The joys (?) of benchwork

So yesterday I streaked out the B. subtilis strain I'm planning to use as a control in my tweezers experiments, and its wildtype parent, first on rich medium (LB) and then (from that, after about 8 hours) on minimal plates with and without the supplements the control strain needs. This morning I found that the parent (call it W) had grown on all the plates, but the control strain (call it Y) had only grown on LB, when it should have grown on the plate with both supplements.

So I wondered whether my supplements (solutions of tryptophan and methionine) might have been harmed by being autoclaved rather than filter-sterilized. So I quickly made up more, this time filtering them, and streaked the right amounts onto the agar of supplemented plates like those that Y hadn't grown on, and then streaked both W and Y on them, from single colonies on the LB plates. Six hours later I could see tiny colonies of both strains on all the new plates. Not just the plate with both supplements, or just on the new-methionine plate, as might be expected if it was the original tryptophan stock that had been ruined by autoclaving. No, strain Y grew on the plate with the new methionine, and on the plate with the new tryptophan, and on the plate with both, as if it was the wildtype strain.

What control did I wish I had done - streaking both Y and W again on the original plate with both old supplements. Maybe I had accidentally streaked strain W in place of strain Y. So I streaked them on that. And then I made some new minimal plates, this time leaving out the trace amount of casamino acids the minimal recipe called for (maybe that had enough tryptophan and methionine???). I spread some of these plates with the new supplements, and streaked W and Y on plates with and without.

Then I wondered if I had somehow taken cells from the wrong vial of my 20-year-old- stocks, so I streaked from the vials again onto LB and old and new minimal plates. Could I have gotten the Y genotype wrong? No, a Google search for its genotype found a page specifying trp and met requirements.

Here's hoping the plates make more sense tomorrow morning.