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Poor agrobacterium growth from glycerols

agrobacterium microbiology plant science media storage

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#1 hall_b

hall_b

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Posted 24 April 2013 - 02:55 AM

Hi all,

I'm trying to grow up Agrobacterium tumefaciens from glycerols. They're from a mutant screen so are in small aliquots (200 microlitres) in 96 well plates. They weren't flash frozen in liquid nitrogen, just put straight in the minus 80 degree freezer, so obviously viability was going to be somewhat compromised but they have grown before.

I had thought it might be freeze/thaw since the last time I used them but I've noticed a couple of my control cultures from larger glycerols that had been flash frozen and definitely don't thaw much when I use them aren't growing either. This is in L broth and L agar. Antibiotics are correct and at appropriate concentrations.

Anyone know what could be going on? All these glycerols have grown before, several times.

Would a different media help? I don't want to use LB because I get poor selection in it. Maybe YEB or SOC?

I seriously hope I haven't 'lost' these stocks. I've got 14 positive candidates from my screen in there Posted Image





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