Methodological insights into fecal microbiota transplantation: Dissecting key approaches for success

Table 3 Stool collection, processing and storage

Processing step	Description	Common variations/methods
Stool collection	Freshly passed stool is collected from a healthy, screened donor in a sterile container[17]	Anaerobic conditions: Certain protocols recommend oxygen-free collection and processing methods to preserve strict anaerobes[14,18,19]
		Pre-collection diet: Some studies suggest a high-fiber diet prior to donation to enhance microbial diversity[20,21]
Stool quantity	The amount of stool collected varies based on the protocol	Typically, 50-60 g of stool added to 250-300 mL of diluent[5,17]. A minimum of 25 g of feces is recommended for lower GI delivery, and 12.5 g for upper GI delivery[14,15]
Diluents/buffer solutions	Stool is diluted with a buffered solution to create a homogeneous slurry, and to a consistency that can be injected through the biopsy channel of scope	Commonly used diluents[5,22]:
		Normal saline (0.9% NaCl)-Most commonly used
		Sterile water (less preferred due to hypotonicity)
		Phosphate buffered saline
		Non-bacteriostatic saline (used to avoid antimicrobial preservatives)
		Milk or milk-based solutions (rarely)
Homogenization	Stool sample is mixed with the diluent to create a uniform suspension, ensuring even distribution of microbiota and removal of large particulate matter before filtration	Done by manual stirring with sterile spatula; laboratory blender. Performed under aerobic or anaerobic conditions[18,19]
Filtration	Large particles (undigested fiber, debris) are removed	Using[5]:
		Gauze filtration
		Stainless steel sieve (250-500 μm)
		Sterile mesh filters
Centrifugation	Further purification or concentration of microbiota; retain microbial rich supernatant	Low (3000-4000 × g), medium (5000-6000 × g) or high (up to 10000 × g) speed centrifugation; relative centrifugal force = 6000 × g for 15 minutes[23]. May be followed by re-suspension in diluent
Washing (optional step)	Component of microfiltration plus centrifugation, called washed microbiota transplantation	Integration of multiple filtration and washing steps, often performed using automated purification systems[16]; automated systems (e.g., GenFMTer) reduces host cells, debris, endotoxins (one-hour FMT protocol)[16]
Storage and handling	Stool preparation is handled under strict sterile conditions to prevent contamination. Can be used fresh or stored	Fresh use: Stored at 4 °C and used within 6 hours
		Frozen use: Stored at -80 °C with glycerol (10%) for long-term use (up to 2 years)

GI: Gastrointestinal; FMT: Fecal microbiota transplantation.