2 replies to this topic

[rnazitto]

Hi Everyone, I am looking to take suggestions on how to grow up some bacteria normally found in the gut of humans. A key aspect of my experiment is first locating a chloramphenicol-metabolizing bacterium from a sewage source that feeds into a lake where I am sampling. I am using an M9 MSM media, replacing any Cl with sulfates in the media to accurately measure metabolites of CAP via HPLC. So far, I take a starting volume of a 1L of lake water and add in the corresponding amounts of MSM. I would then do 1:10/1:100 transfers using 100ug/mL of CAP in all.  But I am having trouble getting the bacteria to grow in the transfer. Is it being incubated at room temp with shaking in the dark.  I also tried flasks with noble agar on the bottom with CAP and also an overlay of Agar with a layer of Agar with CAP. Any help is greatly appreciated!!!

[rnazitto]

Forgot to mention that I was able to obtain a pure culture of a bacterium on MSM Noblem Agar +CAP plates

[Phil Geis]

I'd revert to the original formula for M9 medium.  Your approach seems to be a compromise between CAP as sole source of carbon and energy and CAP metabolism in context of a growing culture.  If you want the former, you should increase the CAP concentration - 100 ppm is hardly substrate level.  Assuming you chose the low level to avoid likely inhibition, it would serve you better to start with a more complete medium with increased CAP then modifying conditions progressively - conceptually shooting for tolerance -> resistance -> metabolism -?> substrate. Diminshing nutrients while increasing CAP.
And why work with such large volumes?  Filter down to something more workable.