Field of Science

No growth in 40 mM arsenate in ANY container!

And excellent growth without arsenate in every container...

As I had planned, I mixed the cells (about 10^5 cfu/ml) with 500 ml of medium, then split the medium in two parts, adding arsenate to one half and the same volume of water to the other.  Then I distributed the cultures among all the containers and put them in the 28 °C incubator for about 36 hr (sitting on the shelf, not agitated in any way).  (The biggest bottles, the orange-capped ones I mixed the initial cultures in, were in the 30 °C room.)

The containers with the blue '-' labels have no arsenate, those with the orange '+' labels have 40 mM arsenate.


The complete lack of growth of the arsenate cultures in the screw-capped glass tubes contradicts my previous results.  Might growth in arsenate depend on gentle mixing as well as on mysterious unidentified factors?  (This shouldn't matter since the cells are motile...)

This is ridiculous.  I don't think I've ever had such blatantly non-reproducible results before.  All I can think of to do next is to once again test growing cultures in glass screw-capped tubes, in medium with and without arsenate.  But even if the cells grow with arsenate in this next test, I won't have any idea why, or why they didn't in this experiment.  And anyway this experiment clearly says the problem isn't the containers.  

Readers, any ideas?

19 comments:

  1. Hello Rosie,

    Do you have frozen or lyophilized stocks made from when you first received the culture? Can you start a new working culture from frozen stocks in case being cultured in the absence of arsenic causes the cells to become sensitive to arsenic?

    Josh Neufeld (Waterloo)

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  2. Dear Rosie,

    Have you checked whether the bacteria in arsenate cultures are still viable? Wash away the arsenate move them to the a rich medium and see what happens.If the revive growth might just have stunted.

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  3. As unhappy as the cells are in arsenate, could the arsenic itself been screwing with their motility in some fashion? That's the only explanation that comes to mind re: mixed v. still cultures.

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  4. From someone totally uneducated in microbiology, but could the stock be impure and have a very small number of cells which can grow in arsenate?
    michiel's suggestion would disclose this.

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  5. Maybe I'm violating a sacred boundary with this suggestion, but I would love to hear Felisa Wolfe-Simon's thoughts on this. She must have done something differently (her error bars in Fig. 1 are tiny!), and we're all tearing our hair out trying to figure out what it is. Have you considered just asking her? Is she too busy to try to help? As a scientist, I'm thrilled when I see that someone has reproduced my results, especially in a different lab. Wouldn't she also want to see this?

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  6. If the cells are not pure culture, some may have plasmids conferring arsenic resistance plasmids.

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  7. The cells have likely mutated, erasing any former capacity for growing in an arsenic-laced environment.

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  8. Is it possible for arsenate to interact with any of the components in the medium?

    And have you been using the same ingredient containers all along? (i.e., have you finished off a container of something and started using a new container of that ingredient?)

    The reason I ask: My experience with trace metal toxicity is that any time we opened a brand new ingredient container we had to test a whole panel of metal concentrations using the new ingredient(s). This is b/c the trace metals would complex with the various organic compounds in the medium ingredients. Since we had to use complex/undefined ingredients (such as casamino acids or yeast extract), the only way to guarantee that consistent bioavailability (and toxicity) of the trace metals would be to use the exact same ingredient containers. Same old container, very reproducible results. New container, new toxicity profile.

    So if you haven't done so already, try talking to a geochemist about whether there could be anything in your medium that could be interacting with the arsenate, either chemically or physically. This might explain why some batches of arsenate medium are more toxic than others.

    If that line of inquiry doesn't pan out, then my next guess would be genetic instability of some type for the arsenate tolerance phenotype.

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  9. Do you have a chemostat? It might be interesting to get them going in continuous culture, and then slowly crank up the arsenic. If the problem is that they've adapted away from arsenic tolerance, that would be one way of trying to get them back on the wagon.

    Even E. coli would start to tolerate arsenic if you took it slow enough. I suggest the following experiment :

    [1] Sequence all available strains of GFAJ-1

    [2] Grow them in chemostats under conditions that would drive them to arsenate tolerance

    [3] When they are able to tolerate enough arsenate to satisfy you that they are adapted, resequence them.

    [4] Align the resequenced genomes with the non-adapted strains

    [5] Design primers to amplify up the affected regions

    [6] PCR up environmental DNA from the original habitat with the primers, make some clone libraries and sequence them (also PCR up sequences from your lab strains, both adapted and non-adapted, just to make sure they work)

    [7] Align the PCR'd sequences with the GFAJ-1 genomes you've got

    If GFAJ-1 ever was arsenate-tolerant, you should see at least some similarities among the lab-adapted adapted strains and the environmental samples supposedly living in arsenate.

    If not, I think we'll all have to notch up the skepticism another couple of pegs.


    Russell

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  10. @Alexa: Wolfe-Simon was "evicted" from her previous lab after all the chaos, so I guess she might very well have lots of free time to give advices..

    http://www.popsci.com/science/article/2011-09/scientist-strange-land

    Actually I think Rosie should hire her. This would give a really positive message about science and women in science.

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  11. NotAnAstrobiologistOctober 18, 2011 at 8:43 PM

    Gah!
    This is a tough one...

    What I would do in your place, start parallel cultures in -As and, say, 5mM arsenate...

    Keep seeding cultures in progressively higher [+As], (and keep the old lower [+As] going as well)...

    See if you can pinpoint the concentration where things blow up...

    Presumably (based on your earlier results) -As should be able to keep growing normally without any problems...maybe at 15mM cells will start to die for magical reasons that can eventually be figured out.

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  12. Our students at CSUSB presented this paper in Journal club, and we had, shall we say, a lively discussion :). One thing that struck me in reading the published back and forth in Science was the proposal that GFAJ-1 uses the high-affinity P transport system induced by arsenate. This was dismissed by FWS out of hand because in her mind it would lead to higher arsenate toxicity (I think that is what she was saying, I am not an expert on this). But it seems to me that if GFAJ-1 has a robust detox mechanism (reduction and sequestration or some such) it could induce the high affinity uptake and still handle the influx of arsenate. Further, it seems like the enrichment strategy they used would select for variants of exactly this type. This variant might be very unstable because expressing high levels of the detox mechanism would be a competitive disadvantage in the absence of As. The analogy that makes sense to me is something like a drug efflux pump. Several other commenters have already suggested methods to deal with this (going back to original stocks, using chemostat culture or serial increase in As) but the more i think about it the more this seems like the simplest explanation, and a testable one at that. Thanks for doing these experiments, this is really a nice way to show students and the public what science should and can be.

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  13. Have you finally added tungsten to the media? Maybe tungsten is necessary for the high affinity P transport system induced by arsenate that Paul Orwin discussed above.

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  14. How about you throw the towel in and stop wasting your own time/money trying to make sense of FWS' bunk science. The paper should never have been published and in these times of tough funding you shouldn't be wasting your time trying to replicate her crappy work!

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  15. @Anonymous, 7:57 PM: The question is, at what point can everyone agree that Rosie has sufficiently demonstrated the "bunkness" of FWS' work? I didn't believe the growth curves even back in December, but what about the people who were willing to give them the benefit of the doubt? These people still exist--you can find them in the comments section of the PopSci article. And what about the authors of the paper, who apparently don't take seriously anything that isn't published in a peer-reviewed journal? I am also stumped as to how Rosie should proceed (at this point, would a journal publish her results?), but at least she is doing something to try to mitigate the rampant confusion and misunderstanding regarding this whole story!

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  16. @alexa, I agree that Rosie should be commended for trying to make sense of the mess that is the FWS paper, but how far will this go? Seems like even the most basic of experiments (growing a culture) cannot be replicated reliably. And people are suggesting sequencing of all the different "strains" of GFAJ-1? Setting up a turbidostat? Funding is tough right now and the cost/burden of replicating/repeating this work ought to be on Oremland and FWS. It still blows my mind that Bruce Alberts hasn't done anything. If Oremland is a respected scientist he ought to try repeating (with better experimental design i.e. gel purifying DNA, MS/MS of the "As-DNA", etc) and retract the paper if they cannot replicate. Hats off to you for trying Rosie, but I hope your real research doesn't suffer from this distraction, although I must admit its been entertaining!

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  17. I disagree strongly with the notion that Rosie should give up (although it's obviously her choice and not mine). Although I think FWS and colleagues misinterpreted their data, I don't think it was fraud, I think they found an interesting bug that they were able to enrich from Mono Lake and coax into growing under high As/low P conditions. The mechanism of that is interesting! I think that the As for P in DNA is incorrect, but that doesnt mean the whole thing is worthless. As an aside, I think any working scientist has to spend a lot of time thinking about practical concerns like getting your next paper published or grant funded, so side projects like this are obviously on a "shorter leash". And why would you think Bruce Alberts or Norm Oremland would want to do this? Finally, of course people in a blog comment section are going to suggest over-the-top solutions (it's alot easier to tell someone else to do something than do it yourself). But maybe someone will have a really great idea! You never know - if you have an idea, suggest it.

    PS- growing a culture is not always "simple", and this set of experiments is good evidence for that.

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  18. I agree with most of the last two comments, but I would like to add some things about the growth experiments. Anonymous at 7:03 said that growing the culture should be considered "basic" and Dr. Orwin countered that growing a culture is not always "simple". I don't think Anonymous was trying to say that it's "basic" in the sense that it's simple or *easy*, I think what he was trying to say is that it's *fundamental* to the claims made in the paper as well as their novelty. If all the authors were able to do was enrich an organism and coax it into growing under an extreme condition, the paper would not have deserved the hype and media attention it received. What would have made the study truly novel would have been the demonstration that the organism was growing without any phosphorus at all, and based on the results in the paper and Rosie's results, it's highly unlikely that it did that.

    It's true that Ron Oremland, Felisa Wolfe-Simon, and all of the other authors are technically under absolutely no obligation to try to repeat their results or help anyone else do so. But if I were one of them, I wouldn't be able to sleep at night because I would be thinking, "huh, I wonder if my cell counts were biased, since I wanted to see growth so badly and Rosie Redfield thinks my method was so unreliable she doesn't even use it?" and "hm, I wonder if the optical density increase I saw was just due to altered light scattering from the inclusions in the cells? Maybe I should redo those experiments."

    So while the authors aren't obligated to unequivocally demonstrate growth and that arsenate replaces all the phosphate in arsenate-grown cells, I can't help but wonder why they aren't interested in at least discussing the work and trying to help others produce these results. I don't think it's "over-the-top" to expect that they would have that interest.

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  19. Hi Rosie,
    Have you considered how the bottles and containers you are using have been washed?
    In the lab I'm in, a guy behind me is working with iron-depleted media; the bacteria will respond and secrete iron-chelating compounds to compensate. Any contamination will result in highly inconsistent results. Extensive washing with chelating agents, HCl and deionized water was necessary.
    Trisodium phosphate is a very common glass cleaner. Surfactant residue can play a big role too; just ask any home brewer of beer.

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