On Tue, 25 Jun 1996, Michael Casey wrote:
> I am studying the rate of growth of Clostridium tyrobutyricum in acid medium
> and have found them to be very sensitive to the presence of oxygen. I follow
> the growth rate by measuring the turbidity at 650 nm. I have tried using
> different techniques to reduce the oxygen content of the medium but with
> little success.
>> Gassing with CO2 does not reduce the oxygen content sufficiently.
>> The addition of cysteine to the medium is also not sufficient but the
> presence of sodium thiogllycolate inhibits growth.
>> The addition of Oxyrase, a commercial enzyme preparation which removes
> oxygen, causes turbidity and prevents accurate measurement of growth.
>> Does anyone have an idea how I could reduce the oxygen content of
> the medium without causing turbity
I used to work with an extremely oxygen sensitive organism,
a Methanosarcina ssp.. What we had in the lab was a manifold attached to
both a vacuum pump and various gasses (I suppose you could use CO2).
The organisms were grown in serum vials that were crimped
shut and contained a rubber stopper. The manifold had several needles
attached to it that could be opened and closed. You would run each medium
through several cycles of vacuum and pressurized gassing.
There are a number of other techniques that are really useful. I highly
suggest reading 'Archaea, A Laboratory Manual' and specifically the
section on the methanogens. There is a really excellent chapter in there
by Sowers and Noll that shows these procedures in detail; both
visual as well as text explainations.
Biological Process Technology Institute
University of Minnesota