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Appl Microbiol Biotechnol. 2020 Jan;104(1):131-144. doi: 10.1007/s00253-019-10242-1. Epub 2019 Nov 28.

Factors determining microbial colonization of liquid nitrogen storage tanks used for archiving biological samples.

Author information

1
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, Braunschweig, Germany. Felizitas.Bajerski@dsmz.de.
2
Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Developmental Genetics, München, Germany.
3
Institute for Multiphase Processes, Leibniz University Hannover, Hannover, Germany.
4
Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Seeland, OT Gatersleben, Germany.
5
Leibniz Institute for Zoo and Wildlife Research (Leibniz-IZW), Berlin, Germany.
6
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Schmallenberg, Germany.
7
BioKryo GmbH, Sulzbach, Germany.
8
German Cancer Research Centre, Heidelberg, Germany.
9
Institute of Physiology and Pathophysiology, University of Heidelberg, Heidelberg, Germany.
10
Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Inhoffenstraße 7B, Braunschweig, Germany.
11
Microbiology, Braunschweig University of Technology, Braunschweig, Germany.

Abstract

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.

KEYWORDS:

Amplicon sequencing; Biobanking; Cryobank; Cryopreservation; Microbial contamination; Risk/quality management; Safe storage

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