This study outlines a method for simulating the anaerobic environment of the human colon to analyze gut microbial metabolism. By using a nutrient-rich medium and fecal samples, researchers can monitor the production of metabolites and short-chain fatty acids.
Begin with a tube placed inside an anaerobic chamber that contains a nutrient-rich medium and a prehydrated fermentable fiber.
The oxygen-deprived conditions inside the tube mimic the anaerobic environment of the human colon.
Next, add a diluted, homogenized fecal sample containing gut microbes to the tube and incubate it.
Regularly invert the tube to mix the microbes with the fiber.
During incubation, the microbes anaerobically metabolize and ferment the fiber, producing short-chain fatty acids and other metabolites, lowering the pH of the medium.
Collect aliquots from the fermenting mixture at regular intervals to monitor metabolite production and check their pH.
Centrifuge the aliquots at low temperatures to separate the microbial biomass into a pellet.
Transfer the supernatant containing the metabolites and short-chain fatty acids into a fresh tube.
Freeze both the microbial biomass and the supernatant in liquid nitrogen.
Finally, store the samples at a low temperature for further analysis.
Keep the inoculated tubes at 37 degrees Celsius inside the anaerobic chamber, and invert gently once every hour to resuspend the fibers and inoculum. After 3, 6, 9, or 24 hours, take out the proper amount of fermented media, and centrifuge the samples at 14,000 times g for 10 minutes at four degrees Celsius.
Immediately freeze the supernatant and pellet in liquid nitrogen, then after snap freezing, store the supernatant for short-chain fatty acids analysis, and pellet for microbiome analysis, at minus 80 degrees Celsius.
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