Microbiological findings in cryocontainers and ultra-low temperature freezers and the source of contamination

Cryogenic liquid nitrogen (LN₂) storage containers are widely used for the long-term preservation of cells and tissues, yet they may represent an overlooked source of microbial contamination. Effective contamination control within cryogenic systems and the surrounding cryobank environment is essential, particularly when supporting clinical cell or tissue transplantation. Contamination risk increases in the absence of adequate air filtration and is further amplified by high humidity condensation leading to ice accumulation, frequent container opening, insufficient packaging of stored materials, and improper handling during loading and unloading procedures. This study evaluated microbial contamination in LN₂ vapor-phase containers (T ≤ -150 °C) using standard cultivation methods and MALDI-TOF mass spectrometry, with emphasis on the potential spread of microorganisms via LN₂ vapor. Contamination levels in LN₂ containers were compared with those in mechanical low-temperature freezers (T ≤ -80 °C), alongside microbial profiles detected on protective gloves used during sample manipulation. Microbial growth (2-10² CFU/mL), based on analysis of the melting ice, was detected in 5 of 8 LN₂ containers. Detected organisms included potential pathogens such as Delftia tsuruhatensis, Acinetobacter spp., Pseudomonas spp., and Staphylococcus aureus, as well as non-pathogenic environmental species. Wall swabs revealed additional bacteria, while all lid swabs remained culture-negative. Mechanical freezers exhibited low-level contamination (1-14 CFU/mL), with microbial profiles overlapping those detected in LN₂ systems. Matching organisms found on gloves indicate operator handling as a major contamination route. Microbial presence in frost and meltwater further suggests possible transmission via LN₂ vapor. These findings underscore the need for stricter hygiene measures, improved decontamination procedures, and routine microbiological monitoring in cryogenic storage environments.