The GFAJ-1 cells I streaked on a plate of my less-than-specified culture medium are growing, and much faster than I had dared hope. After less than 48 hr I can already see tiny (very tiny) colonies. I can't estimate growth rate because I don't know how many cells are the colony,
I was worried that they might not grow at all, because this medium doesn't include the trace-element mix that Wolfe-Simon et al. added to their medium. Instead I simply made up the medium with lovely Vancouver tap water. It also doesn't include most of the vitamins they added - I only had stocks of thiamine and pantothenic acid, so they were the only vitamins I added.
I'm still considering solutions to the cell-counting problem. Flow cytometry of acridine-orange-stained cells won't work very well because some of the cells are in small clumps of 2-10 cells. Collecting known volumes of cells onto black filter membranes would probably work fine but would be a pain and use a lot of the filters. I think the simplest solution is to make a 2-X acridine orange stock that contains a known density of 2 µ polystyrene beads, mix this with an equal volume of culture, and then count both the beads and the cells in random microscope fields-of-view. I'm going to test this now.
Phenotype of the rpoD mutant
10 hours ago in RRResearch