Anarobic culture

David R. Boone boone at
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medium that is injected into the flask while the sample is withdrawn into 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;

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