I have to cast some anaerobic liquid media to check for anaerobic digestion of some pollutants but I have no chance to work inside an anaerobic chamber. How shall I prepare the media without one? Is this possible?
Thanks in advance for answers.
With 1 mL or 500 uL of sterile mineral oil over the liquid culture you can create an microaerophilic environment. I use it when i wanto tuo prepare Salmonella to invade human cell culture
To degas the medium by autoclauving, to use press cap and completely fill the flask with growth medium. After several h from inoculation there is an anaerobic environment.
Anaerobic microbiology experiments can easily be conducted in any lab, without anaerobic chamber. historically people had been working with anaerobes before discovery of all modern equipment also. So, here are some tips from the giants....
For liquid media:
1. Use reducing salts in medium like Na thioglycollate (0.5%)
2. Use reducing aminoacids like L-cysteine (0.25%)
3. Before use, steam the liquid media for 15 to 20 mins and instantaneously cool.
4. Add a red hot iron filing into media just before inoculation.
5. sometimes sheer high salt cntent can create reduced atmosphere.... (Na2CO3, NaCl, KH2PO4- 0.5% each).
And ofcourse, Mineral oil overlay technique.... a classic but efficient technique....
Autoclaving at 121 degrees and purging nitrogen is a tried and tested method for creating anaerobic conditions. Solubility of oxygen at such elevated temperatures is almost zero. So if nitrogen purging is done immediately after autoclaving it ensures anaerobic environment.
Also for preserving anaerobic cultures, a layer of paraffin oil on top of the medium is a good technique.
Hope it helps you. Happy experimenting!!
Robertsons cooked meat broth, thioglycolate broth or any enriched broth with a reducing agent with oil overlayed can selectively isolate anaerobes. ref old mackey 12th edn
It's possible but it requires some extra supplies/effort. I don't know how 'anaerobic' you want your media (sometimes there are traces of oxygen left there but perhaps you don't mind so much?). There are two points to consider: 1/ getting rid of the dissolved oxygen in your liquid media (and headspace of container) and 2/ keeping it out (this is tougher). For the first point you can either a/ degas your media with N2 or N2/CO2 (if you use bicarbonate buffer) for 20-30 minutes (so just bubble out the media with a cannula directly going through the liquid or b/ boil your media under a constant stream of gas (a bit trickier). Keeping O2 out involves capping your glass containers with thick butyl stoppers and aluminium seals. Then all the tests have to be done using syringes/needles so that you dont take the stoppers off. I can send you some literature if you are interested? Good Luck!
The previous mentioned methods would of course work but here is a very simple technique which would not require much of the set up:
Put your liquid culture containing flask in an airtight container/jar and also keep a burning candle besides the culture in the jar. Once the oxygen is consumed the flame of the burning candle would be put off. This would create an anaeorbic chamber for cultivation.
@ Ketna: Is this really working with just a candle? Can I be sure that there is no oxygen inside any more just with the candle going off? Would be great if I could use this method because an airtight container is easy to find here and I could use one of these for longer storage. Thanks a lot.
Yes, It is the method of choice when you are working in constraints. Try it out, no harm.
You can use serum bottle for cultivate anaerobic bacteria. You prepare the medium transfer to serum bottle and flushed with nitrogen gas. Before you flushed nitrogen gas, adding rezasurin for indicate the dissolved oxygen in medium. Finally, closed the bottle with rubber stopper and aluminum cap. In your medium you added L-cysteine for decrease the oxygen dissolved in medium.
Is that mineral oil overlay to the broth culture efficient for grwoth of microaerobic culture? Secondly can we use that broth culture for further identification?
Traditional Robertson's cooked meat broth medium (RCM) over laid with oil. will be suitable for most anaerobic bacteria. liquid medium like the Thioglycollate broth can be used. Addition of reducing substances like the cysteine can increase anaerobic environment. and dont forget that even if you grow them in liquid finally you have to plate them to identify. Antibiotic containing medium can inhibit the growth of aerobic contaminants if any
thio media contain resazurin which is oxygen indicator right?
Reinforced Clostridia Medium (RCM) was prepared under anaerobic condition using Hungate method by sparging N2 gas to cultivate of anaerobic microbes. Addition of reducing substances like the cysteine can increase anaerobic environment. You adding rezasurin for indicate the dissolved oxygen in medium serum bottle techniques.
How can i transfer algal culture to anoxic media? coz i i do the oxygen will enter the vial
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