Field of Science

Vitamins are for wusses (#arseniclife)

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 ยต 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.

5 comments:

  1. I have used Tween 20 to help un-stick cells for counting. It was for flow cytometry, but may also work for microscopy. My cells were fixed in gluteraldehyde and are 2-3um.

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  2. Elyse, how much Tween 20 can be used? I tried 0.01% and it didn't make any difference.

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  3. Based on this post and a previous one it sounds like you're going to try and reproduce FWS' data using different media. If so, won't differences in media be a possible source of any differences in GFAJ-1 behavior that you observe? It seems to me that if you're going to go to the trouble to try and repeat their results you'd do everything as close to what they did as possible.

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  4. @anonymous: You're absolutely right. The RA gets back from vacation tomorrow (from the South of France) and I'm going to have her order all the missing vitamins. (Pathetically, I don't know how to order them myself...)

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  5. We use 0.05% for fixed cells.

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