Dear Hadeel,

I would recommend to put a fresh streak on an LB plate with appropriate antibiotics. Incubate at 28 degree celsius maximum for about 2 days. The optimal growth temperature is between 20 and 30 degrees. It can take 2-4 days until colonies will appear on the plate. Incubation at higher temp. may lead to loss of megaplasmids. I would recommend to redo ist again, and in case you do not see growth at 28 degrees after two days, I would start worrying. Best regards.

Maybe you confused the optimal growth temperature of E.coli, 37°C, with the one of A.tumefaciens, 28°C. The difference is so wide to hamper the growth of the latter.

Albeit the difference between LB and YEB is slight, YEB is better when it comes to grow A.tumefaciens.

The stock is merely constituted by an aliquote of the original culture diluted in glycerol, therefore no treatment is necessary.

I'd let the stock defrost slowly, then plate it on YEB+MgCl2 (http://cshprotocols.cshlp.org/content/2010/5/pdb.rec12216.full?text_only=true ) additionated with proper antibiotics and finally incubate at 28°C static for 2 days.

If you're equipment risks to raise the temperature, it would be better to set it to 26°C.

After that, depending on your needs, you can take an inocule for a liquid culture or another plate, always using YEB+MgCl2 and antibiotics.

The temperature was too high. Use 28C for Agrobacterium if you can. If you only have access to 30C (common for yeast labs), Agrobacterium can grow there also but you will need to increase rifampicin antibiotic (up to 100 ug/ml). I use LB agar plates plus antibiotics. Just streak onto the plate and let recover and grow for 2-3 days.

As others have said the temperature was too high for agrobacterium. In general though I wouldn't be worried if you don;t see anything overnight, Agro glycerol stocks can be quite slow to get going so leave the plates for a couple of days before starting to worry.