Help! Bacteria on agar not growing in LB broth - even without antibiotics... (Sep/24/2009 )

Hi everyone,

Sorry for posting another silly question. I tried searching for answers everywhere but I couldn't find it, so I decided to ask the experts here

A colleague of mine streaked an agar plate with E coli carrying a kanamycin resistance plasmid, around 2-3 weeks ago. However, when I tried to pick clones and grow them in LB, they didn't grow. Initially I thought it was a problem with her electroporation step, but when I tried growing them in kana-free LB, they didn't grow either!

What's going on?? Would leaving the colonies in agar for too long affect their ability to grow in LB?

Thanks in advance!

Let me give you some more info to see if you can spot where I've gone wrong:

I picked clones by touching a 10 ul pipette tip on the colony (sometimes poking into the agar a bit), and then dropping the pipette tip into a 10 ml plastic tube containing 2.5 mL of LB.

Everything is done next to bunsen flame, LB autoclaved (but pipette tips not autoclaved). Then I shake at 300 rpm at 37 C. It's been three days and I still don't see any signs of bacterial growth

Thanks!!

when on plates bugs work well till 4 weeks (if they are kept at 4C ), i dnt mean they dnt work after 4 weeks but their viability may be effected...........Also look if their is satelite formation? r u picking colony or satellite?
always take with a sterile tip and near the bunsen........when u pick a colony with a pipette tip do u streak it (a bit) on another fresh plate? if yes then what about that plate?
what concentration of antibiotic do you use is it ok? try in 10ml fresh LB culture (drop the tip inside the tube) and incubate at 220-225 RPM at 37.........it will definitely work this time.



Dnt poke into agar plate just gently toching the colony is enough.....believe me

I have experienced the same problem. Maybe the bacteria on your plate has died. I suggest you streak a new agar plate and pick the single colony as soon as they are visible.