I do work in plant tissue culture, and recently have been having
problems with an organism that has contaminated a number of flasks. It has
been identified with some degree of certainty as Rhodococcus rhodochrous.
What is unusual about the contamination is that it occurs in unopened
sterile flasks AFTER they have been autoclaved. (I put the culture media
into the flasks, and then autoclave media + culture container together.)
Although I have used this technique for years, this is the first standing
contamination issue I have had.
The culture vessels are glass Mason jars; the lids are metal, and
are perforated and sealed for sterile venting. The contamination occurs
about 3-7 (or often many more) days after autoclaving, appearing as small
red spots on the surface. Most flasks have one single point of
contamination; one or two may have had two, but no more. Because the
contamination has shown up in flasks that haven't even been opened, I
suspect the organism is somehow surviving autoclaving, which is performed
for 20+ minutes at 15 psig at ~ +1,000 sea level. Prior to this recent
spate of contamination, I had been autoclaving at 15 minutes, again with
very rare (less than one per thousand) flasks.
Although contamination is not uncommon with sterile plant tissue
cultures, it is rare to see the same organism again and again. Sometimes
the contamination takes several months to show up, and by this time plants
have been introduced. This one has been a real stumper- I can't figure out
the source, and I have found relatively little information on R.
rhodochrous. Should it be capable of surviving autoclaving for 20 minutes
and up, in vessels holding ~75 mL of solution? My colleague and I have
extensive plant tissue culture experience, and are both stumped over this
one- it doesn't seem to jibe with any one contamination source. Right now,
I'm trying some experiments to see if it is one of the media components
(water, agar, nutrient substrate, etc.), but none seem to match.
I would appreciate any insight anyone might have as regards to
this issue. The e-mail address in the header doesn't work; please reply
here, or to my hotmail.com address (osp001 *at* hotmail). Thanks!