Field of Science

How do I not understand these results? Let me count the ways.

Because I've had inconsistent results growing GFAJ-1 with arsenate and limiting phosphate, I set up a big series of cultures in screw-top glass tubes (the previous cultures had been in small flasks).  The results with the culture tubes are completely different and also internally inconsistent.  Foor convenience I've mainly assessed growth using 'optical density' (OD) to measure the turbidity of each culture, rather than taking the trouble to dilute and plate the cultures and count colonies.

The first surprising result is that the presence of 40 mM arsenate had little or no effect on growth, regardless of the level of phosphate provided (compare the blue, red and green bars for each phosphate level).  Previously I've only sometimes seen very slow growth in 40 mM arsenate cultures with high phosphate (1.5 mM) or with no added phosphate, and other times seen no growth at all.

Second, some cultures with little or no added phosphate grew to much higher densities than previously or than parallel cultures with similar amounts of phosphate (e.g. 4 µM and 5 µM phosphate with no arsenate, and 3 µM and 7.5 µM phosphate with 10 mM arsenate).  Based on colony counts of previous experiments, I expect the 3 µM phosphate cultures to have about 100-fold fewer cells than cultures with 1500 µM phosphate

Third, the culture densities differed dramatically from tube to tube, with little correlation with the amount of phosphate I added. Part of the explanation for this might be that the culture tubes were heavily and variably contaminated with phosphate (they had been previously used, many years ago, and I had rinsed them and their caps well but not acid-cleaned them).  But the contamination would have to have been consistently lower in the tubes that I added no or little phosphate to (because other cultures that differed by only 1 µM added phosphate differed by OD's of 0.2, equivalent to about 5 x 10^8 cells/ml).

(I looked at some of these cultures under the microscope and saw no evidence of contamination with other bacteria, nor did I see any unusual colonies when I plated some of them.)

Fourth, I plated the cells from a few cultures, and one's cfu counts were way lower than expected from its OD.

Fifth, by mistake I initially set up these cultures in base medium with 2 mM phosphate rather than in base medium with no added phosphate.  I didn't throw this set of cultures out but incubated them with the correct cultures.  These grew to higher densities than the low-phosphate cultures, but most didn't grow as dense as similar cultures had previously in flasks.

But again there was substantial tube-to-tube variation in density, and no effect of arsenate at either 10 mM or 40 mM.  Phosphate contamination of the culture tubes can't explain this variation, as all the cultures had ample phosphate.

To rule out effects of contamination with phosphate (or whatever), I'm now going to set up some similar cultures in new plastic screw-top tubes.  I won't bother with 10 mM arsenate and I'll reduce the number of different phosphate concentrations.  I'll also test the effect of filling the tubes to different levels, in case reduced aeration is a factor.

Yes, I have no idea what's going on!


  1. In my limited experience, working with phosphate limited cultures is a nightmare of inconsistent results. Phosphate is everywhere.

  2. I agree with Zachary, it's everywhere - detergents used in the glassware construction contain it and leave traces on the glass, for example. Plastic maybe better? Dunno. What's the carbon source and nitrogen source here? Is it a complex medium or glucose/ammonium? If the former, it's going to have phosphate in it anyway - enough to support growth (E. coli in lysogeny broth, no added P, grows fine).

    One thing that could skew your OD - polyphosphate granules in the cells (or PHB of course) - could make the OD seem higher than it is but with this level of experiment, cell counts are too much hastle.

    If you're using 30-mL(ish) "Universal" tubes, years of experience using them tells me 5mL culture seems optimal, 10mL works fine too but is a bit slower and lower amount of biomass formed - that's with lids sealed. I usually incubate them on a shaker with the lids half closed to let gas exchange, though. I'm sure Pirt worked out some equation for volumes in different size/shape vessels as it's pretty much consistent for different bugs/substrates.


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