The requirement for methanogens are 20% CO2 and 80% N2. How do we obtain this mixture?
we use an anaerobic chamber that allow to use 2 gasses but we cannot control the amounts of gases.
You can get this mixture from the supplier of your gas cylinder. You can actually get any % of mixture of any gas from you gas supplier. You cannot mix a known proportion of gas in anarobic chamber with different cylinder. But i dont think so you can use more than 5% of H2 in anarobic chamber as it is dangerous. I have not heard of any sensor available in anaerobic chamber which doesnot allow you to do so, so technically you can use more than 5% H2 but its dangerous and may lead to explosion, so dont use it.
What you can do is sparge your anaerobic bottle after sterlization with 20% CO2 and 80% H2 outside or using gassing mannifold (presserized)and than grow in anaerobic chamber using normal gas of anaerobic chamber
Best of luck.
I haave found this may be not its place but hope it would be helpful
http://pipeline.corante.com/archives/2010/06/30/how_not_to_do_it_hydrogen_gas_mixtures.php
Yes more than 5% H2 is dangerous to use it anaerobic chamber and even in lab. If you are using in lab than it is done in a seperate made chamber with a facility to sparege gas in ptessure and vent going outsite, along with H2 sensor in lab.
I dint had such facilities in lab. I used a gas cylinder of 75% H2 + 25% CO2 directly supplied by local gas supplied. I used to take the cylinder outside open air, use the gas by sparging it in the required bottle at required pressure directly from cylinder. There is a trick on how to sparge at required pressure directly from cylinder which u should know before trying. Also do wear mask and protective wear in eyes.
If you need further detail and diagram, i can tell you
Best of luck
Thank you a lot Mustapha
I need more details about mixture making, and maintainance of culture uder 80% h2, 20% CO2 and if possible the instrument witch allow to have the amounts.
I have found this one on the attached files.
You CANNOT do this in a chamber! It's DANGEROUS! Very Explosive!
You can do it only in a tube, serum bottle or Oxoid anaerobic jar!!!
what is the better technique to measure the amounts of gas in the chamber (N2, Co2). and to control the precise amount of gas mixture.
The best way for a chamber is to purchase a triple or double mixture gas mix premixed by manufacturer that contain just small amount of H2 to make palladium catalyst working. For mixing it in a tube, one can purchase H2:CO2 premixed in a gas cylinder and add 20 psi to a bottle or tube. For anaerobic jars Oxoid sell packages that create H2:CO2 atmosphere in a jar
For isolation of anaerobic bacteria many Anaerobic chambers are used, but to my knowledge no anaerobic chamber allows use of a gas mixture with 80% hydrogen due to explosive nature of hydrogen if confined in a close area. However 10% Hydrogen, 10% Carbon dioxide and 80% nitrogen mixture cylinder with another nitrogen cylinder is used in Anaerobic Chambers suited for this purpose. For methanogens archae 80% H2+20% CO2 at 2.5-bar is best used in Hungate roll tubes.
You have to use Mass flow controllers where it's possible to maintain gas flow ratios.
thank you Narnepati Krishna Chaitanya . Would you give me an example of one if u ve already used?
Thank you very mutch Narnepati Krishna Chaitanya . It is really very helpful for us.
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