Feb 02, 2025
Standardized Preparation of Fecal Microbiota Transplantation Products for Companion Animals
Peer-reviewed method
- Nina K. Randolph1,
- Hannah Klein1,
- Jenessa A. Winston1
- 1The Ohio State University College of Veterinary Medicine
- PLOS ONE Lab ProtocolsTech. support email: plosone@plos.org
Protocol Citation: Nina K. Randolph, Hannah Klein, Jenessa A. Winston 2025. Standardized Preparation of Fecal Microbiota Transplantation Products for Companion Animals. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl493njgo5/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
Step-by-step protocols for standardized fecal microbiota transplantation (FMT) preparation for dogs and cats. These protocols are utilized by the Companion Animal Fecal Bank at the Ohio State University College of Veterinary Medicine.
Created: July 17, 2024
Last Modified: February 02, 2025
Protocol Integer ID: 103589
Keywords: fecal microbiota transplant, fecal transplant, Dogs, Cats, microbiome therapeutics, companion animals
Funders Acknowledgements:
IHS Foundation
Abstract
Step-by-step protocols for standardized fecal microbiota transplantation (FMT) preparation for dogs and cats, including:
Protocol 1: Standardized FMT Preparation for Double Centrifuged Fecal Slurry
Protocol 2: Encapsulation of Fecal Slurry for Oral Capsular FMT Administration
Protocol 3: Standardized FMT Preparation for Lyophilized FMT Products
Protocol 4: Fecal Lyophilization and Encapsulation of Lyophilized Fecal Slurry for Oral Capsular FMT
Protocol 5: Standardized FMT Preparation for Fecal Slurry without Centrifugation
Protocol 1: Standardized FMT Preparation for Double Centrifuged Fecal Slurry
Protocol 1: Standardized FMT Preparation for Double Centrifuged Fecal Slurry
Standardized FMT Preparation for Double Centrifuged Fecal Slurry
Personal protective equipment (PPE) must be worn at all times while handling feces. Minimum PPE includes a lab coat, goggles, and gloves. A face mask is also recommended to protect the nose and mouth from any possible splash of biohazardous materials while processing feces.
For dogs, the owner collects feces immediately following defecation using a clean plastic bag. Feline owners are instructed to collect naturally voided feces from the litter box within 12 hours of defecation using a clean plastic bag.
The owner labels the plastic bag and stores feces in a standard refrigerator (4°C) within 15 minutes of
defecation (for dogs) or immediately upon collection (for cats). Owners store feces at 4°C for no more than 24 hours prior to delivery to a BSL-2 laboratory.
Owners should deliver refrigerated feces on ice to a BSL-2 laboratory for processing. Feces should be weighed (grams) and processing should be started immediately upon arrival to laboratory (within 24 hours of original defecation).
Note: If processing feline feces, use a metal spatula to remove litter material prior to weighing feces.
Combine all feces from a single donor into a sterile round bottom StomacherTM 400 Classic Standard bagTM (SewardTM, round bottom StomacherTM bag for Model 400, Fisher Scientific, catalog # 14-285-284). Seal the bag using a standard StomacherTM bag clip and place the sealed bag on ice. Gently hand-knead the bag until feces is homogenized.
Note: If additional testing (i.e. pathogen testing, 16S rRNA amplicon sequencing, Dysbiosis Index) of raw feces is desired, use a sterile spatula to transfer 1-gram aliquots of homogenized pooled feces into sterile
cryovials. Store filled cryovials at -80°C until future use.
Add sterile 0.9% saline to the StomacherTM bag with homogenized feces at a ratio of 1:4 feces to sterile saline (4x weight of feces in grams = mL of saline to add to feces).
Example Calculation: If processing 100 g feces
100 x 4 = 400 mL saline added to feces
Carefully hand-knead the saline and pooled feces mixture for 3-5 minutes or until completely
homogenized.
Aliquot 45 mLs of the fecal saline mixture into sterile 50 mL conical tubes until all of the
mixture has been dispensed.
Within the conical tubes, centrifuge the fecal saline mixture at 400g (or RCF) for 30 mins at 4°C (Centrifuge 5810 R, 15 amp version, Eppendorf, Enfield CT, USA). Balance the centrifuge by loading the centrifuge rotor symmetrically and ensuring opposite tubes are the same weight. Store filled conical tubes awaiting centrifugation at 4°C.
After centrifugation, using a transfer or serological pipette, remove supernatant to a 1:0.75 ratio of
fecal sediment to supernatant. Avoid disturbing the sediment layer while decanting.
Example:
45 mL fecal saline mixture with 10 mL of sediment, remove 17.5 mL of supernatant to leave 17.5 mL of solution with sediment in the tube.
Vortex the remaining supernatant with the fecal sediment until the mixture is completely homogenized. Pour the resulting fecal solution into the integrated strainer element of a sterile StomacherTM Circulator Strainer bag (SewardTM Strainer bag for Stomacher TM 400, Fisher Scientific, Catalog # 14-285-22).
Using a fresh clean glove, gently detach the integrated strainer element from the plastic component of the
StomacherTM bag. Clip or hold the end of the strainer element to ensure no spillage of fecal material. Gently squeeze the strainer element until liquid is no longer produced and the retained material feels chalky and dry.
Aliquot 45 mL of the filtered fecal suspension into sterile 50 mL conical tubes and centrifuge the
fecal material at 400g for 15 mins at 4°C.
After centrifugation, using a transfer or serological pipette, remove supernatant to a 1:0.75 ratio of
sediment to supernatant. Avoid disturbing the sediment layer while decanting.
Vortex the fecal sediment with the remaining supernatant until homogenized.
If planning to freeze FMT slurry (at -80°C), add sterile glycerol (Millipore Sigma, Sigma-Aldrich, Catalog # G9012) to the fecal slurry, to a final concentration of 10% glycerol.
Example Calculation: If fecal slurry volume = 40 mL
40 mL x 0.11 = 4.4 mL glycerol
Add 4.4 mL glycerol to 40 mL fecal slurry to reach a final concentration of 10% glycerol
Vortex the final FMT mixture thoroughly.
Note: This final fecal slurry can be: (1) administered immediately via enema, endoscopy, or nasogastric tube; (2) encapsulated and frozen at -80°C for later use as frozen FMT capsules (see Protocol 2); or (3) stored at -80°C for up to 6 months later use as a fecal slurry.
If stored at -80°C as fecal slurry, rapidly thaw FMT slurry in a 37°C water bath for 30-45 minutes and keep warm until administration. Once thawed, FMT product should be utilized within 12 hours.
Protocol 2: Encapsulation of Fecal Slurry for Oral Capsular FMT Administration
Protocol 2: Encapsulation of Fecal Slurry for Oral Capsular FMT Administration
Encapsulation of Fecal Slurry for Oral Capsular FMT Administration
Maintain capsules over dry ice prior to encapsulation to ensure the fecal slurry is kept cold.
Gently separate the two parts of the capsule, then use a repeater micropipette to dispense the fecal slurry into the open capsule.
For dogs: Size 0 gelatin or delayed-release capsules are recommended.
For cats: Size 4 gelatin or delayed-release capsules are recommended.
Over encapsulate the gelatin or delayed-release capsules with a slightly larger commercially available gelatin capsule (i.e. Size 00 for dogs; Size 3 for cats). Continue to maintain the filled capsules on dry ice until ready for long term storage.
Note: This step is important to prevent erosion and degradation of the interior capsule and ensure the integrity of the capsule for oral administration.
Place the filled fecal slurry capsules in a sealed airtight container and store at -80°C until ready for use.
For oral administration, remove from -80°C and place in -18°C to -20°C freezer. Frozen capsules can be easily administered in canned food or manually per os while still frozen. Capsules that reach room temperature will quickly disintegrate, thus compromising capsule integrity.
Protocol 3: Standardized FMT Preparation for Lyophilized FMT Products
Protocol 3: Standardized FMT Preparation for Lyophilized FMT Products
Standardized FMT Preparation for Lyophilized FMT Products
For dogs, the owner collects feces immediately following defecation using a clean plastic bag. Feline owners are instructed to collect naturally voided feces from the litter box within 12 hours of defecation using a clean plastic bag.
Personal protective equipment (PPE) must be worn at all times while handling feces. Minimum PPE includes a lab coat, goggles, and gloves. A face mask is also recommended to protect the nose and mouth from any possible splash of biohazardous materials while processing.
The owner labels the plastic bag and stores feces in a standard refrigerator (4°C) within 15 minutes of defecation (for dogs) or immediately upon collection (for cats). Owners store feces at 4°C for no more than 24 hours prior to delivery to a BSL-2 laboratory.
Owners should deliver refrigerated feces on ice to a BSL-2 laboratory for processing. Feces should be weighed (grams) and processing should be started immediately upon arrival to laboratory (within 24 hours of defecation).
Note: If processing feline feces, use a metal spatula to remove litter material prior to weighing feces.
Maltodextrin (Millipore Sigma, Sigma-Aldrich, Catalog # 419680) and D-(+)-trehalose dihydrate (Millipore Sigma, Sigma Aldrich, Catalog #90210) are dissolved in 0.9% sterile saline at 40°C to respective final concentrations of 18.75% and 6.25%. The solution is cooled on the benchtop to room temperature and filter-sterilized using a 0.22 mm PES membrane (Titan3TM PES Syringe Filters, Fisher Scientific, catalog # 42225-PS).
Example: If the desired final volume of solution is 100 mL
Add 18.75 g maltodextrin and 6.25 g trehalose into a sterile glass beaker. Then add sterile saline to achieve a total volume of 100 mL.
Combine all feces from a single donor into a sterile round bottom StomacherTM 400 Classic Standard bagTM (SewardTM, round bottom StomacherTM bag for Model 400, Fisher Scientific, catalog # 14-285-284). Seal the bag using a standard StomacherTM bag clip and place the sealed bag on ice. Gently hand-knead the bag until feces is homogenized.
Note: If additional testing (i.e. pathogen testing, 16S rRNA amplicon sequencing, Dysbiosis Index) of raw feces is desired, use a sterile spatula to transfer 1-gram aliquots of homogenized pooled feces into sterile cryovials. Store filled cryovials at -80°C until future use.
Add the maltodextrin and trehalose (M:D) solution to homogenized feces at a ratio of 1:4 feces to M:D.
Example: If processing 25 g feces
25 x 4 = 100 mL M:D solution added to feces
Carefully hand-knead the M:D solution and pooled feces mixture for 3-5 minutes or until completely homogenized.
Aliquot 45 mLs of the fecal mixture into sterile 50 mL conical tubes until all of the mixture has been dispensed.
Within the conical tubes, centrifuge the fecal M:D mixture at 400g (or RCF) for 30 mins at 4°C (Centrifuge 5810 R, 15 amp version, Eppendorf, Enfield CT, USA). Balance the centrifuge by loading the centrifuge rotor symmetrically and ensuring opposite tubes are the same weight. Store filled conical tubes awaiting centrifugation at 4°C.
After centrifugation, using a transfer or serological pipette, remove supernatant to a 1:0.75 ratio of fecal sediment to supernatant. Avoid disturbing the sediment layer while decanting.
Example for 45 mL fecal saline mixture with 10 mL of sediment:
Remove 17.5 mL of supernatant to leave 17.5 mL of solution within the sediment tube.
Vortex the remaining supernatant with the fecal sediment until the mixture is completely homogenized. Pour the resulting fecal solution into the integrated strainer element of a sterile StomacherTM Circulator Strainer bag (SewardTM Strainer bag for Stomacher TM 400, Fisher Scientific, Catalog # 14-285-22).
Using a fresh clean glove, gently detach the integrated strainer element from the plastic component of the StomacherTM bag. Clip or hold the end of the strainer element to ensure no spillage of fecal material. Gently squeeze the strainer element until liquid is no longer produced and the retained material feels chalky and dry.
Aliquot 45 mL of the filtered fecal suspension into sterile 50 mL conical tubes and centrifuge the fecal material at 400g for 15 mins at 4°C.
After centrifugation, using a transfer or serological pipette, remove supernatant to a 1:0.75 ratio of
sediment to supernatant. Avoid disturbing the sediment layer while decanting.
Vortex the fecal sediment with the remaining supernatant until homogenized, then immediately freeze at -80°C.
Note: FMT slurry must be frozen at -80°C for at least 48 hours prior to lyophilization.
Protocol 4: Fecal Lyophilization and Encapsulation of Lyophilized Fecal Slurry for Oral Capsular FMT
Protocol 4: Fecal Lyophilization and Encapsulation of Lyophilized Fecal Slurry for Oral Capsular FMT
Fecal Lyophilization and Encapsulation of Lyophilized Fecal Slurry for Oral Capsular FMT
Remove frozen fecal slurry from -80°C freezer, remove lids from conical tubes.
Wrap open end of conical with KimWipe, secure with rubber band, and place in lyophilizer (such as FreeZone Benchtop Freeze Dryer, Labconco, Kansas City, MO, USA).
Lyophilize at -87.5°C and 0.005 mBar for 48 hours.
After 48 hours, remove conical tubes from lyophilizer.
Homogenize and grind the dried FMT material using a clean mortar and pestle at room temperature.
Encapsulate homogenized FMT by filling capsules individually. For bulk lyophilized FMT capsule production, use capsule filling device (Profiller 3600 Capsule Filling System, Torpac Inc., Fairfield, NJ, USA) with the desired capsule type and size.
For dogs: Size 0 gelatin or delayed-release capsules are recommended.
For cats: Size 4 gelatin or delayed-release capsules are recommended.
Store lyophilized FMT capsules at 4°C (or room temperature) until ready for use.
Protocol 5: Standardized FMT Preparation for Fecal Slurry without Centrifugation
Protocol 5: Standardized FMT Preparation for Fecal Slurry without Centrifugation
Standardized FMT Preparation for Fecal Slurry without Centrifugation
Personal protective equipment (PPE) must be worn at all times while handling feces. Minimum PPE includes a lab coat, goggles, and gloves. A face mask is also recommended to protect the nose and mouth from any possible splash of biohazardous materials while processing.
For dogs, the owner collects feces immediately following defecation using a clean plastic bag. Feline owners are instructed to collect naturally voided feces from the litter box within 12 hours of defecation using a clean plastic bag.
The owner labels the plastic bag and stores feces in a standard refrigerator (4°C) within 15 minutes of defecation (for dogs) or immediately upon collection (for cats). Owners store feces at 4°C for no more than 24 hours prior to delivery to a BSL-2 laboratory.
Owners should deliver refrigerated feces on ice to a BSL-2 laboratory for processing. Feces should be weighed (grams) and processing should be started immediately upon arrival to laboratory (within 24 hours of original defecation).
Note: If processing feline feces, use a metal spatula to remove litter material prior to weighing feces.
Combine all feces from a single donor into a sterile round bottom StomacherTM 400 Classic
Standard bagTM (SewardTM, round bottom StomacherTM bag for Model 400, Fisher Scientific, catalog # 14-285-284). Seal the bag using a standard StomacherTM bag clip and place the sealed bag on ice. Gently hand-knead the bag until feces is homogenized.
Note: If additional testing (i.e. pathogen testing, 16S rRNA amplicon sequencing, Dysbiosis Index) of raw feces is desired, use a sterile spatula to transfer 1-gram aliquots of homogenized pooled feces into sterile cryovials. Store filled cryovials at -80°C until future use.
Add sterile 0.9% saline to the StomacherTM bag with homogenized feces at a ratio of 1:4 feces to sterile saline (4x weight of feces in grams = mL of saline to add to feces)
Example: If processing 100 g feces
100 x 4 = 400 mL saline added to feces
Pour the resulting fecal solution into the integrated strainer element of a sterile StomacherTM Circulator Strainer bag (SewardTM Strainer bag for Stomacher TM 400, Fisher Scientific, Catalog # 14-285-22).
Using a fresh clean glove, gently detach the integrated strainer element from the plastic component of the
StomacherTM bag. Clip or hold the end of the strainer element to ensure no spillage of fecal material. Gently squeeze the strainer element until liquid is no longer produced and the retained material feels chalky and dry.
If planning to freeze fecal slurry, add sterile glycerol (Millipore Sigma, Sigma-Aldrich, Catalog # G9012) to the fecal slurry, to a final concentration of 10% glycerol.
Example: Fecal slurry volume = 40 mL
40mL x 0.11 = 4.4 mL glycerol
Add 4.4 mL glycerol to 40 mL fecal slurry to reach a final concentration of 10% glycerol
Note: This final fecal slurry can be: (1) administered immediately via enema, endoscopy, or nasogastric tube; (2) encapsulated and frozen at -80°C for later use as frozen FMT capsules (see section 2.1.1); or (3) stored at -80°C for later use as a fecal slurry.
If stored at -80°C, rapidly thaw filtered fecal suspension in a 37°C water bath for 30-45 minutes and keep
warm until administration. Once thawed, FMT product should be utilized within 12 hours.