DNA, if I can't grow them under exactly the conditions that Wolfe-Simon et al. used?
What mutation rate do I want for my experiment?
10 hours ago in RRResearch