# Elusive Recurrent Bacterial Contamination in a Diatom Culture: A Case Study
## 4 Discussion  
The collected data show that two supposedly axenic _Fragilaria radians_ strains had a very similar history. They were originally successfully axenized and kept in this state for years, only to be eventually contaminated by _Sphingomonas_ sp. The strains were routinely checked for bacterial contamination using DAPI staining, and some contaminated samples were discarded, but _Sphingomonas_ sp. was not detected (presumably due to extremely low counts). At some point the bacteria died out in both strains, although no additional axenization was undertaken. If it was low counts that helped the bacterium remain undetected, they could also have prevented it from being transmitted along with algal cells during passage. It is also possible that _Sphingomonas_ sp. never contaminated the culture itself, instead inhabiting some element of lab equipment. Despite the fact that the vials and the culture medium were sterilized for the cultivation of diatoms, we admit the possibility of bacterial contamination due to poor-quality sterilization of the equipment or contaminated reagents used in DNA extraction and library preparation. Since no observation of either _Sphingomonas_ sp. strain was ever made directly, any detailed discussion of how the algal culture was contaminated or decontaminated would be highly speculative. Yet, we suspect that large-volume growth pipeline may have been a main culprit. Up to 15 L of water for medium preparation were sterilized simultaneously in a high-pressure steam sterilizer at 121 \({}^{\circ}\)C, potentially creating insufficiently lethal  
Figure 4: Maximum likelihood phylogenetic tree built on the positions of 16S rRNA alignment limited to amplicon boundaries. All strains associated with _F. radians_ are shown in blue. All clades except those containing our strains of interest are collapsed (the size of collapsed clades is not to scale); complete tree is available in Supplementary Figure S1A. conditions for some _Sphingomonas_ strains. We also cannot exclude contamination during medium preparation, diatom inoculation or long-term culture growth (normally approx. 30 days). All these stages were carried in a room that was clean, but not sterile. The high-volume growth may also have caused us to miss bacterial contamination in microscopy-based controls. Diatom cells were sedimented from the entire culturing volume on 5 \(\upmu\)m filters, and approx. 50 \(\upmu\)L of diatom biomass were sampled for the microscopy. If bacterial cells form aggregates in the culture, they may be unevenly distributed in this concentrated diatom biomass and thus not present in a small volume used for microscopy. For these two reasons, we have decided to abandon high-volume arsenic diatom culturing, instead growing cells in multiple 1000 \(\upmu\)L flasks. Medium preparation and inoculation are carried out in sterile laminar boxes, and microscopy controls are made before and after biomass sedimentation. The details of these strains' contamination are not very interesting by themselves (except for revising _F. radians_ genome assembly, which is beyond the scope of this paper), but this case demonstrates multiple ways in which contamination goes undetected. Although care was taken to keep the strains axenic, bacterial contamination was noticed neither in culture, nor in read libraries, nor in published genomic data. Thus, it would be useful to describe how these failures occurred and how they could've been avoided. As mentioned above, light microscopy detected no bacterial cells during the entire lifetime of both strains, although each of them has probably contained bacteria for years. Since we do not have any hard evidence of why this happened, we cannot provide reliable guidelines for preventing such occurrences in the future. We can only suggest that microscopy should not be relied upon without corroborating evidence.