In the new batches of media I made, both H. influenzae and E. coli grew in A (our Difco BHI) and in C (borrowed BHI) and D (old HI). They didn't grow at all in B (MBP BHI). Reinoculation of media from these bottles by one of the post-docs and by me (using independent H. influenzae stocks) gave good growth in A, C and D but not B.
But there was surprising contamination in both our cultures. Surprising because previous uses of the identical stocks had no contamination, and the no-cells controls showed no contamination in two control media (indicating that my hemin and NAD supplements were uncontaminated. And an agar-plate streak of my H. influenzae inoculum showed no evidence of contamination. Even the contaminants couldn't grow in medium B, but this would make better sense if another post-doc hadn't previously found that H. influenzae grows just fine in MBP BHI.
To test whether the identify of the person doing the preparation matters, another post-doc made media that should have been identical to my new A, B, C and D. But H. influenzae grew in all of these batches, even the MBP BHI. We can now (almost) conclude that growth results are reproducible for any specific bottle of media we prepare, but not for different preparations made from the same powder. (I write 'almost' because of a couple of pesky exceptions observed last week.)
I'm wondering if there's something wrong with our clean bottles....
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3 weeks ago in Genomics, Medicine, and Pseudoscience
The bottles is an excellent possibility. We had that happen to us a long time ago (long enough that I had forgotten it until you just reminded me). We had a new guy running the autoclave, and through a combination of several small factors the detergent wasn't getting properly cleaned out of all the bottles. Mammalian cells died when medium was made in those bottles. It was a sporadic and frustrating thing that took several weeks to figure out. Increasing the rinses fixed the problem.
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