is my culture got spoiled? - (Dec/13/2007 )

Hi Pals...

I incoluted a 10 ml starter medium (LB) with an old glycerol stock. The growth did not come in 24 hours. But when i checked it on the 3rd day morning, there was good growth.

Because of several reasons i was not able to proceed further with that. its been lying in regrigerator for 7-8 days.

So i have few questions like:

a) Can i use same culture broth as it has been kept at 4 degree C?

Can i use that same broth/starter for inoculating the production medium?

c) Or should i streak from that broth on a fresh plate, inoculate a fresh starter from that plate and then proceed with that starter for expression or what??

Thanks!

i suppose the growth is because the antibiotic is degraded by that time
ur glycerol stock might be contaminated

I would start with your original glycerol stock and streak it onto a plate to get the culture growing properly and then inoculate a new culture. Waiting three days for your culture to grow, you won't really know if its what you want or if its contamination.

Three days is way to long!!! Chances are you have a nice broth of contamination in the fridge right now. I would streak out the glycerol stock onto a selective plate and pluck a nice, new colony from that plate for the starter culture. Also streak out a bit of the glycerol stock onto just an LB plate to see if your stock has been contaminated. Either way, it sounds like you need to make a new glycerol stock.