In article <3v5i3m$53e at oban.cc.ic.ac.uk>,
rsperry at bc.ic.ac.uk (R.S. Perry) wrote:
>I am attempting to grow isolate Gallionella ferruginea. The procedure
is
>lengthy requiring many serial dilutions. The situation is further
>complicated by the fact that growth tubes must be kept in an
atmosphere of
>94%N2, 5%CO2 and 1%O2. I can think of ways of achieving this but
does anyone
>have any practical experience of achieving such. If so I would be
very
>grateful if they could e-mail me informing me of their method.
You need rubber stoppered tubes and vials, at best serum-type crimp
orifice (Bellco). This way you can keep your culteres under a defined
atmosphere. A CO2 incubator will not be any help.
The gas mixture you can either achieve by gassing the vessels with N2
and adding CO2 and O2 (i.e, the appropriate amount of air ;-) with a
syringe, or by mixing the gas from tanks using calibrated flow meters
and needle valves. You can even order commercial gas mixtures ($$$) if
you think that would be more convenient.
The whole story is basically similar to the technique used for
culturing anaerobic bacteria under defined atmospheres (Hungate
technique), but you don't have to go through all the pains necessary
to keep traces of O2 out. The CO2 in the headspace will also allow you
to use a bicarbonate-buffered medium which Gallinella will probably
like better than phosphate buffer.
Read the chapter by Fritz Widdel on isolation of Gram negative
sulfate-reducing bacteria in 'The Prokaryotes' 2nd edn., Vol 4. It
contains a wealth of technical tricks and further references.
Hope this helps for a start.
Regards,
Andreas
Dr. Andreas Brune Phone: +49-7531-883282
Mikrobielle Oekologie Fax: +49-7531-882966
Universitaet Konstanz E-mail: Andreas.Brune at uni-konstanz.de
