AIMS: Clostridium difficile is a common nosocomial pathogen and as such diagnostic and research
methods may necessitate storage of faecal specimens for long periods, followed by subsequent
re-examination. This study investigated the effects of storage conditions upon the viability of this organism
and its toxin.
METHODS: Three genotypically distinct strains of C difficile (two clinical isolates including the UK epidemic
strain, and an environmental isolate) were grown anaerobically at 37°C for 72 hours in a pool
of five faecal emulsions. Aliquots of each emulsion were stored at either -20°C (frozen) or 4°C (refrigerated).
Emulsions were assayed for viable cells, spores, and cytotoxin titre before storage and at days
1, 3, 5, 7, 14, 28, and 56. An aliquot of each emulsion was also removed, assayed, and replaced in
storage at each time point to investigate the effects of multiple freezing/refrigeration/thawing .
RESULTS: Neither storage temperature nor multiple cycles of refrigeration/freezing and thawing
adversely affected the viability of C difficilevegetative cells or spores. Single and multiple exposures of
samples to 4°C had little effect upon the C difficile toxin titre. Toxin titres of multiply frozen and thawed
faeces became significantly lower than for refrigerated faeces (p < 0.01) by day 5 of the experiment
in two of the three strains, and in all strains by day 28. Toxin titres of singly frozen faeces became significantly
lower than for refrigerated faeces (p < 0.01) by day 56 of the experiment in two of the three
strains.
CONCLUSION: Storage temperature and multiple cycles of freezing (refrigeration)/thawing had minimal
effects upon the viability of C difficile or its spores. Storage at 4°C has no discernible effect on C difficile
cytotoxin. However, storage at -20°C has a detrimental effect upon C difficile cytotoxin, and multiple
cycles of freezing and thawing may further adversely effect toxin titres