I inoculate my 100ml M9 culture with cells from a 10ml MDAG - 135 media of 8 hours growth. After overnight incubation at 37degrees I see a good growth of the cells upto OD 2.0 but with also large floating debris/cell clumps. What are these and why do they seem to appear now?
1. BL21DE3-PlysS cells used with amp and chloramp
2. Is the inoculation too strong? i.e should I have incoculated only a few of the cells into the 100ml M9?
3. Is it contamination?
4 Are the cells lysing?
Your input is very much appreciated. Thank you for your time
An example figure is attached here. (I obtained this figure from the net but mine looks similar with larger(4 to 10 times the size) clumps)
Sometimes the cell lysed because you used too much or mixture of antibiotics.I am using this strain and only interval check it on chloramphenicle ( every two or three months)
chloramphenicole is only needed to sure that your host harboring the pLysS plasmid. You can check the presence of this plasmid by culturing the E.coli host on LB+chlor and then delete this antibiotic from your liquid media.
@Ali hatef salmanina.
I prepared fresh chloramphenicol. Thank you for your input. I have decided to continue to use chloramphenicol with correct concentration and hope it will not repeatedly lyse the cells. I shall report if such problem occurs again and then try to reduce the concentration of chloramp if the problem persists.
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