I think you should do a quick control, use your overnight culture to inoculate 4 flasks, one with Amp only, one with chloramphenicol only, one with both and one with neither. That should confirm your result (or not) and tell you what the problem is, such as you lost one of the plasmids or all the cells are dead.

A couple things to consider:

1. Make sure you have enough aeration in your culture. Use a baffled flask if you can, and make sure you don't fill your flask with too much media.

2. Make sure you rinse your glassware thoroughly with DI water before using, especially if you use soap or bleach to clean them.

3. Do multiple cultures in parallel. Sometimes cultures fail for unknowable reasons, and you'll save time by having backups.

Ampicillin is only stable for a couple weeks but if the ampicillin degraded your cells should still grow. More than likely there is an aeration problem or a bacteriophage problem. Probably a bacteriophage problem. To solve it thoroughly clean your glassware and bleach it and wash it and let it dry and autoclave sterilize all your flasks in foil. Clean your bench with ethanol. Only inoculate cultures in the presence of a flame and only check optical density with autoclaved sterile pipette tips in the presence of a flame.

Eventually the bacteriophage problem goes away, but it only seems to happen when you are desperate for data.

Thank you for all the suggestions

1. I have checked the antibiotics (chloramphenicol and ampicillin) both are functioning.

2. I think there is enough aeration (100mL of LB in 250mL of flask).

3. If there is phage contamination in LB broth, E.coli can grow or not?

Because overnight culture grow. The subculture on next day did not grow (I have checked OD for 5 hours) but if I let the subculture overnight, there is grow. I have repeated this for three times still the same.

4. I will clean all the glassware with decon90 this time and prepare new LB. Then, I will repeat overnight culture and subculture. Hope it works.

If there is a bacteriophage contamination, E. coli growth stops at a low optical density. You still have cells in suspension, just most of them are dead.

If there is bacteriophage infection then you should see lots of cell debris and not only dead cells, due to lysis.

If you are not seeing growth in 5 hours but you do see overnight growth, one possible explanation is that most (but not all) cells have lost one of the plasmids. When you have an ampicillin resistant strain it usually degrades all the ampicillin in the medium very quickly, so by the time the culture has reached high density, there is no amp left. So you might have large population of cells that lost that plasmid.

So if 99% of the culture lost the plasmid, instead of making a 100-fold dilution to grow out, you actually have a 10,000 fold dilution of amp resistant cells. So it takes much longer to grow out. I'm not sure this is the issue but worth checking.

Hence if you do the control cultures I described earlier and monitor over the 5 hours you can see if the culture is just not growing, or if the slow growth is due to one or the other antibiotic. You won't actually learn much from the overnight since eventually all the cultures will likely grow.