What is the easiest (and cheapest) way to culture rodent faeces to get as much various gut bacteria as possible? I need to collect the supernatant from this culture for further analysis, so it has to be done in suspension and not on a plate. I understand that most of these bacterias require anaerobic conditions, so what would be the most "low-tech" way to grow them in a lab that has never dealt with bacteria before and doesn't have special chambers for anaerobs.
Thanks in advance
Yulia
You can buy small anaerobic jars and gas packs that fit inside a normal aerobic incubator if you want anaerobic.
There is also always cooked meat medium which is about as low tech as it gets and should allow you to grow aerobes and anaerobes together in liquid without any special kit:
http://www.oxoid.com/UK/blue/prod_detail/prod_detail.asp?pr=CM0081&org=3
https://catalog.hardydiagnostics.com/cp_prod/content/hugo/AnaeroGRO-ChoppedMeatMedia.html
http://jb.asm.org/content/4/2/149.full.pdf+html
If you are just culturing the whole faeces, even in a non selective medium you will find that some of the bacteria grow faster than others and will be overrepresented. Some bacteria may not grow at all.
What is it you are trying to achieve?
The least 'high tech' way of growing bacteria in microaerob or anaerobe athmosphere is to use a plastic bucket with at fitted lid (preferrable air-tight) and add sufficient number of gas-packs from BBL or other companies producing theese. For liquid cultures you willo need to put the bucket on a labmixer to have the liquid circulating. However, I think you will get into trouble when culturing a wide spectra of bacteria from faeces as the bacteria that are most happy with the environment you provided for them pretty fast will outnumber all the rest of the bacteria in your sample. And problably about 70% of the gut bacteria are unaculturable anyway. Anyway, good luck!
Check answers to similar question
https://www.researchgate.net/post/Is_it_possible_to_culture_Bacteroides_fragilis_outside_of_an_anaerobic_box
Fluid thioglycollate media could be used. Just it needed to be filled in tubes more than a half amount of the tube. In that case anaerobic bacteria grow at the bottom and aerobic near the top.
The easiest way is blood agar but you need anerobic atmosphaere (using jars or big glass and into give candel in flame , then strictly cover and without flame will be there anaerobic atmosphare, it is easy and it works, if you nee or to wish to have also anaerobic bacteria.
Thanks for your suggestions! Let me explain what I want to achieve. We want to measure production of Biopterins by gut flora. For that reason we think of culturing mixed population of fecal bacteria overnight in the best conditions possible and then harvest the broth. What would be the most non selective medium that will allow growth of such a mixed population?
Hi Yulia,
Modestas Ruzauskas has mentioned Thioglycolate (TG) broth - this is what I would use for what you want to do. Note that some commerical TG broth comes with Resazurin, an oxygen indicator, not sure if this would affect your analysis, but it might be worth buying broth without rezasurin.
An old (1970s) way to ensure that your TG borth doesn't have excess oxygen was to immerse a red hot nail into the broth just prior to inoculation!
Hope that this might of some help.
You can also use Trypticase soy broth.There you can have Gram-positives and Gram-negatives but not sure if anerobes when resazurin will be not there. So maybe two batches; for common Gram-positives and negatives one and for strictly anerobes second one with resazurin. be also strict with pH of media, of course.
Hi again,
Andrea has made the very good suggestion that TSB is a fantastic broth for growing aerobes, and non-fastdious anareobes will also probably manage to grow up in it too. The rezasurin is simply an indicator of oxygen content, so not necessary for anaerobic growth. TG enhances anaerobic growth because it has a small amount of agar in it (a semi-solid media), so oxygen does not diffuse to the bottom of the tube. You could make TG from basic powdered constituents and leave out the rezasurin (note that the rezasurin might not adverselty affect your results at all, I just don't know what effect it will have).
You could try growing in TG with anerobic atmosphere (keep the lids a little loose to allow gas to penetrate in) - all organisms in the gut are already grwoing anaerobically, you might find you get better recovery of fastidious anaerobes this way. Hope this helps.
Dear Yulia,
All the colleagues have given exceptionally interesting and useful answers above. If i may give an extra advise, a technique you can use to ensure anaerobic conditions and have a rich in cellular biomass cultured broth , is to fabricate liquid media such as TSB or Muller Hiton in glass tubes and flush the tubes and the flask where the medium is dispersed from using a sterile syringe twith nitrogen gas in an effort to remove the oxygen completely, a detailed description of the technique can be found in Demain and Davies Manual of Industrial microbiology, you will need to seal the tubes with butyl rubber stoppers and wheaton (alumina) seals, the photos here might help you. http://figshare.com/article/Development_of_a_novel_growth_strategy_for_bacteriocin_production_and_extraction_from_selected_strains_of_lactobacilli/681658.
You can autoclave the tubes normally. This technique however will not help you to identify which bacteria exactly are growing in your sample , however you will be able to monitor the growth using a spectrophotometer and then calculate doubling time and growth rate.
To get a more targeted answer to your needs it would be useful to describe what you want to achieve from such an experiment.
Best wishes,
Myrto
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