Curiouser and curiouser

So yesterday I did almost exactly the same test of medium A I had done the day before. The H. influenzae results were the same - cells grew in sLB but not in sBHI. But this time the E. coli didn't grow in sBHI either (it did grow in sLB), whereas yesterday it had grown well in both media. The growth/no growth distinctions were made by both microscopic examinations and turbidity checks. This confirms that the post-docs' earlier result of E. coli sometimes not growing in sBHI was not due to something odd they had done on that day. So whatever the problem is, it can affect both E. coli and H. influenzae.

The only differences I'm aware of are that I inoculated the cultures with fewer cells (both E. coli and H. influenzae), and, it being a different day, I used cells taken from newly grown colonies on agar plates. I had the same results with the other batches of media the post-docs had tested (B, C and D), as did the post-docs. And I had the same result with medium batch E, freshly prepared by one of the post-docs.

I also tested survival of the cells by plating the inoculum (H. influenzae only) before it was added to the test media, and the cultures at different times (H. influenzae after 30 minutes and 3 hours, and E. coli after 3 hours). I used both old plates that were known to work fine and new plates made with batch E BHI, so this will also tell me whether the problem occurs only in liquid medium.

Today I'm going to test whether the size of the inoculum makes a difference. This will require lots of plates, so I hope the test batch E plates worked fine. It would be hard to do this test properly using just the microscope and turbidity checks. Inoculum size can make a difference - I remember once when we had to use a borrowed shaker for our culture tubes (our roller wheel being broken) we found that a too-small inoculum of H. influenzae gave no growth in sBHI. It was as if the medium contained a limited amount of something toxic that was removed from the medium when it was absorbed by the cells, so that when only few cells were present each received a toxic dose of this hypothetical substance, whereas when more cells were present each received a proportionally smaller and thus non-toxic dose. We didn't bother tracking down this mystery because we stopped using the borrowed shaker as soon as our roller wheel was fixed.

The plating I've done should tell me whether the BHI kills the cells or just doesn't allow them to grow. I may also do the 50:50 (sLB:sBHI) mixing test, which I didn't have time to do yesterday - maybe even testing different ratios of the two media.

I may also seek out another source of pure water (from another lab), just in case something has gone weird with either our still or the carboys we store its output in.