I isolated the DNA of yeast strains obtained from the Sauerkraut brine and subsequently performed the PCR-RAPD for the strains. Before sitting down to PCR-RFLP, I would like to get busy with PCR-RAPD for the bacteria (hopefully mostly LAB) and here is the thing - what ways of ensuring anaerobic conditions can you suggest, besides using airless chamber or wrapping test tubes with foil?
I haven't experienced big difficulties in cultivating so far, yet I would appreciate it if you could provide me with some solutions in case I'd have to modify something. Thanks a million in advance!
Why not try securing inoculum contained tubes in a jar with anaerobic sachet in it
There are many options to grow anaerobes, all depending on what ones, why you want them and how much biomass. 'Easy to grow' anaerobes can be cultivated on appropriate plates in an anaerobic jar. More strict things require more rigorous medium preparation. Sounds like anaerobic broths, dispensed under appropriate head space, into sealed bottles (serum bottles would do the trick). If the latter sounds like what you are after, let me know and I can refer you to many papers
What I'm looking for is an effective and an easy-to-do way to provide anaerobic conditions in the case of growing microorganisms on MRS agar slopes. These slopes are meant to provide bacteria for growing them later in broth, in order to isolate the DNA for RAPD and RFLP. I'd appreciate it very much if you were kind enough to refer me to the papers you mentioned.
You may employ an easy and cheap method. Take an empty can of metal of biscuits, (after you have eaten the biscuits) with a piece of paraffin candle placed inside. After seeding Petri dishes, place the plates inside, light the wick of the candle and close the lid of the can. The candle will go out when the oxygen is depleted and already we have anaerobic environment. It's simple, easy and, above all, very cheap.
In my lab there's a nitrogen tank and I purge with that before sealing the anaerobic space. I liked the candle idea too.
You can also get those plastic bags with a zip lock, put your cultures in, and extract all the air with a syringe before sealing the zip, like here
http://nanologix.com/images/white-agar-534.jpg
Hi
you can also use a glass bottle filled with media and tightly packed with a rubber stopper (cap). after inoculation just eject air from the empty space on the top of medium, and add some nitrogen to fill the the vacuum again.
Hope this will work for you
Many years ago, Robert Hungate first described methods for culturing microbes from the rumen. Over the years, there have been many modifications, but the basic principles apply. Degas your medium, dispense under O2 -free gas (often N2, but occasionally a gas mix with CO2), use reducing agents as necessary (from things like thioglycollate to sulfide), stopper you vessel with impervious tops or stoppers. You have to make a call on the reducing agents. For your interest, check out the work of these microbiologists of the past. I also suggest checking out Hungate and Macy (73) who describe the roll tube method. Yoy dont need to do exactly that method, but they tell you why you do certain things. BCK Patel published a simple method to dispense anaerobic medium (1985) and I used that to make medium to grow everything from clostridia to sulfate reducers (strict anaerobes)
Thank you all for quick response! I definitely feel more comfortable, now that I have some solutions in case of any difficulties.
Gavin Rees has given very useful suggestions. Thioglyclolate and cysteine are reducing agents that may be added to adapt a medium to anaerobic conditions. rezazurin indicator will turn pink if oxygen penetrates the environment but will remain colorless if the environment is anaerobic
There are many options, depending on your resources and anaerobic requirements. I work with strict anaerobes, but the concepts apply to facultative anaerobes.
-A rigorous method is given here: Hungate, R. E., & Macy, J. (1973). The roll-tube method for cultivation of strict anaerobes. Bulletins of the Ecological Research Committee, 123-126.
-A more recent paper for liquid media prep: Wolfe, R. S., & Metcalf, W. W. (2010). A vacuum-vortex technique for preparation of anoxic solutions or liquid culture media in small volumes for cultivating methanogens or other strict anaerobes. Anaerobe, 16(3), 216-219.
Sounds like you want simple, however, proper anaerobic culturing requires some work. These papers should give you a start and provide background on what to look out for. Good luck!
The Hungate method listed above works very well for maintaining strictly anaerobic conditions. I can send you my group's modifications of this method. The only "specialized" equipment that you would need is either Hungate tubes or serum bottles, butyl rubber septa, and crimping equipment in addition to access to either nitrogen or nitrogen/CO2.
Happy cultivating!
I'll have to agree with Gavin, Samuel and Sean on this one - highly recommend cultivating anaerobes in serum bottles according to the Hungate method.
I also agree with these methods the above researchers mentioned.
Besides putting the flasks or plates in an anaerobic chamber, you can also cultivated anaerobic bacteria in serum bottles covered with butyl rubber septa and crimped with aluminum cap. Of course, the bottles should be filled with either N2 or N2/CO2 for some time, to substitute the remaining O2 and sterilized before inoculation.
Good luck!
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