Not the size of the inoculum, and not the water...

I tested the effect of inoculum size by trying to grow serial dilutions of both E. coli and H. influenzae in a variety of media, with 10-fold dilutions ranging from 10^-2 to 10^-9 of a turbid resuspension. All of the E. coli dilutions grew fine in LB and none grew in BHI or sBHI (testing the effect of hemin and NAD on E. coli in BHI). And all of the H. influenzae dilutions grew fine in sLB but not at all in sBHI. So the growth failure doesn't depend on the number of cells inoculated.

So today we made up BHI using water obtained from various other labs nearby (labs with fancy water-purification systems) and from tap water. None supported growth of H. influenzae, and they only weakly supported growth of E. coli. Because I had realized that the LB that always supported growth had been made up about 6 weeks ago, we also made fresh LB with our current stock of distilled water. It supported growth just as well as the old LB. So the problem isn't something in our water.

My tests of the different agar plates confirmed that media that doesn't support growth in broth does support growth just fine when solidified with agar. And plating of the previous day's E. coli 'didn't grow' cultures showed that the broth contained about 10^6 cfu/ml, which is not very different from the cfu it was inoculated with.

One of the post-docs has set up a test of whether the growth state of the inoculated cells matters. She took H. influenzae cells left from last-night's test (the same ones that failed to grow in the various water tests but did grow in sLB), and from the newly-growing sLB culture.

I've now set up overnight tests of whether the agar-solidified medium supports H. influenzae growth if the cells are embedded in a layer of top agar (more dilute agar) on top of the sBHI, or are embedded in 10ml of sBHI agar. I tested both top agar made with medium E and made with half medium E and half LB.

And to test whether added sLB restores growth to H. influenzae cells in sBHI, or whether added sBHI poisons cells in sLB, I've inoculated cells into various mixtures (5:0, 4:1, 3:2, 2:3, 1:4, 0:5).

The situation is getting scary, as we're fast running out of variables to test.