Advice sought for working with M. nematophilum

Hi everyone. About two months ago I ordered M. nematophilum strain CBX102 from the CGC for use in infecting some strains. I followed the methods laid out by the Hodgkin lab’s many papers on the subject and have had problems getting the worms to exhibit the Dar phenotype to the extent reported in the literature. I got to thinking maybe the bacteria is just not doing too well. I tried to re-streak it on LB plates with and without antibiotics and it just isn’t growing any more.

Has anyone found this strain, or the bacteria in general, to be particularly fickle (other than its reported slow growth)? Any input would be greatly appreciated. Thanks!

We routinely use M. nematophilum and did sometimes find that cultures failed to give the Dar phenotype.
We find the cultures become less “infective” over time so we do the following…
regularly (at least monthly) streak new CBX102 onto an NGM plate from the glycerol stock and use colonies from this plate to grow cultures
grow small volume cultures (50ml or less) for 48 hours at 37oC and only use them for up to 3 weeks (after this time we observe decreased penetrance of the Dar phenotype)
seed NGM plates (no antibiotics or nystatin) with 10% CBX102 diluted in OP50 and use the plates within 48 hours.
This consistently gives us approx 98-99% Dar animals with N2’s
Any fungal or bacterial contamination (In your cultures, on your infection plates or the plates you pick from) will significantly decrease the penetrance of the Dar phenotype. If we see any contamination we throw everything away and start again.
Starvation alters the penetrance because the Dar phenotype is influenced by serotonin signalling (see
Infected adults will not become Dar we typically look in the F1 or infect synchronised L1’s

Thanks for the advice! I’ve been infecting them as synchronized L1s, so it may be due to the age of the bacteria. Additionally, I had been using agarose plates rather than NGM, as we work with strains that have a tendency to burrow into agar. I’ll switch that up this go-around and see how it goes.