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:
- 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.
- I did the same, this time into new tubes that I'd washed thoroughly with deionized water (no detergent) before sterilizing them by autoclaving.
- 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.