Growing cells and making RNA (...tomorrow...)

Today I'm actually doing something in the lab!

As my latest contribution to the sub-inhibitory antibiotic effect collaboration, I'm growing wild type cells with and without a sub-inhibitory concentration of the antibiotic rifampicin (aka rifampin), collecting the cells in log phase, and making RNA from them.

The plan (discussed previously) is to get the cells growing in their normal medium and then transfer them to parallel flasks with and without the antibiotic. The cells will be allowed to grow and divide for at least two hours (four cell doublings), diluting the cultures with fresh medium as needed to keep the cell density low enough that the cells continue to double exponentially. This will make sure that the cells have fully adjusted their metabolism to the availability of abundant medium with and without antibiotic.

Then I'll collect samples of cells at OD = 0.1 and OD = 0.2 (OD is optical density, a measure of the turbidity of the culture and this of cell density). I'll pellet the cells in the microcentrifuge and freeze the pellets, so I can do the RNA preps later (tomorrow?). Two 2 ml tubes of each OD = 0.1 culture and one of each OD = 0.2 culture, should give me plenty of RNA.

Fine plan, but one immediate hurdle. I meant to inoculate a starter culture of the cells last night, from a frozen stock, but forgot. So this morning I used some frozen cells prepared for just such an eventuality. But after more than two hours the OD of this culture isn't increasing. First thought - maybe my culture medium was no good. H. influenzae grows in brain-heart infusion medium supplemented with hemin and NAD, but I used the old stocks of hemin and NAD I had in my fridge. Maybe they were too old. So I got fresh stocks, supplemented some more medium, and inoculated some more cells from more of the same frozen cells. After an hour their OD hadn't increased either. Then I remembered the great freezer meltdown of 2005. Check the date on the frozen cells - yes, January 2005, probably these stocks went through the meltdown so that most of the frozen cells in those tubes would have been dead. Probably my old medium was fine, but the very small numbers of viable cells in the frozen stocks means that most of the OD I was reading was turbidity due to dead cells.

Finally the ODs are starting to increase. But there's a seminar I really want to go to at 4pm (Peter and Rosemary Grant - very famous evolutionary biologists), so I think I'll have to just let these cells grow overnight and use them as the starter for proper cultures tomorrow.