What medium is in your plates? Are they LB or similar or a minimal medium?

Secondly, DH5a is a RecA- strain (as are most E. coli cloning hosts). These do not survive long on plates, maybe 2 weeks max. So I think everything is dead on your 6 week old plate.

However I don't understand why the frozen stock did not grow, which is why I asked about the medium you are using. You might grab a small chunk of the -80 stock and put in liquid broth to let it grow then streak out for single colonies. If you get it to grow then I would make a new -80 stock.

Thank you, Dr. Michael, for your time.

The medium I used was LB.

Regarding the frozen stock, I am concerned that freeze-thaw cycles could affect the viability of the bacteria. Even though I placed the stock on ice, partial melting occurred while transferring it from -70°C to my work lab. I took one loop from this stock, which is equal to 10µl, and streaked it on an agar plate.

From your answer, I understand that taking a small chunk of the -80°C stock might mean that one loop of that stock wasn't sufficient for the bacteria to grow! Therefore, I will follow your advice to grab a small chunk of the -80°C stock and put it in liquid broth to let it grow, then streak it out for single colonies.

I will also prepare a new stock as well.

Dr. Michael, I have another question if you could assist with it.

I tried to grow DH5α cells to prepare a new batch of competent cells for the second time, to use these cells in bacterial transformation. With the previously prepared competent cells, I did not observe any colonies in either the positive control or my samples. However, after two nights, I noticed a few colonies only on the pUC19 plate. I am concerned that two aspects of the DH5α preparation might be reasons for the absence of colonies. First, the OD reached 0.569 before I diluted it to the optimal density of 0.4. Is it correct to dilute the bacterial culture this way?

Second, the ampicillin used had been stored at 4°C for almost 9 months, raising doubts about its effectiveness. Despite this, the absence of bacterial growth in the plates without ampicillin indicates that the ampicillin is still active.

What do you think, Dr. Michael, about my interpretation of the absence of colonies?

Thanks a gain for your time

If a bacterial culture doesn't grow after subculture, several factors could be responsible, including contamination, suboptimal growth conditions, viability issues with the inoculum, inhibition by antibiotics or toxic compounds, genetic mutations, and handling errors. To troubleshoot, repeat the subculture with proper aseptic technique, ensure appropriate growth conditions, check for contamination, verify the viability of the bacterial stock, and consider alternative growth media or conditions. Consulting experienced microbiologists or laboratory technicians may be helpful if the issue persists.