Which bacterium? Which antibiotic?

HHmmm... the bacterium is R. baltica, and the antibiotic is chloramphenicol. The MIC would be 24ug/ml on agar plates and 50ug/ml in liquid culture.

It's a gram negative. I think that you can use Cmf as in the way of a simple gram negative like E.coli.

Agar plates at 25 microg/ml as in the liquid culture. I think that 50 microg/ml is too much for Cmf, is a strong antibiotic by itself.

I tried 25 ug/ml, and for months nothing grew on my plates.... but lately I have started to get colonies on my negative control plates... makes me want to cry. But if I use much higher concentrations like 50 ug/ml I think they cannot cope with it though they are resistant...

I think you got colonies in your negative plates 'cause Cmf after few days is not longer usefull, well, you cannot trust plates after a couple of days if they aren't stored at 4°C. Or maybe they are just satellite colonies which get resistance to Cmf by theirselves in someway.

Even if you know the MIC, I suggest you to take a positive clone (trasformed only with the empty plasmid) and try (in double or triple) to let'em grow at different Cmf concentration 20- 30 - 40 - 50 ug/ml.

And make your choice.

You are right... I am totally aware of it but I have not much choice because R. baltica reaches maximum plating efficiency only after 2 weeks of incubation at 28 deg and there is no way around this. I actually have tried growing them at different concentrations, but at one point they just die. I will try transferring them to plates rather than liquid culture and see if they still grow with higher concentrations.

Thank you so much!!! If there are even more suggestions, they are more than welcome.

Hey, It's me again... I have done as you suggested, but I have a little problem, still... When I use 30 ug/ml Cam, I get both transformants and colonies with no plasmid, but on the other hand when I use 35 ug/ml, I don't get any colonies at all:(

Do you think it is worth playing around between 30-35 ug/ml (I am wondering if there is a concentration in between where only transformed colonies grow) or should I completely switch to another antibiotic?

Logically, there doesn;t seem to be much point growing them on plates. The transformants (containing plasmid) will be breaking down the chloramphenicol in the plate in the growth region, and so the local concentration will decrease to nothing. Because you wont be selecting for the plasmid anymore, the bacteria will lose it, and then probably die as they spread into chloramphenicol richer surroundings. Maybe you could try liquid and continue to supplement or, continue to subculture over time.

Just my two cents....

Hhhmm... So that would mean I should inoculate them after transformation and recovery directly into Cam containing liquid medium and not on plates? Actually I thought of that, on the other hand the bad thing with these bacteria is that if they are diluted out too much (which will happen after the untransformed cells are dead) they don't grow at all:( I was thinking if I use Tetracycline for example it would be pumped out rather than degraded, and perhaps I could get more consistent results then?

I would inocculate straight into liquid, and grow and split them like you would tissue culture cells. That way, you can be sure they are being selected for in fresh Chlor, they are in fresh media, and you will continue to dilute the non transformed cells. Remember, chloramphenicol doesnt directly kill bacteria, but inhibits their protein synthesis.

I am not sure i understand the "on the other hand the bad thing with these bacteria is that if they are diluted out too much (which will happen after the untransformed cells are dead) they don't grow at all:( "

They dont grow? or take too long to grow? They should certainly grow faster in liquid, especially if you shake them well. I'm not sure about the Tetracycline reference too....? Is your plasmid chlor/tet?

Well, first of all the plasmids I used all have Cam resistance, but I guess I could also modify them for Tet resistance. As far as I know the protein encoded by the tet resistance casette binds the ribosome rather than the drug itself, therefore unlike Chlor., Tetracycline would not be degraded hopefully since I have to incubate them for such a long time.

The cells have a doubling time of 12h in liquid medium and for sure they grow faster than on plates... On the other hand when I inoculate them too dilute they don't die but they also do not build up any biomass no matter how long I wait. I will still try as you suggested, for sure, maybe it does work better than plating:) Just there bugs are really unpredictable...

Sounds like a pain in the butt ... good luck with it! Let us all know how you go :)