An In Vitro Batch-culture Model to Estimate the Effects of Interventional Regimens on Human Fecal Microbiota

Considering the emerging role of gut microbiome in various human diseases, several gut microbiome modulators like probiotics, prebiotics, diets and drugs can help in ameliorating gut microbiome-associated health ailments. However no quick and affordable screening system are available to predict the effects of such gut microbiome modulators, but this system is simple enough to predict how particular interventions can impact gut microbiome that ultimately can impact the host health. This protocol is simple, affordable and practical for laboratories investigating the gut microbiome modulators and their effects on microbiota and host health.

Maintenance of aseptic and anaerobic conditions is crucial for this experiment. Also the fecal specimen should be used afresh, immediately upon collection. Otherwise if the experiment is planned later, the sample should be stored at minus 80 immediately upon collection, without any exposure to ambient temperature and air.

Handling accuracy during aliquot prepping and pipetting is also important for maintaining accuracy and reproducibility. It will take less than 12 hours in the daytime. We can build a culture overnight if they need the samples in 24 hours.

Then, followed by the short-chain fatty acids and the microbiome determination. This process will be demonstrated by two great uni students, Shokouh Ahmadi and Rabina Mainali from my lab. To prepare fermentation media under a fume hood, in a reagent bottle on magnet mixture, mix solution A, solution B, trace mineral solution, water soluble vitamin solution, folate biotin solution, riboflavin solution, hemin solution, short-chain fatty acid mix, and resazurin.

Then add into the bottle yeast extract, sodium carbonate, cysteine hydrochloride monohydrate, and trypticase. Add 296.1 milliliters of distilled water, adjust the pH with one normal hydrochloride or sodium hydroxide to ensure it is around 7.0. Transfer the beaker to an aseptic workstation, and use a vacuum filter with pore size at 0.22 micrometers to filter the media for sterilizing.

To prepare one liter anaerobic dilution solution, dissolve five grams of sodium chloride, two grams of glucose, and 0.3 grams of cysteine hydrochloride monohydrate in the ionized water. Autoclave the reagent bottle with cap at 121.1 degrees Celsius for 15 minutes, and store it inside the anaerobic chamber. Before the start of the fermentation experiment, keep all the materials, solutions and tools needed for the fermentation experiment inside the anaerobic chamber for at least 48 hours, to ensure that any residue oxygen is removed.

At least 24 hours before the fermentation experiment, weigh 300 milligrams of inulin, and transfer it to a 50 milliliter tube, which is then placed inside the anaerobic chamber. Add 26 milliliters of sterilized fermentation media into the tube, and vortex to mix. Allow hydration of fiber for approximately 24 hours.

The day of the fermentation experiment, prepare fecal inoculum. First, weigh five grams of fresh fecal sample in a 50 milliliter conical tube. Add anaerobic dilution solution to the tube to reach a final volume of 50 milliliters.

Vortex for 15 minutes, or until completely homogenized. 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 three, six, nine 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. This protocol is used to demonstrate the effect of a specific prebiotic. The feces of healthy human subjects, following treatment with inulin, showed relatively stable pH for nine hours, while dropped dramatically after 24 hours.

The inoculation of inulin significantly increased the levels of short-chain fatty acids and lactate in inulin-treated fecal specimen is compared to non-treated fecal microbiota culture. Principal coordinate analysis shows different microbiota composition to the weighted and unweighted UniFrac measures of beta diversity between inulin treated and untreated samples. Indices of alpha diversity versus phylogenetic diversity through PD whole tree, species richness, operational taxonomic units, species evenness, as well as relative abundance of major phyla and genera, all present different gut microbiota signatures between inulin treated and untreated samples.

Taxonomic cladogram, derived from linear discriminant effect size analysis of 16S sequences, represents the differentially abundant taxa between different groups of samples. Follow all the steps carefully. Most important thing is to maintain strict aseptic and anaerobic conditions.

The supernatant and from the experiment can be used to treat Short-chain fatty acids level changes as a result of the fermentation based of fibers. Additionally, the in the pellet can be used for analyzing the changes in gut microbiome composition. Some chemicals used for making the culture media are potentially hazardous.

Please the MSDS sheet and take precautions accordingly.