Here are the results of my latest GFAJ-1 growth-in-arsenate experiment, for the cultures in glass tubes. The blue lines and points are for cultures grown with no arsenate, and the red ones for cultures with 40 mM arsenate. Oh, the X-axis is growth time in hours, and the Y-axis is cells/ml.
One arsenate culture failed to grow, but the others all grew to the final densities I expected from the amounts of phosphate in the medium (light blue and light red, no added phosphate, medium blue and red, 3 µM phosphate, and dark blue and red, 1500 µM phosphate).
The most interesting result is that, although the no-arsenate cultures grew well right from the beginning, all the arsenate cultures had a ~ten-fold initial drop in cell density, after which they grew at about the same rates as the no-arsenate controls. The cells used as the inoculum were not pre-grown in arsenate medium, and this drop suggests that, although some of them can cope with the arsenate, many are dying.
So, two changes to try:
- Inoculate cultures with cells that had been pre-grown in medium containing 40 mM arsenate. (I had tested and discarded this idea once before, but the cells I was using had a complex history so the result may not have been reliable.)
- Inoculate cultures with larger densities of cells. I have been deliberately using small inocula (10^3-10^5 cells/ml) to ensure that the growth truly reflected the culture conditions (especially the limiting phosphate), but now I'm going to use about 10^6/ml.