Bacterial contamination- need some help

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!

Cheers,

-AJHicks
Chandler, AZ

"Aaron Hicks" <[Only registered users see links. ].net> wrote in message
news:[Only registered users see links. ].net...


Aaron,

I think the first thing you need to look at is the autoclave. Do the
temperature and pressure gauges accurately measure what is actually going on
in the chamber? When was the last time you had the autoclave validated? Do
you run spore control strips? Has the chamber ever been mapped for cold
spots?

Second, see if you can locate the source of the contaminant. Where do you
store the autoclaved jars? Are the jars stored in a high traffic or high air
flow area? What are you using to seal over the holes in the metal lids? Is
the material more than steam permeable--what is the pore size? Do you
regularly wipe down the area where you store the autoclaved jars? What
amount of condensation are you seeing on the jars?

If you find out that the autoclave is not performing according to how its
gauges indicate, can you autoclave the medium for more than 20 minutes with
out degradation of nutrients in the broth? If you can, extend the autoclave
time to 45 or 60 minutes. I just reread and saw you have agar in your media,
this means that you really can't autoclave it more than 20 minutes.

How corroded are the metal covers on the jars? Could the rust be harboring
or protecting the organism? Can you switch away from using the metal covers
and just use a steam permeable barrier?

You may want to consider taking air and surface samples in your storage and
prep areas, the autoclave, and of your jars and other prep materials before
and after autoclaving. You are probably going to find the organism in the
work area but you may see it more in one area than in another. If and when
you autoclave the contaminated jars with the medium and culture intact does
autoclaving kill it? It should, but trying this may tell you something.

I am sure I have missed something but, to sum up, I would check the
functioning of the autoclave first and then look at how and where you store
the prepared medium. If you have any questions email me at lmank at comcast
dot net. I hope you figure it out soon.

Laurn

"Aaron Hicks" <[Only registered users see links. ].net> wrote in message
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A very common source of contamination is ventilation system in the building.
I also suggest you to wash your glassware with not only detergents, but also
some harsh chemicals like 1M HCl or even rinse with chromerge (cromium
dioxide in concentrated sulfuric acid - handle with extreme care though as
it might severely burn you). Also, it might be a good idea to check the
source of the water used in autoclaves for possible contamination. Some
stand alone autoclaves I've seen use refilable water bottles, and in case of
being contaminated with the stubborn spore-forming species, they might might
contaminate your glassware and media. Autoclaving under conditions you
described does not kill 100% of "tough" spores.
- Emir

You don't say what your "sterile venting" procedure looks like. I
assume your bottles are open or vented during autoclaving. You won't
kill some bacteria/spores in sealed containers. Are you sure the
venting seals the bottles from the environment? Are they 0.2u
filters?

Have you checked your autoclave for temperature? Small changes in
temperature around 121C have dramatic effects on kill efficiency.
Test the autoclave with spore samples which can be cultured for
growth.

"Aaron Hicks" <[Only registered users see links. ].net> wrote in message
news:[Only registered users see links. ].net...

As others have said, the first thing I'd do is check the autoclave. You can
buy (or make) strips of filter paper impregnated with Bacillus spores to put
through the system and then incubate. I'd also look at the loading of the
autoclave - I've seen people packing as much as possible into one, which
does not allow for free passage of the steam, allowing "cool" spots to
remain. If your medium is particularly rich or particulate, it's a good idea
to up the temp a bit - we sterilize mineral media at 110C for 15 min, but
high protein media go in for 20 min at 120C. I would also take a look at the
vent seals - dry sterilization requires much higher temperatures and if the
bugs have found themselves a niche where they can avoid the steam, they'd
just sit there.
I must admit that I would not have considered Rhodococcus to be particularly
heat resistant, so I would also look at the procedure for unloading the
autoclave - how is the venting down and what happens as the flasks cool
down? Is it always the same flask/lid combination? Try completely different
vessels (eg putting some medium in in conical flasks with cotton plugs) and
see what happens.
We had this trouble with a spore former in our fermentors once - we
eventually found that it was lurking in the rubber seals around the
electrodes and one student thought that as the fermentor only had mineral
medium in it, she'd be kind to the electrodes and sterilise at 110C. Since
we started putting all fermentation stuff through at 120C, the problem has
vanished.
Lesley Robertson
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