I have been preparing anaerobic media in the traditional way (and as mostly outlined in DSMZ recommendations) and adding additions to the vials post autoclaving. This has worked well in that I've never had issues with contamination and my cultures always grow. However, this means that the vials are not technically replicates and require individual vial pH amendment at this point. It also is more labour intensive. I have the equipment for the Widdel flask method, however I am a little concerned with how best to avoid contamination and also when additions are added to the main media (post autoclaving and during sparging) do I then pH adjust the media at this point?
Any advice or help is greatly appreciated.
Ciara
Hi Jan,
Thanks very much for the advice (again!). I had that chapter actually it has been a lot of help plus a chapter from David Stahl's group. I had worried about contamination but from your experience it was not a problem in this method? And you found it remained oxygen free? I will be using N2/CO2 to sparge and will be adding bicarb, reducing agents, trace elements, K2HPO4 solution, and vitamins post autoclaving. I will give it a shot this week, thank you very much for your help!
Best wishes,
Ciara
Hi Ciara,
I was using "widdel flask method" with slight modifications for ~ more than 15 years.
Normally I did not experienced serious troubles with it.
You may just follow Jan s recommendations!
Best, Alexander.
I have extensive experience of Widdel Flask and used this system for 4-years without any contamination. I generally add all the heat sensitive component like vitamins , amino acids post autoclave and used filter sterilized gas for flushing the vials and head space. I never experienced any contamination in any Hungate tube or serium vials. Just take precaution and follow aseptic techniques.In case of bicarbonate buffer sufficient saturation of medium with N2: CO2 require and it takes some time
- Badges
- Science topic