The design of this experiment is described in the previous post.
The upper graph shows that, in the absence of arsenic, growth is phosphate-limited and nicely reproducible. The red lines are replicate cultures with no added phosphate (two in glass screw-capped tubes, one in plastic (square symbols) ); the blue lines are replicates with 3 µM phosphate added (three in glass, one in plastic) and the purple ones are replicates with 1500 µM phosphate added (two in glass, one in plastic).
The lower graph shows that growth is much less reproducible in the presence of 40 mM sodium arsenate. The blue, red and purple lines are the same phosphate treatments as in the left panel; again square symbols indicate cultures in plastic rather than glass tubes. The most striking result is, as seen before, that cultures in the plastic tubes (the three lowest lines) grew very little, even with abundant phosphate. The two no-added-phosphate cultures in glass tubes (red) grew identically and slightly better than their no-arsenate controls, as did two of the three 3 µM phosphate cultures (blue). The third 3 µM phosphate culture grew to only half the density of the others. The two 1500 µM phosphate cultures (purple) grew to high density, one slower than the other.
The growth differences between replicates are unlikely to be due to differences in inoculum size, since all cultures began with 10^5 cells/ml.
I have no idea why growth in polypropylene tubes makes cells arsenic-sensitive. Googling 'arsenate' plus 'polypropylene' didn't suggest anything.
Maybe now I'll try cultures in glass flasks.
I'm absolutely sure you've considered this, but since you didn't mention it: your data seem to be consistent with the possibility that the arsenate is adsorbed onto the glass, away from the cells. Arsenic does seem to be a component used in the manufacturing of some glass, raising the possibility that it does interact with silica in some way.
ReplyDeleteThis hypothesis predicts that adding silica particles (or ground up glass, or diatomacious earth) to the plastic tubes would also rescue the growth in the presence of arsenate...
Perhaps, as Anonymous suggested, the glass is detoxifying the arsenate. Calcium ions can immobilise arsenate. Most glass contains calcium. Perhaps you could make up a solution in glass without bacteria then test for arsenate in the solution after 24 hours?
ReplyDeletehttp://pubs.acs.org/doi/abs/10.1021/es980998m
ReplyDeleteI've been assuming that the glass tube has far too little surface area to sequester all the arsenate from a 40 mM solution. Could I be wrong about this? How much arsenate might a square mm of glass bind?? Better turn this comment into a post.
ReplyDeleteI guess I could leave the medium in glass tubes for day, then transfer it to glass and plastic tubes with cells...
Showing that glass tubes sequester all the arsenate would certainly create problems for the Wolfe-Simon paper...
I was the "anonymous" commenter from your previous blog post.
ReplyDeleteMy hunch continues to be that the issue isn't plastic somehow making arsenate more toxic, but that the bioavailable arsenate concentration in glass is lower than the total arsenate concentration. My knowledge of arsenate chemistry is poor, but we all know that calcium can make phosphate less bioavailable, so Pat's suggestion of calcium doing the same for arsenate sounds like a promising lead.
One way to test the general idea would be to incubate arsenate-containing medium in your glass tubes and in your plastic tubes (basically an uninoculated control) for 24-48 hours. Then transfer that medium to fresh polypropylene tubes (no glass for anybody!) and inoculate. If my suspicions are correct, then the medium that was pre-incubated in glass will permit growth following the transfer to plastic, but that medium pre-incubated in plastic will not.
If this pans out, then I would repeat my suggestion that plasticware be used rather than glassware for these experiments b/c your ability to know with certainty the concentration of bioavailable arsenate in glass is less than ideal. This would also mean that you'd probably have to do a series of experiments to determine the limits of arsenate resistance GFAJ-1 in plastic culture vessels in your laboratory (rather than assume the resistance limits described in the FWS paper will hold true in your plasticware cultures).
If all of this holds true, then this raises additional questions regarding the reported FWS results and conclusions... but one step at time.
Ah, I see that you read my mind while I was typing my last comment...
ReplyDelete"I've been assuming that the glass tube has far too little surface area to sequester all the arsenate from a 40 mM solution."
ReplyDeleteYou don't need to sequester *all* of the arsenate... just enough to bring the bioavailable concentration below the threshold of toxicity.
This reminded me a little of some work on dialysis when silicon was found to protect against aluminium toxicity. Example publication: "Does silicon protect against aluminium toxicity in dialysis patients?" doi:10.1016/S0140-6736(94)90856-7
ReplyDeleteIf silicon or boron (if the tubes are borosilicate) was leaching from the glass (particularly after acid washing) it might have some kind of protective effect against the worst effects of the arsenic. There seem to be some papers discussing the idea that plants uptake silicon to detoxify arsenic. I would be curious to know what the silicon concentration is in the growth media and whether it is higher in the glass tubes than the plastic ones. Limited access to journals so can't dig anything plausible up.
Wait, let me get this straight...
ReplyDeleteYou tried GOOGLING it and it didn't answer your question? How could that be? Isn't that what you are promoting as the new scientific literature?
The influence of glass on the activity of arsenic in the growth medium is a potentially important finding.
ReplyDeleteOne suggestion would be to sacrifice a glass tube; break it into little pieces small enough to fit into your polypropylene vials. This may complicate your turbidity measurements but if you can measure the turbidity with a small aliquot you may be able to prove disprove the selective partitioning of the added arsenic in the presence of glass.
In addition to PP, I would also try using teflon vials to absolutely rule out any container driven contamination - again same issue here - you will not be able to perform the quick turbidity measurements since teflon is opaque.