# Wastewater Treatment with Bacterial Representatives of the _Thiothrix_ Morphotype
## 3 Methods for Identification of _Thiothrix_ in Wastewater Treatment Plants  
The significant contribution of the _Thiothrix_ morphotype bacteria to the operation of wastewater treatment plants has long been known, leading to the development of methods for their identification using light microscopy (Figure 2). The application of these methods, along with detailed morphological characterization, led to the identification of filamentous bacteria in wastewater treatment plants that were assigned to the genus _Thiothrix_ and described as Type 021N, _Thiothrix_ I and _Thiothrix_ II [6,7]. Therefore, these names were retained in many subsequent articles written by non-taxonomists dealing with microorganisms in wastewater treatment plants [13; 14; 15; 16; 17; 18]. The original classification of these bacteria outside of the genus _Thiothrix_ was further confirmed by [PERSON] and [PERSON] [10], who proposed placing _Thiothrix flexilis_ in the genus _Thiothrix_ and _Thiothrix__disiformis_ and _Thiothrix__eikelboomii_ in the genus _Thiolinea_. To monitor the population dynamics of _Thiothrix_ in wastewater treatment plants, species-specific fluorescence in situ hybridization (FISH) probes are often used, in addition to light microscopy [13; 18; 26; 74; 75; 76; 77]. Current phylogenetic data suggest that some of the probes used could also be suitable for representatives of the genera _Thiolinea_ and _Thiofilm_[18; 75; 77]. Since the phylogenetic heterogeneity of _Thiothrix_ was long known, sets of different probes were developed to identify all known species of the genus known at that time. Their use showed that mixed populations of the _Thiothrix_ morphotype are often found in wastewater treatment plants, and bacteria now assigned to the genus _Thiolinea_ can also be found in these mixtures [31]. Figure 2: Progress of methods for identification of representatives of _Thiothrix_ morphotype from time perspective: light microscopy morphotype identification [6; 7]; DAPI staining to confirm bacterial morphotype [71] and identify Poly-P inclusion [47]; FISH for identification of _Thiothrix_, _Thiofilm_ and _Thiolinea_ genera and some _Thiothrix_ species [31; 72; 73]; PCR using _tilS_ and _rpoB_ probes [19] and high-throughput sequencing with _tilS_ profiling [19] for _Thiothrix_ species identification. The capabilities of the FISH method and the PCR method when using probes for 16 SRNA in both methods are current at the time of writing the review and are indicated for the taxonomic groups of _Thiothrix_, _Thiofilm_ and _Thiolinea_ revised in 2018 [10], with no consideration of the taxonomic status of these groups before 2018. * Not applicable for species identification of _Thiothrix_ Clade I, the composition of which is listed in Figure 1, at the date of the review’s preparation. Quantitative PCR has been used less frequently to study _Thiod thrix_ populations. For Type 021N, primers were developed and used in combination with Sybr Green to quantify these bacteria in laboratory tests [78]. Genus-specific primers were developed for _Thiod thrix_ 16S rRNA, which showed that, in the wastewater studied, the largest number of bacteria similar in morphotype to _Thiod thrix_ belonged to the species _Thiod thrix eikelboomii_ (= _Thiodinea eikelboomii_) [14]. [PERSON] et al. also showed that the ratio of _Thiodinea eikelboomii_ to the total number of bacteria (TH/TB) (%) was a better predictor of the secondary effluent volume and quality than the _Thiodinea eikelboomii_ abundance alone [14]. However, the rapid development of high-throughput sequencing technologies in recent years has led to the increasingly frequent detection of _Thiod thrix_ by community profiling against 16S rRNA genes [24; 52; 79; 80]. Moreover, representatives of the genera _Thiod thrix_[56; 81] and _Thiodinea_[11] were identified in activated sludge by metagenomic analysis. Although it is known that a mixture of several _Thiod thrix_ species can occur simultaneously in wastewater treatment plants, the identification of these species has so far been a challenge.