If you have access to an anaerobic chamber, it is best to use one of these. If not, there are commercial systems which produce anaerobic conditions for use in bacterial agar plate jars (eg: Anaerogen from Oxoid). These re very simple to use and require no catalyst. I sugegst that you include a P. aeruginosa (strict aerobe) control and a Clostridium (strict anaerobe) control. I use Clostridium because your control can be kept in a cooked meat broth and sub-cultured daily, the spores do not die for months, whereas there is a risk of losing your control organism if you use a Bacteroides or similar. You can buy rezasurin anaerobic indicators, these are white when the jar is in anaerobic condition and pink when oxygen is present, helps you to monitor if your anaerobic conditions generator has worked if you use a galss or clear plastic jar. Remember to grease the seals on your jar with vaseline regularly in order to keep it air tight. Most anaerobes can be safeylt manipulated on the bench in air for three or four hours without dying, but you must put them back in anaerobic conditions after that or they start to drop off. Finally, keep the incubations conditions moist (put a damp paper cloth in the bottom of the incubation jar) - they don't like drying out.

you have to grow them in a environment with Nitrogen

in a fermentor you have to bubble Nitrogen till the dissolved oxygen concentration becomes zero. you have to them maintain that flow rate and partial pressure of Nitrogen

here is an interesting link I found

http://onlinelibrary.wiley.com/doi/10.1002/9780471729259.mca04fs12/pdf

for small scale culture you need hypoxic chambers and anerobic culture hoods

Good luck

Murali

you need special anaerobic jars, in which you can keep your culture plates and tubes, along with special anaerobic packs,, close it and incubate the jar..

Such jars and packs are commercially available..

Both the above mentioned methods are good, if your cultures can survive in higher nitrogen content, nitrogen packs can be used to replace oxygen or anaerobic jars can be used, with commercially available packs, which aid in removing oxygen .

We used rubber stopper systems, mostly tubes or big jugs with stoppers. We used big glass carboys that were autoclaved and the water purged with argon or nitrogen after it is heated, and then cooled down. lots of sodium dithionite added. If you need a specific protocol, I can look at my pH.D thesis. But the water has to have dissolved oxygen removed as noted already above. The carboys were used to feed the fermenters. I have no idea about high tech stuff, as I did things about 25 years ago.

If you have access to an anaerobic chamber, it is best to use one of these. If not, there are commercial systems which produce anaerobic conditions for use in bacterial agar plate jars (eg: Anaerogen from Oxoid). These re very simple to use and require no catalyst. I sugegst that you include a P. aeruginosa (strict aerobe) control and a Clostridium (strict anaerobe) control. I use Clostridium because your control can be kept in a cooked meat broth and sub-cultured daily, the spores do not die for months, whereas there is a risk of losing your control organism if you use a Bacteroides or similar. You can buy rezasurin anaerobic indicators, these are white when the jar is in anaerobic condition and pink when oxygen is present, helps you to monitor if your anaerobic conditions generator has worked if you use a galss or clear plastic jar. Remember to grease the seals on your jar with vaseline regularly in order to keep it air tight. Most anaerobes can be safeylt manipulated on the bench in air for three or four hours without dying, but you must put them back in anaerobic conditions after that or they start to drop off. Finally, keep the incubations conditions moist (put a damp paper cloth in the bottom of the incubation jar) - they don't like drying out.

For liquid, Degas liquid first (vacuum), and THEN purge with H2 or N2 as stated above, it simply more effective (i.e. bubbling with N2 may get you to less than 15ppm but not the rest of the way down to less than 1ppm. For plates, if you have the kit, use the sachet system inside an glovebox like an M-Braun that can go down to 1ppm. No flames in there, so you have to have very good aseptic technique using a sterile toothpick for colony selection.

The anaerobic chamber is definitely the best choice and removing dissolved oxygen prior to inoculating your bacteria is critical for very sensitive anaerobes. Of course you can use gas packs, but you will be limited in the type of work you can do. For example, if you need to monitor your cultures by spectrophotometry (OD), the gas packs will be of little use. That said you could use the tubes equipped with rubber stoppers as suggested in previous answers. Moreover, not only the incubation under anaerobic atmosphere is essential, but also the time bacteria spend under aerobic conditions. For example, when preparing culture dilutions, centrifuging and resuspending bacterial pellets, picking colonies and spreading on new agar plates, etc. Any source of oxygen might be detrimental to your bacterium if it is a very sensitive species/strain. If you only need to have bacterial growth, that might not be critical, but if you need a specific number of viable cells, then traces of oxygen can become a problem. Richard Bradbury (above) mentioned that bacteria can remain on the bench for up to 3-4 hours which can be fine for some anaerobes, but our experience with Clostridium difficile showed us that some strains are more sensitive than others to oxygen and leaving cultures on the bench more than one hour results in significant bacterial mortality, and yet this is not the same for all strains of Cdiff. So if you don't know exactly how strict your anaerobe is, try to keep your cultures away from oxygen as much as possible.

I would highly recomment that you contact Prof. Dr. Friedrich Widdel

MPI for Marine Microbiology

Celsiusstr. 1

D-28359 Bremen

Germany

Phone:+49 421 2028 - 702

Fax:+49 421 2028 - 790

e-mail:fwiddelmpi-bremen.de

Thanks for the reference. It is really helpful.

Ashfaq

Emory University

Hi,

I am running an ex-vivo fermentation using colonic digesta. I read your points on anaerobic condition, which was really helpful. I was wondering if any one knows if I can get a premixed media (I need it to be carbohydrate free). I've heard there are some but I don't know what they are called or why I can't find them! thanks in advance.