I came across your email on a blogspot by chance and thought that you might
be able to offer help with my problem. My lab work mainly involves
mycobacteriology and using 7H11 agar for plating is somewhat routine. Very
recently, I have been facing serious problems with contamination on unused
agar plates and I am rather perplexed as to how this could be. Of course,
contamination on agar is quite common a phenomenon but having said that, the
nature of the contamination seems puzzling. There happen to be grain-like
bacterial colonies seeded evenly within the agar, with a substantial number
of morphologically different colonies growing on the surface of the medium.
They are all white in colour, though the surface-growers have a
characteristic 'shiny, appearance. I am also told that this type of
bacterial colonies are also known as 'pressed-coin'. I haven't been all that
successful in deciphering this issue online and so, I wonder if you could
help me at all. Also, I must point out here that everyone in the lab where I
work uses the same autoclave machine. In addition, the supplementary
reagents that I add to molten agar prior to pouring plates are as well those
used for preparing liquid medium (7H9 broth) and I find not contamination
Have you tried subculturing these colony types to another type of
general laboratory medium, like TSA or TSA with 5% sheep blood to see
what they are? Have you tried staining these to see what they are? Are
they acid fast? I would think that 7H11 would grow regular bacteria, as
I do not believe that it has any antibiotics in it to inhibit non-acid
fast bacterial growth. If they are some sort of Bacillus sp. then your
medium is not being properly sterilized by your autoclave.
Have you run controls on your autoclave to make sure that it is getting
to the right temperature? You can buy commercial autoclave controls in
various formats. How long are you sterilizing for?
Are the plates you are pouring into sterile? Are you pouring into plates
that have had the package opened for quite some time?
Are you somehow introducing something nonsterile into your medium after
you autoclave but before you pour? You're not stirring the medium with
anything that is not sterile, are you? Do you cover your flask in the
autoclave? We used to put gauze over the top of the flask and seal with
autoclave tape. This let the steam out but kept stuff from falling into
the media while it cooled off in the 56 degree water bath until we
poured. Hopefully your flask is not open to the air in the time between
autoclaving and pouring.
If it is down in the agar then it is a sterilization problem. I've never
made 7H11 - always used commercially made plates - so don't know how
tricky it is to make. If your supplements don't produce contamination in
your 7H9 broth, then the only explanation is that your autoclave is not
functioning correctly and your media is not properly sterilized or you
are introducing a contaminant post-autoclaving.
Judy Dilworth, M.T. (ASCP)
"martin rao" <[Only registered users see links. ]> wrote in message
news:[Only registered users see links. ]. net...
A plant pathologist I know, who brings plants into the lab and
cultures microbes from them, had a long-unexplained, un-inoculated
Petri dish contamination problem until he started storing plates in
deep, sterilized plastic tubs. Turns out mites, so small you don't
normally see them, were crawling around, carrying microbes into
everything. Choose tubs big enough so mites can't climb into them.
Good luck, Tom McCloud
On Wed, 16 Jul 2008 14:33:39 +0800, "martin rao"
<[Only registered users see links. ]> wrote:
From what you have described, it is clear that either bacteria in your
agar are not being killed by the autoclaving or you are introducing
contaminants by additions to the agar after autoclaving. Those are the
only ways you are going to see both surface and sub-surface colonies;
the bacteria have to be distributed throughout the agar when it is
poured to produce the pattern you describe. Others have suggested ways
to check autoclave operation, although I note that you say others use
the same autoclave and (apparently) are not experiencing contamination
problems. You also state that you use the same supplements in broth
without any growth occurring. Since I am not familiar with the media
you are using, I ask whether *all* supplements are used in both agar and
broth? Is the *only* difference between agar and broth the presence of
agar? If so, that really leaves only the autoclaving as the possible
culprit (unless you are doing something really strange, such as possibly
using non-sterile pipets, etc, to add supplements to the agar; that
really makes no sense but it is all I can come up with as an additional
Larry D. Farrell, Ph.D.
Professor Emeritus of Microbiology
Idaho State University
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