I'm using an anaerobic lap system to change the headspace of culture vials that have been made using the Widdel process, however after I run the lap system program my vials have gone blue/pink indicating air has gotten in. Has anybody come across this issue before or has any advice?
Thanks in advance,
Ciara
The process requires speed and accuracy. You need to do it as fast as possible to avoid any entrapment of even of a little air. Secondly the temperature should be maintained
Thanks
you can use serum vials with rubber stopper and aluminium cap. After that you sparge with N2 or CO2 gas. Some time may use reducing agents.
Thank you Dr. Nrior, I don't think that is the issue with the anaerobic lap system as there is no means for entrapment of air and the temperature is room temperature as the anaerobic lap system is used after media preparation. Thank you Dr. Gandu, my serum vials are sealed with butyl stoppers and crimped with aluminium seals and I have used reducing agents. The media is prepared using the Widdel set up and sparged and aliquoted into serum vials. The vials are clear at this point after being left for an hour for the reducing agents to work. Then when I use the anaerobic lap system to give each vial a desired pressure of N2/CO2 the vials become pink.
Dear Ciara,
Why are you left of experiments for an hour for reducing agents? why not are you sparging immediately after media preparation?
Dear Bharath Gandu, I think you misunderstand me. I use the Widdel process to aliquot the media + sparge the vials. Then I leave the vials to go completely colourless and I use the anaerobic lap system to give the desired volume of N2/CO2.
Dear Ciara Keating,
I hope you are doing fine.
I would also recommend the same following answer,
you can use serum vials with rubber stopper and aluminium cap. After that you sparge with N2 or CO2 gas. Some time may use reducing agents.
Besides, see the attached PhD thesis on setting up the BMP assays.
I hope you will get the answer.
Have a wonderful day.
with regards,
Nizami
Dear Ciara,
I have being working with Widdel Bottle and also in Widdel Lab for quite a number of years. Normally when I was preparing bicarbonate - buffered medium, then I dispensed the medium from Widdel Bottle to serum vials at the same time flushing the serum vials with N2/CO2. Then I did not need to replace the gas atmosphere.
1) I apologize but I do not understand "anaerobic lap system". If that is the system for vacuuming/flushing vials, then before starting to work with the system I flushed all tubings with the anoxic gas to remove air from tunings before inserting needles via rubber stoppers into vials.
2) My additional personal trick was that I normally added into the medium in Widdel Flask a little bit of very strong reductant - dithionite (a few small crystalls). Just to be sure to that medium is a bit redox buffered... that after some manipulations the medium stays for sure in the reduced state.
3) Resasurin is also pH reactive compound - Have you checked pH in coloured vials?
4) If during N2/CO2 treatment only a very tiny bit of oxygen is penetrating into vials - then it may completely react with the redox indicator, and the vials is still quite anoxic or in another words the vials may be fitting to grow some anaerobes...
I hope that some points would be helpful,
Alexander.