martin rao wrote:
[snip]
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
> there, whatsoever.
[snip]
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
possibility).
--
Larry D. Farrell, Ph.D.
Professor Emeritus of Microbiology
Idaho State University
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