Methicillin revelation

Phillip San Miguel pmiguel at purdue.edu
Wed Jun 23 11:43:29 EST 1999


Okay, maybe it's only a revelation to me:  The combination of
methicillin and ampicillin in plates greatly reduces the numbers of
satellite colonies yet, E coli strains such as DH5alpha are resistant
to methicillin.

I was introduced to methicillin by this group:

http://www.bio.net/hypermail/METHDS-REAGNTS/9407/0318.html

    I wasn't trying to reduce satellite colonies, per se.  But had a
protocol for generating gamma delta insertions in plasmids for
sequencing that would produce lawns (of bacteria that would not grow
in amp/LB liquid culture) if I plated at a density that produced more
than 20 or so transconjugants per 100 mm plate.  The amp 20/ meth 80
plates worked perfectly.  I could get 100's of transconjugants on a
single plate without "lawning it out".
    Then methicillin was no longer available from distributors.  We
had recently bought a decent size bottle so we would be okay for a
while.  Plus, a benefactor known to us only as "Mike" provided the
following information to this newsgroup:

http://www.bio.net/hypermail/METHDS-REAGNTS/9703/0454.html

 That is, we might be able to use oxacillin or nafcillin in place of
methicillin.  I had always presumed that methicillin just worked
better than ampicillin.  This resulted in a post I made to this group
last week:

http://www.bio.net/hypermail/METHDS-REAGNTS/9906/0314.html

    It turned out that none of my putative recombinants had any
plasmid in them--I grew them on methicillin plates and then in
methicillin liquid culture.  I wasn't sure why, but "Mike's" post
(above) suggested that E coli would be resistant to methicillin and
that it was through some obscure (I see in at least one version of the
FAQ, this question is disputed) mechanism that adding methicillin to
an amp plate makes it less satellite-prone.
    So I actually checked this yesterday.  I made plates with the
following antibiotics in them: "m" stands for methicillin 100 ug/ml;
"a" ampicillin 20 ug/ml, "N" nafcillin 100 ug/ml; "o" oxacillin 100
ug/ml and "n" naladixic acid 15 ug/ml.  (DH5alpha is resistant to
naladixic acid.)  The plates were n, mn, on, man, oan and Nn.
pBluescript in DH5 alpha grew on all these plates.  My transformation
that had yielded false positives on meth plates before produced a lawn
on everything except man and oan.  (Plated 50 ul of a 1 ml
transformation).
    So DH5alpha does appear to be resistant to nafcillin, oxacillin
and methicillin even at high concentrations.  The definitive
experiment would be to streak out DH5alpha itself on these plates.
But I'm guessing that, indeed, most lab strains of E coli are
resistant to them.

Phillip San Miguel




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