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

F Bajerski,J Schenkel,B Glasmacher,A Bürger,K Mühldorfer,K Müller,H Rüdel,E R J Keller,T Müller,J Overmann,M Nagel

doi:10.1007/s00253-019-10242-1

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.

Highlights

The long-term storage of biomaterials is a precondition for modern life sciences, enabling follow-up scientific investigations, medical diagnostics, biotechnological applications, and the conservation of genetic resources and diversity (Overmann 2015; Overmann and Smith 2017; Schüngel et al 2014; Stock et al 2018)
Variance analysis of gene copy numbers between the different sample types showed that samples taken directly from the liquid nitrogen (LN) phase could not be distinguished from the negative controls (p = 0.158, ANOVA, Table S4), indicating that the microbial entry in the LN samples is below the detection limit determined by the negative controls and reference samples
Bacterial cells and 16S rRNA gene copy numbers were hardly detected in controls and LN samples, while cell numbers in ice or in debris samples significantly surpassed the values of LN samples and the determined detection limit

Summary

Introduction

The long-term storage of biomaterials (biobanking) is a precondition for modern life sciences, enabling follow-up scientific investigations, medical diagnostics, biotechnological applications, and the conservation of genetic resources and diversity (Overmann 2015; Overmann and Smith 2017; Schüngel et al 2014; Stock et al 2018). Specific cryopreservation protocols have been established for different organisms and cell types. Living biological material may be prepared for cryopreservation under both, sterile or unsterile conditions. The biological materials themselves as well as the storage facilities may contain additional, accompanying organisms. LN and other liquefied gases are commonly manufactured in so-called air separation units which separate the atmospheric gases at very low temperatures. During this process, the air is filtered and dried. Tests conducted by one manufacturer of liquefied gases using validated methods could not detect any pathogens (personal communication Dr Carsten Pilger, AIR LIQUIDE Medical GmbH)

Methods

Results

Conclusion