My wildtype H. influenzae cells seem to grow reproducibly in the 'RM' medium that's a mixture of two published defined growth media RPMI and MMB (neither of which gives reproducible growth in my hands). But I'm not entirely happy with this medium yet, for several reasons.
One is its arbitrariness, its lack of a scientific rationale. Is it unnecessarily complicated? Because I don't know why it works, I don't trust it. The other day I tried making a more streamlined version of it, supplementing RPMI with each of the components that distinguish MMB from it (glutamic acid, glutamine, glutathione, higher concentrations of vitamins), but the cells didn't grow at all.
Another is that I haven't yet tested whether H. influenzae cells can grow indefinitely on it. So far I've been starting each RM culture with cells that have been grow in in rich medium. Today I'll start cultures from cells that were grown in RM. But inoculating liquid cultures from liquid cultures is not a very good long-term test, because contamination can creep in.
What I really need to do is to make agar plates of RM (or whatever defined medium I want to test, streak cells on them, and pick single colonies for continued growth in RM broth. But to make RM agar I either need to have a 2-fold concentrated RM liquid to which I add a 2-fold concentrated solution of agar in water, or I need to autoclave the RM liquid with powdered agar. But the ingredients of RM take up too much volume - I can't make it 2-fold concentrated. (I could buy some RPMI powder rather than the ready-to-use solution I have now...) And I'd rather not autoclave it, as I suspect it's not entirely heat-stable.
Today, in addition to my time course of competence in sBHI and RM ± inosine, I'll try to fit in more medium tests.
Welcome to the 4th Reich part 1.
4 hours ago in Angry by Choice