Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples
Creators: name: Bajerski, F.
name: Bürger, A.
name: Glasmacher, B.
name: Keller, E.R.J.
name: Müller, K.
name: Mühldorfer, K.
name: Nagel, M.
name: Rüdel, H.
name: Müller, T.
name: Schenkel, J.
name: Overmann, J.
Publisher: name: Heidelberg : Springer Verlag
Year: 2020
Subjects: subject: Amplicon sequencing
subject: Biobanking
subject: Cryobank
subject: Cryopreservation
subject: Microbial contamination
subject: Risk/quality management
subject: Safe storage
subject: Bacteria
subject: Cytology
subject: Debris
subject: Diagnosis
subject: Fungi
subject: Genes
subject: Ice
subject: Liquefied gases
subject: Liquid nitrogen
subject: Medical applications
subject: Quality management
subject: RNA
subject: Storage (materials)
subject: Storage management
subject: Amplicons
subject: Cryo-preservation
subject: Tanks (containers)
subject: bacterial RNA
subject: liquid nitrogen
subject: RNA 16S
subject: Acinetobacter
subject: amplicon
subject: Article
subject: bacterial cell
subject: bacterial colonization
subject: bacterium detection
subject: Bacteroides
subject: cell count
subject: controlled study
subject: Elizabethkingia
subject: epifluorescence microscopy
subject: gene dosage
subject: high throughput sequencing
subject: Janthinobacterium
subject: marker gene
subject: Methylobacterium
subject: microbial community
subject: Mycoplasma
subject: nonhuman
subject: Pseudomonas
subject: species richness
subject: Staphylococcus
subject: storage
subject: Streptococcus
subject: Bacteria (microorganisms)
subject: Dewey Decimal Classification::500 | Naturwissenschaften::570 | Biowissenschaften, Biologie

description: The availability of bioresources is a precondition for life science research, medical applications, and diagnostics, but requires a dedicated quality management to guarantee reliable and safe storage. Anecdotal reports of bacterial isolates and sample contamination indicate that organisms may persist in liquid nitrogen (LN) storage tanks. To evaluate the safety status of cryocollections, we systematically screened organisms in the LN phase and in ice layers covering inner surfaces of storage tanks maintained in different biobanking facilities. We applied a culture-independent approach combining cell detection by epifluorescence microscopy with the amplification of group-specific marker genes and high-throughput sequencing of bacterial ribosomal genes. In the LN phase, neither cells nor bacterial 16S rRNA gene copy numbers were detectable (detection limit, 102 cells per ml, 103 gene copies per ml). In several cases, small numbers of bacteria of up to 104 cells per ml and up to 106 gene copies per ml, as well as Mycoplasma, or fungi were detected in the ice phase formed underneath the lids or accumulated at the bottom. The bacteria most likely originated from the stored materials themselves (Elizabethingia, Janthibacterium), the technical environment (Pseudomonas, Acinetobacter, Methylobacterium), or the human microbiome (Bacteroides, Streptococcus, Staphylococcus). In single cases, bacteria, Mycoplasma, fungi, and human cells were detected in the debris at the bottom of the storage tanks. In conclusion, the limited microbial load of the ice phase and in the debris of storage tanks can be effectively avoided by minimizing ice formation and by employing hermetically sealed sample containers.