# Elusive Recurrent Bacterial Contamination in a Diatom Culture: A Case Study
## 3 Results
Insufficient sensitivity of light microscopy with DAPI staining was already shown by the fact that bacterial contamination went undetected in 2011-2012, while DNA-based methods are thought to produce false negatives (i.e., detect no contamination when it is present) only in rare cases [29; 30]. Three DNA samples were isolated from strain Ax BK280 using frozen cells from March 2017 and fresh culture produced in November 2020 and February 2021. Complete list of taxonomic assignments for generated OTUs is available in Supplementary Table S1. However, the essential result is that the samples from 2020 and 2021 do not contain any abundant bacteria at all. The reads that were identified as non-organelle bacteria are scattered among numerous singletons and small phylotypes with single-digit read counts. In our opinion, these represent noise (for comparison, rates of 0.02-0.06% of library for the bacteria known to be absent from sample were previously documented for MiSeq 16S rRNA amplicon sequencing [31]), and the absence of any single major source of bacterial reads implies that the diatom culture is free from bacterial contamination. In contrast, the sample from 2017 contains _Sphingomonas_ as a single dominant phylotype (besides plastids), confirming that this strain was also contaminated by the same bacterium as strain G9, or at least its relative. Thus, a series of 2011-2012 454 libraries of strain G9, as well as strain Ax BK280 in 2017, contain some _Sphingomonas_ DNA. The bacterium itself was never observed, despite using DAPI staining to monitor culture axenicity. Microphotographs of cells from corresponding cultures are shown in Figure 3.  
To find out whether the same bacterial strain was present in 2011-2012 and 2017, we have built ML phylogenetic trees including 4 OTU consensus sequences, 16S rRNA fragment extracted from genomic assembly, and _Sphingomonas_ sp. strain baik7s previously co-isolated with F. radians [25]. Trees were built both using the complete alignment (Supplementary Figure S1A), and only using positions corresponding to V3-V4 16S rRNA fragment boundaries sequenced from 2017 samples (Figure 4, Supplementary Figure S1B). Both trees show that two contaminants represent different _Splingomonas_ species. In the tree built on the positions limited to the amplicon boundaries (shown in simplified form in Figure 4), three _Splingomonas_ groups are scattered over the tree with multiple reference clades between them (all OTU consensi are sister to each other). In contrast, the tree built on complete alignment recovers contaminant from 2011-2012 as sister to strain baik7s, but these two leaves are shown as basal relative to the rest of the tree, and the former has a very long branch. Thus, we interpret this result as a long-branch attraction artifact probably caused by low-quality alignment, and conclude that three separate _Splingomonas_ strains were colonizing _F. radians_ cultures at different times. Since most sequences in SILVA (as well as closest BLAST hits against NCBI nr) are environmental and lack taxonomic identification below genus, we do not make any claims regarding the precise taxonomy of both contaminants. Figure 3: The microphotographs of DAPI-stained _F. radians_ cells. (**A,B**) strain G9, February 2011; (**C**) strain G9, July 2012; (**D**) strain G9, October 2012; (**E,F**) strain Ax BK280, 2017. Blue: DAPI specifically binding to DNA and nonspecifically binding to polysaccharides; red: chloroplast autofluorescence; yellow: DAPI nonspecifically binding to polyphosphates. Green fluorescence is produced by a mixture of DAPI-stained polysaccharides and polyphosphates.