Field of Science

Maybe it's the water? Or the tubes?

I thought of two (three!) more easily testable explanations for why the GFAJ-1 cells grow on agar plates but not in the liquid media.

First, maybe there's something in the water.  When making up my first batch of the culture medium for GFAJ-1, I didn't go to the trouble of making up the special trace-element mix that Wolfe-Simon et al added their culture medium.  This seemed reasonable because I'm using cheap 'Regent Grade' chemicals (as did Wolfe-Simon et al.), and these are likely to contain traces of all the needed elements.  Just in case they didn't, I made up the medium with tap water rather than the usual distilled water; Vancouver's tap water is very pure, but it still probably contains traces of everything cells might need.  The stock salt solution (4X) was made with distilled water, but the final liquid medium is 75% tap water.  But the agar plates are mainly distilled water (only about 10% tap water), because the 2% agar stock I used was made with distilled water.  So maybe my GFAJ-1 cells aren't growing in liquid medium because tap water contains something that inhibits growth.

Second, I used brand new glass culture tubes for my liquid cultures, because I didn't want growth to be affected by traces of past cultures or of phosphate detergent that might have adhered to the surfaces of our usual tubes.  I didn't wash the tubes before I used them, because I didn't want to risk leaving detergent on them.  But maybe the new tubes are coated with something that inhibits growth.

Third, Wolfe-Simon et al. used screw-cap glass tubes for their cultures, but my tubes have loosely fitting caps designed to admit air.  Maybe the air is inhibiting growth - that would be consistent with the good growth in parafilm-sealed petri dishes.

So this morning:

  1. I resuspended a single colony of cells from an agar plate in 1 ml of medium, and inoculated 0.2 ml of the cells into each of five tubes containing distilled-water medium with different amounts of phosphate.
  2. I did the same, this time into new tubes that I'd washed thoroughly with deionized water (no detergent) before sterilizing them by autoclaving.
  3. I asked the RA to check the prices of screw-cap tubes.  They're expensive and available only in large quantities, so we won't order them unless the other tests don't solve the growth problem.

15 comments:

  1. Can you make a fair and proper comparison if you don't replicate their work exactly and to the letter? I guess if anything interesting/strikingly different from Wolfe-Simon et al. found, happens, you will repeat everything using their methods to-the-letter, so as to be able to actually make a comparison?
    rich

    ReplyDelete
  2. Tap water is chlorinated and that might be your problem. It is often a problem encounter by homebrewers and slow growing yeast. Boiling the water usually fixes the problem if it is chlorine but not if chloramines is added at the water plan.

    ReplyDelete
  3. @Rich: Absolutely right. I now have the complete trace elements mix, and all the vitamins should arrive this week. I'll do the final experiments with medium containing these even though the cells don't seem to need them.

    @Anonymous: The online info about Vancouver's tap water is out-of-date. I'm now testing distilled water too.

    ReplyDelete
  4. why should we believe your data over Felisa ?

    ReplyDelete
  5. because Felisa prooved to everybody to do poor science.

    ReplyDelete
  6. That is not really true...... Have you actually read her paper? Her papers are well written and the science is actually solid. These results are preliminary. Analytical techniques are subject to interpretation. She realizes this and so should you. Please do not give blanket statements....they mean nothing. Actually Felisa is a very talented young scientist. We should be encouraging young women and men to go in science, instead of being so negative.
    By the way, it is easy to disprove a theory, really hard to prove it. The really question is felisa observed something in this material. How do we explain it ? Whether her theory is right or wrong, does not really matter. A young scientist had the ability and talent to challenge a theory and we ridicule her and treat her poorly (RR called her an alien) RR you should be ashamed of yourself and you call yourself a leader in science. So be a leader and be a good example.

    ReplyDelete
  7. NotAnAstrobiologistJune 29, 2011 at 8:43 AM

    The paper is written carefully, but I think you are wrong to say the science is "solid".

    That is kind of the key point about this whole thing, and seems to be the position of Rosie as well.

    ReplyDelete
  8. Where did you learn to do microbiology using tap water as your "defined" medium? For an experiment such as this the exact elemental composition of the water must be known exactly. Do you even know how much background P there is in the tapwater for your "no-P" amendments? If you think that it is acceptable practice to use tapwater in such an experiment, then good luck with all of the other complexities that this experiment will involve. You mentioned that you were going to make a 5X stock of 1 M NaCl, isn't that above the salt saturation limit? You are obviously way out of your experience level on this, but through your blogging you have everyone convinced that you are THE authority who will show what all of the well respected extremophile microbiologists in the FWS paper did wrong. Good luck, this should be amusing.

    ReplyDelete
  9. 5M NaCl is ok, perfectly soluble. Try it at home.

    ReplyDelete
  10. The blog post didn't say tap water was her "defined" medium. Read it carefully and you'll see that she said she didn't have the trace element mix to add to the media so she used tap water as it is likely to have trace elements. As long as she's not publishing any experiments from this growth, I don't see the problem.

    Read the post before getting so fired up.

    ReplyDelete
  11. @ Anonymous 4:38

    If Rosie is "not publishing" these results as you say, she will surely be blogging them and commenting to any media outlet who will listen about them so why not follow accepted procedures? Its true that tap water likely has some vitamins in it but what else does it contain? The media composition for these experiments must be tightly contrained using DDI water with known amendments. Its one of the major criticisms that Rosie heaped on FWS when she declared her a bad scientist.

    ReplyDelete
  12. and by the way 5 M NaCl is pretty much at the practical limit of salt solubility at room temp, so does that sound convenient to work with to you @4:21?

    ReplyDelete
  13. You'll find 5M NaCl stocks on the shelf of a LOT of biomedical research labs.

    ReplyDelete
  14. NotAnAstrobiologistJune 30, 2011 at 7:07 AM

    5M NaCl is a standard stock.

    Me thinks @5:23 and associated posts is trolling for dollars here...

    Would recommend you put a bit more nuance into your posts @5:23, would make everyone happier...which is the point of trolling anyway.

    ReplyDelete
  15. I have a 5M NaCl stock on my shelf. So does everyone else and their Mom.

    ReplyDelete

Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="http://www.fieldofscience.com/">FoS</a> = FoS