Coating LB plates with tet after pouring?

Chris Boyd chrisb at festival.ed.ac.uk
Fri Jul 21 04:50:00 EST 1995


Nathan O. Siemers (nathan at protos.bms.com) wrote:

: I only occasionally use tet or tet-kan plates, mainly for maintaining
: pili in E. coli.  Problem is, the tetracycline goes bad quickly.  Can
: you spread a tet solution over the plate, allow it to dry (and
: diffuse), then spread or streak your bacteria?  If so, what
: concentration of tet is reasonable to use?

Yes, this is possible with tetracycline, but there are potential
drawbacks.  One is that spreading alcoholic solutions over plates
uniformly is difficult to achieve.  Also, the overlay tends to diffuse
inefficiently around the periphery of the plate, so non-resistant cells
will grow there.

I get round these problems as follows:

I make a stock solution of Tc (Sigma) at 10 mg/ml in EtOH (stored at
-20 degrees in a light-tight container) and use it at 10 ug/ml in both
liquid and solid media. Therefore, on an average 25 ml agar plate, you
need to spread 25 ul of stock.  If you try to do this directly, you'll
get the problems I mentioned. What I do is dilute the 25 ul in 200 --
300 ul of L broth and spread this immediately on the plate forming a
shallow lake.  Then allow to dry on a certified flat surface (in a 37
degree incubator if possible). This can take upwards of 10 minutes,
depending on the initial dryness of the plate.

Remember too that magnesium ions antagonize Tc activity, so use Mg-free
media!

Finally, I seem to remember that if the Tc resistance is Tn10 encoded,
it helps to induce the resistance operon prior to plating by growing
the cells in a subinhibitory concentration of Tc, but I've no details
on that.

Best wishes,

Chris Boyd          | from,  \MRC Human Genetics Unit / Western General Hospital
chrisb at hgu.mrc.ac.uk| not for \        Crewe Road / Edinburgh EH4 2XU / Scotland



More information about the Methods mailing list