The dilution test showed that adding as little as one part sBHI to 4 parts sLB was enough to prevent H. influenzae cells from growing. The plating test showed that H. influenzae cells would form colonies on the surface of an sBHI agar plate, but wold not form colonies if they are embedded in a layer of sBHI top agar. None of these results make any sense.
So we decided to go back and retest all the variables, in the assumption that at least one of our previous tests gave a misleading result. The first thing to retest was the effect of different sources of BHI powder, because this is the most likely culprit. We have (A) the Difco/Bacto stock we've been using (a nearly-empty 2.5kg tub), (B) a newly opened bottle of Marine BioProducts brand, and (C) a Difco BHI bottle from another lab (dated 2003). We also have (D) an ancient bottle of Difco heart infusion powder (HI) that I bought when I started the lab in 1990 but never opened.
I made 200 ml of each, weighing the powder directly into the bottle (no weighboat) and filling the bottle directly from the water carboy (no measuring cylinder). I inoculated 5 ml of each with 200 ul of H. influenzae cells from the LB overnight. As controls I used the reproducibly toxic BHI stock E I had been previously using, and the LB that the cells reproducibly grew in.
After a few hours it was clear that the cells were growing slowly in medium A, faster in media C, D and LB, and not at all in B and E. The growth in A is unexpected, as A should be identical to E. We also inoculated E. coli into these media - we'll see the growth results this morning.
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