How should fecal culture samples be stored to maintain bacterial viability?

Fecal Culture Sample Storage for Bacterial Viability

For optimal bacterial viability in fecal culture samples, refrigerate at 4°C (±2°C) and process within 6 hours of collection; if longer storage is required, freeze at -80°C with 10% glycerol as a cryoprotectant, avoiding repeated freeze-thaw cycles. 1

Immediate Short-Term Storage (≤6 Hours)

• Refrigeration at 4°C is the preferred method for samples that will be processed within 6 hours, as this maintains bacterial viability without significant alteration in microbial composition 1
• Samples should be transported to the laboratory as soon as possible after defaecation to ensure manipulation and storage occurs within this 6-hour window 1
• Refrigeration at 4°C causes no significant alteration in fecal microbiota diversity or composition when samples are stored for up to 72 hours 2
• For samples that cannot be immediately refrigerated, they should not be exposed to temperatures above 20°C if processing will exceed 24 hours 1

Extended Storage (>24 Hours to 2 Years)

Freezing Protocol

• Freeze samples at -80°C for long-term storage, which is the gold standard for maintaining bacterial community structure 1
• Add a cryoprotectant (glycerol) to a final concentration of 10% before freezing to enhance bacterial survival and maintain viability 1
• Samples should be frozen within 2 days of collection to minimize changes in microbial community structure 1
• Fecal suspensions can be stored for up to 2 years at -80°C, though preferably use within 1 year as microbial viability may decline after 9 months 1

Critical Pitfall: Freeze-Thaw Cycles

• Avoid repeated freeze-thaw cycles, which are more deleterious to bacteria than duration of cryoconservation 1
• Freeze-thaw cycles reduce bacterial viability to approximately 23% even with cryoprotectant use, and significantly impact microbial cell integrity 1
• Aliquot fresh stool samples before initial freezing to facilitate subsequent analyses without requiring multiple freeze-thaw cycles 1
• Products exposed to temperature excursions that might affect quality must be discarded 1

Processing Considerations for Viable Bacteria

• Process fecal samples in an anaerobic chamber when the goal is to transfer viable bacteria, as most colonic microorganisms are strict anaerobes and oxygen exposure is detrimental to their survival 1
• Homogenize the fecal slurry in autoclaved reduced PBS and glycerol supplemented with L-cysteine hydrochloride to support maximal bacterial recovery 1
• A minimum quantity of 25 g of feces is recommended for lower gastrointestinal applications and 12.5 g for upper gastrointestinal use 1

Thawing Protocol

• On the day of use, thaw frozen fecal suspensions in a warm (37°C) water bath or at room temperature 1
• Infuse or process thawed samples within 6 hours from thawing to maintain bacterial viability 1

Pathogen-Specific Considerations

For specific bacterial enteropathogens in diagnostic culture:

• Shiga toxin-producing E. coli (STEC) and Salmonella maintain excellent viability (≥97.5% recovery) for 7 days with or without transport media 3
• Campylobacter, Shigella, and Yersinia show better recovery with modified Cary-Blair transport medium (82.9%, 85.7%, and 93.8% respectively) compared to storage without transport media 3
• Transport media may not be required for STEC or Salmonella but should be considered for other enteric pathogens 3

Storage Temperature Monitoring

• Storage facilities must use -80°C freezers with connected alarm notification systems to ensure appropriate temperature maintenance 1
• Temperature range and duration of any out-of-range periods must be recorded and controlled 1
• Samples must be stored in biosafety level 2 facilities with standard operating procedures for safe handling of human material 1