Coating LB plates with tet after pouring?

Ole Skovgaard olesk at RUC.DK
Thu Jul 20 05:34:20 EST 1995


In article <heller-1907951837230001 at mac3.ifr.ing.tu-bs.de> heller at ifr.ing.tu-bs.de (Marcus Heller) writes:
>From: heller at ifr.ing.tu-bs.de (Marcus Heller)
>Subject: Re: Coating LB plates with tet after pouring?
>Date: Wed, 19 Jul 1995 18:37:23 +0200

>In article <NATHAN.95Jul17125346 at protos.bms.com>, nathan at protos.bms.com
>(Nathan O. Siemers) wrote:

>> Hello, World.
>> 
>> 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?
>> 
>> Thanks,
>> 
>> nathan
>> 
>> --
>> Nathan O. Siemers, Ph.D.        
>> Bristol-Myers Squibb Pharmaceutical Research Institute
>> 3005 First Avenue, Seattle, WA 98121
>> (206) 727-3741          siemers at bms.com
>> "You want fries with that?"
>Hallo Nathan,

>it is possible to bring tetracycline or any other other antiboitic to agar plates after pouring. Just store the antibiotic solution filtrated with 0.2µm filters at - 20°C in eppendorf cups. Advice for stock solutions and concentrations in medium you can 
>ind in maniatis, handbook of molecular cloning. Thaw the volume needed and distributed it on the plate e.g. with a sterile pipette. Let it dry for a few minutes. Then you can inocculate your plate as usual.

>Good luck.
>Tanja
>GBF Braunschweig

Hello Nathan and Tanja,
It is not that Tanja's reply is wrong, but used in unscilled hands it may lead 
to errors.
What you do, when you are spreading on top is to expect an diffusion of your 
stuff - in this case Tet. - into the plate. It happens, but takes time. 
Especially Tet. is quite slow in diffusion - on minimal plates you can see the 
yellow color diffuse slowly. Another proplem is that a "not too wet" plate may 
absorp the stuff too fast to get an even spreading. Add first a little sterile 
medium, water or whatever, and then your stuff in the samme droplet. That 
prevents that your stuff is sucked into the plate at the application spot. 
Even with this precaution the outer 0,5 cm of the plate wil have a lower conc. 
than the rest. For instance you can see Tet-S colonies after transformation.
This procedure will always be an emergency proc. Inclusion into the plate and 
use withen shelf life is preferable.
yours
ole

******************************************
****   Ole Skovgaard                  ****
****   Dept. Life. Sc. & Chemistry    ****
****   Roskilde University, DK        ****
******************************************



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