TABLE 1.

16S and 23S rRNA-targeted oligonucleotide probes and hybridization conditions

Probe	Sequence (5′ to 3′)	FA (%)a	Specificity	Reference
EUB338	GCTGCCTCCCGTAGGAGT	—b	Most Bacteria	1
EUB338 II	GCAGCCACCCGTAGGTGT	—b	Planctomycetales	7
EUB338 III	GCTGCCACCCGTAGGTGT	—b	Verrucomicrobiales	7
ALF968	GGTAAGGTTCTGCGCGTT	20	α-Proteobacteria, except Rickettsiales	22
BET42ac	GCCTTCCCACTTCGTTT	35	β-Proteobacteria	19
GAM42ad	GCCTTCCCACATCGTTT	35	γ-Proteobacteria	19
GNSB-941	AAACCACACGCTCCGCT	35	Phylum Chloroflexi	9
CFX1223	CCATTGTAGCGTGTGTGTMG	35	Phylum Chloroflexi	4
CF319a/b	TGGTCCGTRTCTCAGTAC	35	CF cluster of the Bacteroidetes	20
Nso190	CGATCCCCTGCTTTTCTCC	35e	Ammonia oxidizers	21
Ntspa662	GGAATTCCGCGCTCCTCT	35	Genus Nitrospira	8
Comp Ntspa662f	GGAATTCCGCTCTCCTCT		Competitor for Ntspa662	8
NIT3	CCTGTGCTCCATGCTCCG	40	Nitrobacter spp.	43
Comp NIT3g	CCTGTGCTCCAGGCTCCG		Competitor for NIT3	43

^aFA, formamide concentration in the hybridization buffer.

^bThe probe can be used at any formamide concentration.

^cUnlabeled probe GAM42a was used as a competitor to enhance specificity.

^dUnlabeled probe BET42a was used as a competitor to enhance specificity.

^eAlthough the original description of the use of Nso190 indicated that 55% formamide should be used, we experimentally confirmed that 35% formamide was sufficient to discriminate ammonia-oxidizing β-Proteobacteria in our autotrophic nitrifying biofilm, which is consistent with other descriptions of the use of Nso190 at much lower stringencies than those originally described (32).

^fUnlabeled probe Ntspa662 was used as a competitor to enhance specificity.

^gUnlabeled probe NIT3 was used as a competitor to enhance specificity.