The main goal of this publication is to present a method for normalizing the microbiome of rodents. In this demonstration, the effect of environmental enrichment on the microbiome is assayed during colon tumor genesis. This protocol shows how normalizing the microbiome prior to an environmental enrichment intervention improves the experimental reproducibility.

We also show how to reduce animal stress and improve interactions using an enriched environment. Although we have focused on the effect of environmental enrichment in a colon tumor model, the method can easily be applied to better understand the microbiome's role in the etiology of disease. Set up the cages under sterile conditions working under a ventilated hood.

The controls for this experiment consist of conventional cages with bedding and lab tissue for nesting, but otherwise lack enrichment devices. To set up the pup rearing or environmentally enriched cages, connect two large autoclaved cages with a sterilized grommet-secured tunnel and include a sterilized platform in each cage to increase floor space. For EE, also provide sterilized running wheels, tunnels, igloos, huts, crawl balls, and nesting material.

Place both the enriched and the control cages in the same rack to provide equal ventilation. For breeding, combine one sire and two potential dams in normal cages. Every morning, check for vaginal plugs which indicate that the animal has mated during the night.

If a plug is not seen, resistance to vaginal probing can detect an internalized plug. If a female has a vaginal plug, transfer her to a large pup-rearing cage. Up to 12 pregnant dams can be housed in the environmentally enriched cages with platforms provided they are all expected to deliver during the same week.

When the pups are 21 to 28 days old, distribute the males and females by genotype into control environments or enriched environments. Make sure to keep an proportion of each genotype in each cage. Non-enriched cages can support up to five mice whereas enriched cages require at least 20 mice for social stimulation and can support up to 41 mice.

Start collecting stool one or two days before euthanizing the mice. Always collect at the same time of day. To collect stool from live animals, carefully scruff the animal over a clean cage.

Then collect the defecated stool using sterile forceps and transfer to a sterile microfuge tube. If an animal does not immediately defecate, place it into a clean cage and wait for the animal to defecate which can take up to an hour. To collect stool from a euthanized animal, dissect it to access the colon.

Then cut the colon just below the cecum and lay the colon out on a piece of filter paper. Then use forceps to lift the top of the colon tube and open the lumen using scissors. Cut longitudinally distal to proximal to splay open the colon lengthwise.

Then collect stool from the distal colon into a sterile microfuge tube using sterile forceps. Store the stool samples at minus 80 degrees Celsius. In addition to the stool, collect other tissue samples from the colon and small intestine, microsomes, adipose tissue, and so forth.

Utilize a commercial kit to isolate microbial DNA from the stool following a stool pathogen detection protocol. When doing so, transfer the samples from the minus 80 degree Celsius freezer on dry ice and weigh them. Next, set up a PCR that amplifies the microbial DNA as described in the text protocol.

Then clean up the reaction products using magnetic beads. For the clean up, first briefly centrifuge the amplicon PCR plate to pool the reaction products. Next, vortex the magnetic beads to evenly disperse them and transfer 20 microliter aliquots to each reaction well.

Mix the beads with the solution thoroughly by pipetting the entire volume slowly 10 times. Then let the beads settle for five minutes. Next, place the PCR plate on a magnetic stand and wait two minutes for the supernatants to clear following magnetic collection of beads.

Then remove and discard the supernatants. Next, with the plate still on the magnet, fill each well of beads with 200 microliters of fresh 80%ethanol and wait 30 seconds. Then carefully remove the supernatant.

Repeat this wash step once and let the beads air dry for 10 minutes. Now elute the reaction products from the beads by removing the plate from the magnetic stand and adding 52.5 microliters of Tris to each well. Completely mix the beads into solution by pipetting the entire volume slowly 10 times.

Then collect the beads on the magnetic stand as before and transfer 50 microliters of the supernatant to a clean PCR plate. Cover the plate with a clear adhesive and store it at minus 20 degrees Celsius for up to one week. Use the collected supernatant to perform an index PCR to attach bar codes to the adapter sequence.

Then purify the products and prepare the indexed library for sequencing. In mice, environmental enrichment is known to improve lifespan and tumor wound repair. To define the role of microbiota in this phenotype, breeding animals were integrated into the mouse colony for at least one month to normalize the microbiota.

Then they were bred and their progeny were raised in an enriched environment or a normal environment. The progeny were also tumor bearing or wild type. After 16 weeks, the animals were euthanized and stool samples were collected to isolate microbial DNA.

Next, PCR was used to amplify the 16S ribosomal subunit in the samples to survey the microbiome components. The reaction products were then identified using bar code indexing. Interestingly, enhanced environments do not improve biodiversity in wild type animals, but they vastly increase biodiversity in tumor-bearing animals.

This demonstrates the sensitivity of the method. This increase in biodiversity was attributed to an increased presence of the phylum Proteobacteria with significant increases in the classes alphaproteobacteria and betaproteobacteria and a decrease in pathogenic gammaproteobacteria. The largest increase in the betaproteobacteria class was in the genus Sutterella, a likely commensal involved in secreted immunoglobulin A degradation.

After watching this video, you should have a good understanding of how to alter husbandry practices to normalize the microbiome between experimental animals prior to commencing with your interventions of interest. While performing this procedure, remember that animals have established their territory within the environment. Because of this, changes to the environment can cause stress that will affect the animal's microbiome.

A sudden change in the animal's behavior is a physical sign of stress. Following this procedure, specific microbes can be isolated and further characterized to test the sufficiency of the identified microbe on disease progression.