I am doing sub-culture of my bacteria (WT) every 16 h in 5 ml LB broth with antibiotic for 30 days. For whole genome sequencing, I should change the LB broth sub-culture 5 ml to 100 ml to collect pellet from 10th day, 20th day and 30th day. I also carried spread plate technique (5 days once) for checking the morphology of bacteria. I did not find any variant (mutant) upto 9 days on LB plates. When I changed my culture volume 5 ml to 100 ml, I found WT bacteria (80%) as well as variant (20%). After 15 days, I found WT bacteria (20%) and variant (80%). After 20 days, I found complete variant on LB plates. Can anyone suggest the reason (nutrient starvation, LB volume, oxygen depletion) please?
Hi are you sure there are no bacteriophages present? It would explain that a resistant variant survives while the wild type is fading, however your shift happens very slow. Have you been checking for CFU concentration over time? Another reason could be the formation of biofilm and/or the VBNC state..
Dear Hani Anton,
I am working in rice plant pathogen (Burkolderia glumae : quorum sensing bacteria) for experimental evolution study. (Rifampicin 100 ug/ul)
I am doing 30 days (I am changing LB media every 16 h) for checking the mutation rate. The WTs are yellowish with crystals (oxalate) whereas variants are white with no crystals. Crystals are observed in center of the WT colony. After 10 days, both variant as well as WT were observed.
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