MacConkeys instead of Xgal/IPTG selection?

john markwell markwell at unl.edu
Thu Aug 26 15:48:28 EST 1993


denomme at FHS.CSU.MCMASTER.CA (Greg Denomme) writes:



>On 26 Aug 1993, Robert Rumpf wrote:

>> I've heard that the alpha-complementation-based color selection using 
>> lacZ-phagemids, lacZ deletion host cells, and Xgal/IPTG plates can also be done
>> using the same phagemids/host cells but plated onto MacConkey's agar.  Is this 
>> true, how is it done, and what's the mechanism for color selection?  I 
>> appreciate any help you can give...
>> 

>  MacConkey plates are commonly used in Microbiology for plating out stool
>samples, E. coli grow well because of a few goodies like bile salts and
>some sodium chloride.  MacConkey agar also contains lactose and methyl red.
>  If your E coli strain has a phagemid with a proper construct for
>lactose utilization then lactose is consumed, anaerobic glycolysis
>produces pyruvate and lactate, the colony is acidic and therefore turns
>red on MacConkey agar.  If the phagemid has a cloned gene that interupts the
>lac gene then lactose is not utilized and is less acidic and appears white.

>I use MacConkey agar routinely and is available ready-to-use from Difco
>(dehydrated).  The only problems are that 1) the colonies grow a little
>slower so you may need to incubate the plates longer than with LB agar and
>2) the plates are a slight pink so the red colonies are hard to see when
>they are very small.  BTW you may see colonies that are white with
>red centres as has been discussed just recently with X-gal selection (white
>with blue centres).  For about $50.00 you can make 9 liters of MacConkey agar.

>Hope this is sufficient for your needs,
>Cheers,
>Greg
>denomme at FHS.McMaster.ca

>I have no affiliation with Difco Laboratories whatsoever.
Actually, MacConkey agar contains neutral red (pKa about 7), not 
methyl red (pKa about 5).  Selective media have been employed with the
latter indicator, but they require much more acid production to cause 
red colonies.


John Markwell
Biochem., Univ. Nebraska

--
John Markwell			Phone: 402-472-2924
Dept. Biochemistry		FAX:   402-472-7842
University of Nebraska		Internet: markwell at unl.edu
Lincoln, NE  68583-0718



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