David R. Boone
boone at ese.ogi.edu
Mon Feb 26 11:12:59 EST 1996
Ben Long wrote:
> I am trying to set up an anaerobic liquid culture of cyanobacteria but
> I have very little experience with anaerobic culture. Can anyone offer
> some suggestions as to a simple method for keeping my culture under
> anaerobic conditions for 12 - 24 hours but still allowing hourly sampling
> of the culture medium.
In the absence of light, cyanobacteria will quickly consume the dissolved
O2, so the only thing you need to concern yourself with is re-introduction
of O2. Wrap the vessel with aluminum foil to block light. Fill your
culture vessel all the way to the top, eliminating all headspace. For
instance, if you use a stoppered vessel, insert a needle through the stopper
so displaced liquid can be expelled as the stopper is inserted.
> I currently use a 5.0 litre bottle for culturing my bugs and would like
> to modify this system to an anaerobic one if possible. I have been told
> that sparging the culture with Nitrogen will provide the required
> conditions, but how can I be sure that the culture is anaerobic and how
> can I continually sample from it? Any suggestions welcome.
Sparging would remove dissolve O2 but is probably unnecessary. The lack of
O2 can be easily monitored by using a redox indicator (methylene blue or
[better] resazurin--either of these will be colorless under anoxic
conditions) that indicates low redox potential.
To remove samples you must introduce anoxic liquid while removing a sample.
This can be accomplished with two syringes having their needles inserted
through the stopper. One syringe is filled with anoxic water or culture
medium that is injected into the flask while the sample is withdrawn into
the other syringe. To prevent short-circuiting of injected culture medium
to the sample removed, use a longer needle (you can buy 15-cm needles from
Popper & Sons) for liquid injection than the one you use to remove sample.
The rubber stopper can be surface-sterilized with ethanol before inserting
needles, or you can leave the needles in place, removing the sample syringe
and replacing it with a fresh, empty syringe.
Shaking would not be satisfactory for mixing a culture which is full to the
top. Instead use a magnetic stirrer.
David R. Boone, Professor of Environmental Microbiology
Oregon Graduate Institute, Portland; 503-690-1146
boone at ese.ogi.edu; http://www.ese.ogi.edu/ese_docs/boone.html
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