First, how many arsenate ions would it take to completely coat the inner surface of one of the glass tubes I'm using? The tubes are about 10 cm long, with an inner circumference of about 2 cm, so that's about 20 cm^2 of glass surface. This paper says that the ionic radius of arsenate ions is 0.248 nm, so I'll assume a diameter of 0.5 nm. If the ions were to pack squarely side-by-side onto the glass surface, there would be about 2x10^3 per µm, or about 2 x 10^6 per mm. That's about 4 x 10^12 per mm^2, or 4x10^14 per cm^2. So if the inner surface of a glass tube were densely coated with a monolayer of arsenate ions, it would sequester about 8x10^15 ions.
Next, how many arsenate ions are in 5 ml of a 40 mM solution?
6x10^23 ions/mole times 0.04 moles/liter times 0.005 liter = 1.2x10^20 ions.So less than 0.01% of the arsenate ions in my medium could be tightly packed in a monolayer on the inner surface of a glass tubes. The assumption of dense packing is very conservative, so I don't think sequestration of arsenate by the glass can be the explanation for the GFAJ-1 growth I'm seeing. But thank you to the commenters for prompting me to do the calculation.
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ReplyDeleteThe arsenate levels are really very high in the media. But perhaps the arsenate ions form something other than a monolayer on the silica, maybe, as a previous commenter mentioned, mediated by positively charged ions in the media?
ReplyDeleteThis paper discusses aggregates of phosphate and calcium ions in solution - might it be applicable to arsenate, too?
http://onlinelibrary.wiley.com/doi/10.1002/zaac.200400151/abstract
After all, don't silica-based miniprep columns take advantage of silica's affinity for phosphate? - perhaps the interaction is synergistic in some underappreciated way, and works for arsenate too?
There is an assumption there that the glass surface is perfectly smooth. Can you be sure of that after acid washing? Is there an assay for arsenic that can be used on old growth cultures?
ReplyDelete"There is an assumption there that the glass surface is perfectly smooth." My understanding is that at the approximately nm scale, glass is far from perfectly smooth. I do not, however, know how the sizes of the glass surface features compare with the size of arsenate ions.
ReplyDeleteEven so, I would still say a case can be made that worrying about the mechanism at this point in time is a little premature. The mechanism is secondary to demonstrating whether or not the "arsenate in glass" phenomenon (as opposed to the "arsenate in plastic" phenomenon) occurs in the first place. The test for this is really simple and straightforward (see RR's comment in the previous post), so I would suggest taking "the Nike approach" (in other words, just do it). If it was hard to do, then I would feel very differently about it. But the proposed experiment should be easy to carry out.
I readily agree that the "arsenate in glass" idea does not appear to have an obvious mechanism, but the fact also remains that obvious mechanisms for "arsenate in plastic" don't seem to be readily forthcoming either.
Worst case scenario: the proposed experiment reveals that the "arsenate in glass" phenomenon is clearly incorrect and everybody knows not to put energy into worrying about it (and focus on the plastic question instead). IMO that is not an unreasonable cost/benefit ratio.
@Andy: Conceivably, but that would be a PhD project in chemistry, far outside of my expertise.
ReplyDelete@acleron: I considered the issue of glass roughness, but doubt that it could increase the (purely hypothetical) binding by a factor of >10,000.
@GH: Since my goal is not to understand all the science (this is only a little side project, I have a shitload of other stuff to get done) I'm not going to focus on the plastic question either. but on finding conditions giving reproducible phosphate-limited growth in 40 mM arsenate. But I will do the simple test, just to satisfy our curiosity.
If you had a way to measure arsenic levels (atomic absorption?), then all of this would be moot, right?
ReplyDeleteThen you could measure arsenic before incubation, in the spent media, etc. etc.
With the understanding that the time and resources you have for this project are limited...
ReplyDeletePlastics and glass will both carry along weird contaminates that might not be so easy to clean off. You might want to talk to a geochemist or someone else who specializes in finding weird stuff in weird places about figuring out what's in/on your labware. It's possible that there's something else in your glass that's mopping up the arsenate. Maybe not all of it, but enough to get it below a toxicity threshold? Alternatively, maybe there's something in the plastic that's raising havoc in a +As environment.
As far as cleaning procedures go, my understanding of acid washes is they don't actually damage the surface of the glass (as opposed to KOH etches). Since I'm not a microbiologist and am not familiar with what sort of acids you guys use, maybe you need a more aggressive method? Aqua regia? Piranha? I know it sounds like overkill but if you think you have a contamination issue and the normal approach isn't working, it's time to get scary.
Haven't seen a link posted to the pop sci article on Felisa:
ReplyDeletehttp://www.popsci.com/science/article/2011-09/scientist-strange-land?page=1
Pretty fairly written overall I'd say. It does leave you feeling a little sorry for FWS. A touch cruel that she was 'evicted' from Ron Omerland's lab...surely her adviser has some culpability here.
I am certainly excited to see Rosie's experiments continue, but it's hard to imagine that FWS's conclusions are correct given the picture the article paints.
@ NotAnAstrobiologist
ReplyDeleteNo need to feel sorry for FWS. There are only two possibilities: she is a bad scientist or she is unscrupulous. In any case, she does not deserve to have a job as a scientist.
The article you link is far from fair. It claims that FWS's paper "is not so flawed that it should not have been published" and that FWS just "found herself" in a difficult situation beyond her control. The truth is FWS put herself into this situation by submitting a flawed paper. Not even the most inexperienced of my students would try to submit a paper in which there is not enough evidence to support the conclusions and to rule out reasonable alternative explanations.
You can try to be balanced about matters of opinion but statements about facts are either true or false.
I didn't catch the "not so flawed" quote...agreed that really misrepresents basic fact.
ReplyDeleteStill, I feel bad for her.
@NAA: Thanks for the link - I hadn't seen this article (though they did fact-check parts of it with me).
ReplyDeleteFWS made a couple of big mistakes, but she was also very poorly served by the more senior colleagues who should have been watching her back. Sadly, I don't think I've seen any of them admit to bearing any responsibility for this mess.
Glass v. plastic. One thing that comes to my mind is that glass is not a static container. When we need to grow yeast under iron limiting conditions, we use plastic. You can also use acid washed glass, but the acid only strips iron off the surface of the glass. If said acid washed glassware is not used within a few days enough iron leeches to the surface to promote growth again. I highly doubt arsenic is leeching into the glassware and being removed from the growth medium at much beyond the 0.1% you measured for potential surface binding, but maybe something is leeching out from the glass to help protect the cells.
ReplyDeleteI like the idea of trying a run with acid washed glassware. If you see increased 'sensitivity' to arsenic that suggests whatever is contributing to the phenomenon is acid soluble. Although a negative result does not help much.
@Lorax: I did acid-wash these glass tubes. (2% HCl for 48 hr)
ReplyDeleteBut you also autoclaved the glass after you acid-washed it, effectively re-introducing stuff (e.g., metal traces carried with the steam) to your glassware.
ReplyDeleteGeochemists have always told me that it is better to sterilize first, then acid wash in a sterile laminar flow hood.
As an aside, the lab I worked in avoided glass whenever their experiments required precise metal concentrations. This was b/c glassware will have various metal contamination issues that can not be reliably removed with acid washes. That lab used acid-washed polycarbonate when metal-clean and/or metal-precise was important. They always autoclaved first, then acid-washed.
Rosie - would you mind dropping me a quick email? I can't find your email address sorry - I've some expertise with metals/glassware in microbiology - I work on bugs that are very sensitive to minute traces of copper so we have to be very careful. I've also worked on heavy metal metabolism, which is along a similar vein.
ReplyDeleteEmail: b o d e n r i c h AT @ g m a i l DOT c o m
Many thanks - I think I may be able to help a bit on this one.
Bear in mind that I'm only using 'reagent grade' chemicals, so any trace metals that might be on the glassware are probably also in the medium.
ReplyDeleteHow can you try to do these experiments with reagent grade chemicals after you crucified Wolfe-simon for not using pure enough reagents to avoid P contamination in her As salts? You, madam, are a hack.
ReplyDeleteAnd the medium contains a 'trace metal' supplement, so if trace metals are causing the reproducibility problems then they're intrinsic to the experiment...
ReplyDelete