Field of Science

Cells behaving badly (is it the medium's fault?)

Yesterday I did a time course experiment, to see how the 'spontaneous' development of competence in the rich medium sBHI differed between wildtype cells and cells with either the sxy-1 hypercompetence mutation, the purR::kan knockout mutation, or both.  But I had to give up halfway through because the cells stopped growing.

The graph above shows the densities of the four cultures as a function of time, with the purple line showing what I had expected based on the many previous time courses I've done.  (I deliberately started with cultures at slightly different densities, to space the sampling out a bit.)  They were all growing in medium from the same bottle, and they all stopped growing at about the same time. 

Just before I had taken the first samples I had diluted all the cultures in medium from a new bottle, one that had been prepared on a different day, so I wondered if there had been something wrong with this batch, or if I might have forgotten to add one of the needed supplements to it (NAD).  I quickly added more NAD to each flask (at time = ~150 minutes), but that didn't boost growth.

Then I tested several different batches of medium, including the the remaining of the second bottle I had used, as well as two different batches of NAD.  Unfortunately the first bottle I had used was the last bottle of its batch, so I couldn't test it.  I inoculated each with the same amount of cells, and let them all grow overnight.  The second graph shows that there are substantial differences between different batches of medium, and that none of them gives the amount of cell growth I'd expect from previous time courses (labeled as 'years ago' because I haven't done one recently).

I don't think the problem is just how long the bottles of medium had been sitting on the shelf, as the components are typically quite stable.  Instead I'm wondering if we might be using medium from a different supplier.  In the past I'd noticed substantial differences in how well different brands of BHI supported cell growth, and had sworn to use only the best (Difco), but I know we were recently given some BHI from another supplier.  Tomorrow I'll repeat the time course, with the March 1 medium.

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