Hello.
I am trying to culture Agrobacterium GV 3103 and EHA105 in 15 mL LB media with the antibiotics kanamycin and rifampicin (100 ug/mL & 20 ug/mL)(added 200 uL of previously normally cultured Bacterial solution) . But, for some reason, after 12 hours of agitation (200 rpm), some bacteria don't develop. When I repeat the experiments, sometimes one specific sample grows normally and the other does not. So far, I haven't seen any consistency in my attempts, for example, I have alfa and beta, one day alfa develops normally while beta doesn't , but the very next day only beta does and alfa is not developed. I suspected that it was because of the 50 mL plastic tube, so I changed to Erlenmeyer flask, but the trouble persists. Even in some samples, I centrifuged the LB to pellet and check if any bacteria was therein, but sometimes I have got not any pellet. I suspect this problem is probably a basic laboratory trouble. Still, I would appreciate any suggestion that can help me to find out how to get a consistent growth of my bacterial. Thank you. (PD: The bacteria I am trying to cultivate was previously isolated from solid LB (with antibiotics) and was chosen after PCR verification).
Yes, both are known to be resistant to those antibiotics. The KAN is the selectable marker. Indeed the strain that is giving me more trouble is the one EHA105.
Try using a slightly larger amount of cells to get your culture started, not all the cells in any given colony will be alive. Keep a close eye on the shaker temperature too, Agro is sensitive to too much heat, maybe try a growth temperature closer to 25 than 30 just in case. I know my lab has overnight temperature fluxes and those have been problematic for Agro cultures.
Thank you for your answer. I have tried what you have said and the results up to now are ok. It seems that the proportion AgroCells/LB is an important point. I have tried, first, to culture in 5 mL LB (with antibiotics as usual) and the growth has been good, even faster (less than 10 hours). So far, it seems that the strain EHA105 is more sensitive to these nuances of proportion than GV3103.
I'm glad things are currently going well! It does seem that Agro needs a larger amount of starting inoculum than other bacterium such as E. coli.
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