TABLE 2.
Oligonucleotide probes used for CSLM-FISH in this study, probe sequence, target sites, target organisms, and formamide concentration in the hybridization buffer
| Probe name | Labeling dye | Stringencya (% [vol/vol] formamide) | Target siteb (16S rRNA positions) | Target organism(s) | Reference(s) |
|---|---|---|---|---|---|
| PLA886 and competitor | FLUO/Cy5 | 35 | 886-904 | Most Pirellula, Planctomyces, Isosphaera, and Gemmata spp. | 33 |
| PLA46 | Cy5 | 25 | 46-63 | Gemmata, Isosphaera, Pirellula, and Planctomyces sp. and the Anammox branch (Brocadia and Kuenenia spp.) | 33, 38 |
| NSO190 | Cy3 | 35c | 190-208 | Ammonia-oxidizing β-Proteobacteria | 30 |
| AMX820 | Cy3 | 25 | 820-841 | Brocadia anammoxidans | 42 |
| KST1275 | Cy3 | 25 | 1275-1292 | Kuenenia stuttgartiensis | 38 |
a
The formamide concentration used during hybridization.
b
Escherichia coli numbering.
c
Although the original publication on the use of NSO190 indicated the use of 55% FA in FISH applications, we experimentally confirmed that at 35% half of the probe signal was removed with Nitrosomonas europaea as the test organism, consistent with other publications describing the use of NSO190 at much lower stringencies than originally published (4, 26).