I have PEG in bacterial culture as osmoticum. I find the difference of half in term of O.D of stress versus control culture. On dry mass, there was difference of 10-15mg/100ml culture. Substrate consumption is equal in both samples. No effect on enzyme level of substrate oxidation. On HPLC, I could not see much difference. What else could be the reason for low O.D or what other parameters i could try to find the effect of osmoticum?
The relationship between OD and colony forming units (cfu) for most bacteria is dependent on the osmotic strength of the solution. Under high osmotic strength conditions bacteria can become smaller and more spherical and therefore diffract less light (lower OD). Also many bacteria have evolved sensory mechanisms that modulate protein expression and cell growth in response to changes in environmental osmotic strength.
Are you monitoring OD over the full time course of the growth curve of your bacteria? Is the doubling time decreased for the bacteria grown in high osmotic strength conditions? Does the osmotically stressed culture reach stationary phase at a lower OD?
I am measuring O.D for 24 hours, Control cultures reach at stationary phase at O.D 1,8 and stressed culture at 0,8-0,9 O.D. However both of these cultures have almost same doubling time. I am confused how and transcriptomic analysis of these cultures also showed very little fold change difference. maximum to 3 fold, most of them are transposons.
Cell shape change is also under stress control, it means cell have an effect of osmotic stress then how they keep same doubling time and equal substrate consumption???
The shape chage may be partially under stress control but I would think the main driving force is the osmotically driven efflux of water from the cell.
I would also guess that the 0.8 OD observed for the culture grown under high osmolyte concentration corresponds to nearly the same cfu as the 1.8 OD observed for the culture grown under low osmolyte conditions.
Are you comparing transcription of cultures in exponential growth phase? At least for E. coli I can list a few genes that will have significantly enhanced expression, >>3-fold.
yes, I isolate RNA 6 hours later after applying stress from control and from stressed culture. I used pellet from 20ml preculture to inoculate 100ml culture with and without stress. Doubling time for my organism is around 90-100 min. So after 6 hours 3 -4 divisions have been passed. Because original number of cells (preculture)is high, so at the time of harvesting (6hours later), O.D is quite high like 0.8-0.9.
Are you sure the culture is still in mid log phase growth? At that high of an OD I would be concerned that the cultures have already entered into stationary phase and have down-regulated expression of many genes.
Have you tried to innoculate from liquid culture? I've previously grown cultures in low osmotic strength media to early log phase and then made a 1:1 dilution into media containing a 2X concentration of osmolyte ( final1X concentration of osmolyte). I'm always concerned about innoculating a culture from pelllet since the cells are hyperconcentrated during pelleting and quorum sensing stress responses may be activated.
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