For growing anaerobic bacteria on solid agar plates, I am using an "anaerobic jar" with "gas pack" that consumes all the oxygen inside the chamber after it is sealed. We usually seal the petriplates containing solidified agar medium after inoculation, to avoid contamination (which works well with aerobic cultures). Should we do the same for growing anaerobic bacteria in anaerobic chamber?
I doubt that if we seal the plates, the oxygen inside the plates will be trapped, while the gas-pack removes the oxygen from the chamber. And therefore making it difficult to maintain anaerobic condition inside the plates. But if we do not seal, how to avoid contamination?
If you are using an anaerobic chamber you need not seal your pertiplates. Anaerobic chamber is more or less like a laminar air flow bench equipped with anaerobic gases to maintain oxygen free environment. However during incubation in a gaspack you can seal the petripates with parafilm to avoid chances of contamination. However there are number of oxygen scavenging compounds such as cysteine HCl, sodium thiosulfate can also be added in the medium in minimum concentration to remove the oxygen from the said medium thereby preventing contamination from aerobes....
I assume you use laboratory film for sealing the plates. Most likely there will be some oxygen inside the plate if you seal it. You can ensure whether the conditions are aerobic or anaerobic by adding anaerobic indicator strip (at least Merck and Oxoid provides these, most likely you find other suppliers too) on one test plate. Seal it and then put it in anaerobic jar with gas pack. The indicator strip shows whether sealing of the plate affects to development of anaerobic conditions.
Its important to seal the petriplates...use cling films or parafilms for it...u can use indicator strips too...Jars with gas packs could be a better choice
@Dr. Jouni Heikkinen. Thanks for the response ! I use laboratory film or paraffin film to seal the plates, but have not used the indicator strips till now. I will certainly try to use it. Thanks for the suggestion again.
@Dr. Armando Hernandez. Yes, thats what I think too. But my doubt is whether leaving the plates unsealed would lead to contamination from the air inside the chamber.
Petri plates are designed to provide sterile environment for the growth of microbes on solid media. There is no need to seal the petri plates as all aseptic operations are performed strictly under qualified Laminar air flow (LAF) cabinet. Microbes won’t enter the petri plates in any way when they are closed and handled properly. To be extra precautious some microbiologists will follow this practice but it not an absolute requirement. So, you need not to worry that unsealing of plates may lead to contamination.
I create anaerobic conditions in a very simple cost-effective manner by using glass fish tanks as candle jars. Position the plates (inverted) in the tank, light candle, place aluminum foil over the top of the tank, and seal foil tightly with the tape. Tanks should not be filled to more than 50% capacity to ensure consistent and adequate growth.
I handled lot of bacteria under anaerobic condition by following the above simple technique without using gas-pak system.
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Hello Shiv the gaspack is a good option for facultative anaerobic MOs, for the usage of more strict anaerobics, you need to degas your materials, means you have a dissolved oxyygen molecules in plastics, Silicone and in media that you need to get rid of first. The more sensistive your organisms are, the more acurrate has to be your degasing process....this can take many days for strict anaerobics. You can try some heavier-than-air gasesm CO2 is a simple way, if you have access to argon: this is more accurate
To Mr. Frank
I think you have mistakenly written my name. The author of this question is Mr. B Chandrasekhar and this answer should have directed to him.
@Mr. Frank. Wow. That is a good piece of information. I didn't know that. Thanks..!!
Hello there!! I also grow anaerobic bacteria like you, in an "anaerobic jar" where I have sparged CO2 just before putting the plates inside, or with the "gas pack" and it usually works quite well. I have also tried to grow them without the anaerobic jar, sealing the petriplates with parafilm and although this is not the best method, I also had good results. I don't know if it's the case, but have you tried antibiotics? My strain's wild type is naturally resistant to kanamycin so when I'm having troubles I grow it with a little kanamycin and my contaminants disappear.
Hello Ms. Margarida
Thanks for the comment. I have never used antibiotics, but it is a good idea if you are working with antibiotic resistant bacteria. How do you put the plates in after sparging the "anaerobic jar" with CO2? How do you grow anaerobic bacteria without using the jar? Do you use anaerobic medium?
Hello again :)
When I use the anaerobic jar I just put the plates inside the jar, close it and then connect it to the CO2 bottle and sparge it through the lid opening. But when I do not use the jar I supply the medium with NaHCO3 and my strain consumes the CO2 from the medium, although it takes 2 to 3 days to grow some isolated colonies.
You are following the best and easiest way to grow anaerobes. I had used these jars for growing C.diff. isolates. Anaerobic chamber/hood is another option for continuing experiment.
Well i agreed with Frank and Shiv. Have used both the systems for growing anaerobes..However i have made strict anaerobic conditions in growth media as well, by using ammonia and sulphur residues.
About pyrogallic acid + NaOH as oxygen scavenger ― check my post at: https://www.researchgate.net/post/Can-somebody-recommend-a-method-for-making-48-96-well-plate-anaerobic
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