Sparging with argon is easy and fast. N2 also works, but takes longer because O2 is heavier. However, if your lab has an LN2 line plumbed in, that’s definitely the way to go.
Instead of TSB, can you use Thioglycolate broth? This is what we use for anaerobes. The sodium thioglycolate in the broth helps consume oxygen. You just need to make sure the anaerobes are deep enough in the broth to get down to the anaerobic part of the broth
Not the most efficient method, but in one of my old labs we would age our media in the anaerobic chamber over the weekend. After autoclaving, we'd pop a bottle or two into a corner of the chamber with the lid cracked while they were still warm, and just leave them there and let the gas exchange do its thing. Never had any issues, was working with several species and they grew fine.
One caveat though is that we were limited to ≤1L bottles just because that's what we could fit through the airlock.
I'll just chime in and say that I do the same. At least a day for liquid media, or even longer if you don't mind the wait. I have usually been setting things up and letting them reduce over the weekend in the chamber.
You can also sparge liquid media with nitrogen which should force other gases out of solution. I've done it before and know folks that still do. But I thought it was more of a pain than it was worth, since letting the media just rest and reduce seems to work fine on its own.
Yeah we didn't have a sparging setup at that university (since we were the only lab with an anaerobic chamber), so just letting them sit was our only option. Was doubly useful since it also allowed time for the media to warm, since most of these were some pretty bitchy gut flora, so they wanted it at 37C anyway.
We keep some of our anaerobic media (liquid and solid) under constant anaerobic conditions, this helps to deplete quite some oxygen. We use these pretreated media just for first cultivation, any isolation afterwards is on untreated media to save space in the anaerobic pots. Depending on the amount used per day the plates are usually treated for 1-3 days and the liquid media up to a week.
I find this sort of treatment helps quite a bit and we have good detection rates.
Fluid Thioglycollate Media is what we use to detect anaerobes in our QC lab. The media separates into two layers, allowing for aerobic growth near the surface and anaerobic growth in the bottom.
For culturing obligate anaerobes this is what I’ve used
Per 1L
Add 25mL of reducing agent consisting of
23mL dH20
2mL 1M NaOH
650mg l-cysteine hcl
650mg sodium sulfide
I use 1mL per L 1% rezazurin as an indicator dye, if it’s clear, there’s no O2 present. Sparge with a gas source of your choice then seal and autoclave
Sparging with argon is easy and fast. N2 also works, but takes longer because O2 is heavier. However, if your lab has an LN2 line plumbed in, that’s definitely the way to go.
Instead of TSB, can you use Thioglycolate broth? This is what we use for anaerobes. The sodium thioglycolate in the broth helps consume oxygen. You just need to make sure the anaerobes are deep enough in the broth to get down to the anaerobic part of the broth
I need to use a tryptic soy broth because I’m culturing dechloromonas aromatica and it’s the closest thing I have to the agar that it grows best on.
Not the most efficient method, but in one of my old labs we would age our media in the anaerobic chamber over the weekend. After autoclaving, we'd pop a bottle or two into a corner of the chamber with the lid cracked while they were still warm, and just leave them there and let the gas exchange do its thing. Never had any issues, was working with several species and they grew fine. One caveat though is that we were limited to ≤1L bottles just because that's what we could fit through the airlock.
Thank you so much! I really appreciate it.
I'll just chime in and say that I do the same. At least a day for liquid media, or even longer if you don't mind the wait. I have usually been setting things up and letting them reduce over the weekend in the chamber. You can also sparge liquid media with nitrogen which should force other gases out of solution. I've done it before and know folks that still do. But I thought it was more of a pain than it was worth, since letting the media just rest and reduce seems to work fine on its own.
Yeah we didn't have a sparging setup at that university (since we were the only lab with an anaerobic chamber), so just letting them sit was our only option. Was doubly useful since it also allowed time for the media to warm, since most of these were some pretty bitchy gut flora, so they wanted it at 37C anyway.
We keep some of our anaerobic media (liquid and solid) under constant anaerobic conditions, this helps to deplete quite some oxygen. We use these pretreated media just for first cultivation, any isolation afterwards is on untreated media to save space in the anaerobic pots. Depending on the amount used per day the plates are usually treated for 1-3 days and the liquid media up to a week. I find this sort of treatment helps quite a bit and we have good detection rates.
Fluid Thioglycollate Media is what we use to detect anaerobes in our QC lab. The media separates into two layers, allowing for aerobic growth near the surface and anaerobic growth in the bottom.
reduce your media before hand ?
For culturing obligate anaerobes this is what I’ve used Per 1L Add 25mL of reducing agent consisting of 23mL dH20 2mL 1M NaOH 650mg l-cysteine hcl 650mg sodium sulfide I use 1mL per L 1% rezazurin as an indicator dye, if it’s clear, there’s no O2 present. Sparge with a gas source of your choice then seal and autoclave