Field of Science

Preliminary test of viability in MIV (and other stuff)

Yesterday I looked at changes in the numbers of viable cells (cfu, colony-forming units) after cells of different genotypes (wildtype, purH, purR) had been transferred to the starvation medium MIV.  I also did this after transfer of wildtype cells to the defined medium cMMB without inosine.

The experiment had a couple of problems.  I'd wanted to compare survival of sxy- cells, but the only vials of frozen cells we have date from before the big freezer meltdown of 2004, and the culture I started from one vial remained resolutely non-turbid all day.  However I streaked it out and some colonies grew up overnight, and I've now confirmed that they are indeed kanamycin-resistant (the knockout is an insertion of miniTn10kan). So I can test them tomorrow.  I also had intended to use RM- rather than cMMB-, but my new stock of the 10x base for MMB smelled very nasty after autoclaving, and had a large precipitate, so I suspected that the glutathione I'd included had broken down (though the published instructions say to autoclave it).  So I threw it out and made fresh without glutathione, but this has also thrown a big precipitate.  Now I wonder if the problem is that this time I carefully adjusted its pH up before autoclaving, which the instructions don't say to do.  The solution started to become a bit cloudy when the pH got above 7.0, so I adjusted it down a bit, to 6.8, before autoclaving.  I'll try again tomorrow.

Anyway, both the mutant strains survived in MIV just as well as wildtype, with the cfu increasing 2-3-fold in 60 minutes.  I left the cells incubating overnight and plated them again this morning, so we'll  see if there's any differences in longer-term survival.  The wildtype cells transferred to cMMB didn't divide at all, nor did they become even a tiny bit competent. 

Now that I know roughly what to expect, I can do a more detailed test, and include the sxy knockout mutant.

No comments:

Post a Comment

Markup Key:
- <b>bold</b> = bold
- <i>italic</i> = italic
- <a href="">FoS</a> = FoS