Anaerobic Incubation Solutions

[AT-JeffT]

Hey All,

I'm wondering if any of you have some low-budget solutions for anaerobic incubation of plates. A local brewery was kind enough to give me some anaerobic indicators and I tried a candle in a mason jar. That didn't consume enough o2. I also added some co2 fittings to some tupperware and flushing it with co2. No luck their either.

Anyone have a lower-budget solution than the gaspak systems?

Thanks, Jeff

[Yeast]

Got to the last sentence and then my suggestion got shot down. Gaspak is expensive but yet one of the more affordable options

[Noble31]

2.5 gal corner keg if you have a big enough incubator.

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[AT-JeffT]

2.5 gal corner keg if you have a big enough incubator.

Sent from my SGH-M919 using Tapatalk

I could see this working. What's your SOP for purging? In through the liquid post and vent through the gas post?

[wailingguitar]

Hey All,

I'm wondering if any of you have some low-budget solutions for anaerobic incubation of plates. A local brewery was kind enough to give me some anaerobic indicators and I tried a candle in a mason jar. That didn't consume enough o2. I also added some co2 fittings to some tupperware and flushing it with co2. No luck their either.

Anyone have a lower-budget solution than the gaspak systems?

Thanks, Jeff

That's interesting. I have had several labs tell me to use candles in jar, I generally light it, let it burn out, open, re-light, and seal. We recently hired a microbiologist and he didn't see anything wrong with it. I will ask him if he has any other suggestions/tips.

EDIT: I spoke with him and he said that was the method he always used with good results. He suggested checking the seal. Are you getting a good 'pop' when the candle burns out and the air inside cools? He said maybe try putting some Vaseline around the rim to help the seal. Another possibility being that the indicators themselves are bad.

[rdcpro]

I could see this working. What's your SOP for purging? In through the liquid post and vent through the gas post?

When I worked for a company that made gas purifiers (SAES Pure Gas), I found that cycle purging a vessel at least 3 times (and up to 5 times, if you're really serious) gets the O2 (and anything else) down to essentially undetectable levels. We would do this with pressure vessels cycling from 0-120 psi. We'd also do this with a piping system to remove the air prior to running whatever gas we're going to end up with. If the piping system was really long (like from one end of the building to the other), we'd pressurize from near one end, and vent from the other. But in a vessel like a corny keg, it's not necessary.

If you simply try to purge from the bottom and vent through the gas post, you won't remove all the air, due to turbulence. CO2 is heavier than air, but they still mix (otherwise, we'd all asphyxiate here on the surface of the earth).

Regards,
Mike Sharp

[AT-JeffT]

That's interesting. I have had several labs tell me to use candles in jar, I generally light it, let it burn out, open, re-light, and seal. We recently hired a microbiologist and he didn't see anything wrong with it. I will ask him if he has any other suggestions/tips.

EDIT: I spoke with him and he said that was the method he always used with good results. He suggested checking the seal. Are you getting a good 'pop' when the candle burns out and the air inside cools? He said maybe try putting some Vaseline around the rim to help the seal. Another possibility being that the indicators themselves are bad.

If a candle in a jar works, that would be great. I only tried it (single burn, no relight) with the indicator which indicated it was over .5% o2. I'll try the relight method you described and try and culture some anaerobes. What type of vessel are you using?

I'm using a wide mouth mason jar and I'm getting a good seal. Definitely negative pressure when opening it.

[wailingguitar]

If a candle in a jar works, that would be great. I only tried it (single burn, no relight) with the indicator which indicated it was over .5% o2. I'll try the relight method you described and try and culture some anaerobes. What type of vessel are you using?

I'm using a wide mouth mason jar and I'm getting a good seal. Definitely negative pressure when opening it.

12 oz mason jars

[AT-JeffT]

12 oz mason jars

Do you use 60mm diameter plates?

[wailingguitar]

Do you use 60mm diameter plates?

These were small LMDA plates I got from BSI. Not sure the exact size, but 60mm sounds about right.

[MJMurphy]

Years ago we had a few make-shift incubators. It consisted of a length of PVC pipe (4" diameter?) with a cap at one end with a plastic gas disconnect threaded into it. At the other end was the type of plumbing test plug that can be put into place and removed easily. Near that end on the side of the pipe was another plastic gas quick-connect threaded into the pipe.

Inside was a wire deal that held x-number of plates in place. After putting the plates into the device, you would seal it and purge it with CO2 for so many minutes.

By the time I began in the lab we have moved onto the GasPack system and never used it, but the devices were still around.

If the budge allows, the GasPack system is a pretty good option.

We have moved to a CO2 incubator but instead of the unit regulating the CO2, we keep a constant flow - so many cc's - when the unit it in use. The unit does measure the CO2 so we can verify the %, but it goes out of range at a point.