Field of Science

Inconsistent effects of 40 mM arsenic

OK, I've now done the same experiment four times and gotten two different results, twice each.  Each time I set up six 10 ml cultures of GFAJ-1, each inoculated with about 10^4 cells from one frozen stock (three times) or from a previous culture (one time).


Most of the results are consistent.  Each time, the cultures with no arsenate grew as they had previously, to about 2x10^9 cfu/ml (#1), 2x10^7 cfu/ml (#3), and 0.5-1x10^7 (#5).  Each time, the cultures with 40 mM arsenate and 1.5 mM phosophate or no added phosphate grew little or not at all in the first two days.  YThe two times I've done a longer follow-up they both grew very slowly, to final densities a bit lower than their no-arsenate controls.

But the results with 40 mM arsenate and 3 ┬ÁM phosphate (#4) are very inconsistent.  Twice they behaved like the other arsenate cultures, growing very little at first and, in the one follow-up, slowly growing to a density slightly lower than the control.  But twice they grew as fast as the control and to about double the density.  I have no idea why.

I think I should consider the fast growth of two #4 cultures to be the anomaly, as the failure of the #4 cultures to grow the other times is entirely consistent with the behaviours of the other arsenate cultures (#2 and #6).  But how to investigate why?  Rather than just repeating the cultures again and again, I should try varying the conditions a bit.  Screw-cap tubes instead of foil-capped flasks?  Slightly different amounts of phosphate?  Less arsenate?

If I had more time I'd test all these variables.  But my big new genetics course starts in two weeks, I'm going to London next week for a week (part brief visit to family, part Science Online London), the CIHR grant proposal needs, by Sept 15, both revision and new supporting data (some of which I'm trying to generate), and the postdoc and I still need to finish his uptake-specificity paper by then (he still needs to send me his new analyses).

Setting up the cultures isn't a big deal, but monitoring them takes time every day, because I'm diluting and plating them to get colony counts.  Maybe if I just monitor growth visually (by turbidity) and not by plating.  Wolfe-Simon et al. grew all their cultures in screw-capped glass tubes, so maybe I'll just set up a lot of these, with more diverse concentrations of phosphate and arsenate.

3 comments:

  1. I wonder if this could relate to the behavior of arsenate in this medium? Could it have a reaction with another media component that sequesters it, maybe something that is pH dependent? I haven't thought this thru all the way, but similar things (not with arsenic!) have happened to me, and when it did, it was always pH...

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  2. So the +As cultures sometimes grow to twice the density of controls, and sometimes more slowly but still eventually reaching desities similar to no As controls. And yet the blog-o/news media only picks up on the fact that in some cases you have trouble getting them to grow, and claim a refutation of the idea that these bacteria are even As-tolerant? The Science paper never said that they grew quickly.

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  3. I would suggest inoculating the problematic tube (40mM/3uM) with many different dilutions of the O.N culture. I have no explanation to this, but sometimes a pattern emerges and a range of dilutions behaves differently. I hope you'll solve the problem. Good luck.

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