Aug 01, 2025
Fecal Carmine Red Protocol
Forked from Fecal Carmine Red Protocol
- Adam Hamilton1,
- Ian Krout1,
- Tim Sampson1
- 1Emory University
Protocol Citation: Adam Hamilton, Ian Krout, Tim Sampson 2025. Fecal Carmine Red Protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vz9yq8gx1/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 01, 2025
Last Modified: August 01, 2025
Protocol Integer ID: 223850
Keywords: ASAPCRN, GI Transit, Gut, Motility, fecal carmine red protocol, bright red fecal pellet, large intestinal transit, whole gut transit time, bright red carmine dye, assay, volume of bright red carmine dye, pellet expulsion, time the dye, dye, gastric emptying, time between gavage, mice, gi tract, length of the gi tract, fecal carmine red protocol this assay
Funders Acknowledgements:
Aligning Science Across Parkinson’s
Grant ID: ASAP-020495
Aligning Science Across Parkinson’s
Grant ID: ASAP-000375
Heritage Medical Research Institute
Abstract
This assay is used to determine whole gut transit time. Mice are given an oral gavage containing a known volume of bright red carmine dye. Mice are placed into empty cages and are then observed in 15-minute intervals until they produce a bright red fecal pellet. The time between gavage and pellet expulsion is the time the dye takes to travel down the length of the GI tract, which is influenced by gastric emptying, and small and large intestinal transit.
Materials
Carmine Red Solution
6% w/v carmine red (Sigma cat #C1022),
0.5% w/v methylcellulose (Sigma cat #M7027) in water.
Sterile Cages
Gavage needles
Troubleshooting
Pre-protocol
Prepare Carmine Red Solution (see materials).
Autoclave Carmine Red solution, if sterility is required. (i.e. microbiome analysis)
Shake well or stir on low heat to homogenize
Shake/vortex each right before gavage.
Day of Assay
3h
Orally gavage mice with 100 µL carmine red solution. Generally, soft-tipped, disposable (Instec FTP-20-30, or similar) feeding needles, attached to a 1 mL slip-tip syringe.
Equipment
Polypropylene Feeding Tubes for Rodents
NAME
Gavage Needle
TYPE
Instech Labs
BRAND
FTP-20-30
SKU
LINK
Record time of gavage for each mouse.
Following gavage, place animals back into home cage with cage mates, food, and water.
Allow animals to rest with food, water, in home cage for 03:00:00
3h
Separate individual mice into single-housed, clean (sterile, if needed) cages, with no bedding, which will interfere with the observation of a red fecal pellet. Cover with cage top.
Assay can be set up with or without food as long as all comparable runs are done the same way. If
adding food, place 1-2 food pellets into a portion cup with water to create moistened chow (sterile if needed). Moistened chow reduces the risk of mice spilling water in the cage. Glass dishes can be used as cups to prevent flipping of the cups.
Every 00:30:00 , check cages for a bright red pellet. Can observe through sides (with pen light if needed) or by opening cage top. Placing the cages on a white sheet of paper, rather than a
black benchtop, will also make visualization easier. If unclear, a pellet can be taken out of the cage and smeared on a napkin to determine if dye is present. If food was provided, check in the portion cups for red pellets as well. Ensure that whichever method of observation is used, all animals are disrupted similarly (ie. all animals have their cages opened).
30m
Record time at which the first red pellet is observed for each mouse. For a normal, healthy adult mouse, this will be ~3-4hrs following gavage, but can range from 2-8+ hrs. Assay maximum time is set to 5 hours, but can be altered as needed, but can be altered as needed up to 10hrs.
To prevent stress associated with single-housing of mice, limit the single-housed portion of the assay to 6-8hrs, putting the maximum recordable transit time at 8-10hrs post-gavage.
Once a red pellet is observed, return that mouse to its home cage.
If the maximum allotted time is reached without production of a red pellet, return the mouse to their
home cage, and record their transit time as the maximum.