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

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.

Fig. 1

Liquid nitrogen (LN) storage tanks and sampling procedure of LN and ice. (a) Sampling of the LN phase using a reaction tube and grip tongs. (b) Sampling of the ice phase formed underneath the lids (and rim)

Fig. 2

Epifluorescence photomicrographs of SYBR Green I-stained samples and algae autoflourenscence. (a) bacterial cells detected in the negative control NC-B-2 containing 2 × 10² cells per ml, (b) bacterial cells detected in the ice sample B-4-2 containing 8 × 10³ cells per ml, (c) eukaryotic cells of sample G-20-1, confirmed by PCR with Line1 primer, filaments of Cyanobacteria in sample I-30-3 (d) autofluorescence of microbial cells (excitation 425 nm, emission 630 nm) and (e) overlay with phase contrast. Sample code, institute-identity number-replicate; Scale bar, 5 μm

Fig. 3

Correlation of gene copy numbers and cell counts. A clear separation of ice (circles) and LN (triangles) samples could be observed. The arrows indicate the gene copy numbers of the negative controls. A clear separation of ice (circles) and LN (triangles) samples could be observed. The negative control (diamonds, NC_B) with the highest cell number determined the threshold for the detection limit, illustrated by a vertical dashed line. The axes are log₁₀-scaled. The LN samples are in the range of the negative controls. The debris samples (squares) had the highest gene copy numbers and cell counts. The concentration is calculated per ml evaporated LN, thawed ice, air volume reaction tube (NC_B)

Fig. 4

Weighted UniFrac distances of bacterial sequence variants at species level. Shown are the distances grouped by the samples phase: debris, ice, liquid nitrogen (LN) and the negative controls (NC)

Fig. 5

Bacterial community structure shaped by environmental parameters determined by a constrained analysis of principal coordinates (CAP) of a selected data set. The NC samples and all samples with < 277 cells ml⁻¹ were excluded from the analysis. OTUs that do not appear more than 5 times in at least 1% of the samples were removed. The CAP was calculated based on weighted UniFrac distances. The parameter cells_ml x + condition + storage + copies + in_use + open + storage_device + material were used as constrained variables. Black colored shapes depict samples encoding for different institutes including the ID shown as numbers. Colored circles depict the selected taxa encoding for the 20 most abundant genera