Rapid and Sensitive Quantification of Anammox Bacteria by Flow Cytometric Analysis Based on Catalyzed Reporter Deposition Fluorescence In Situ Hybridization

The quantification of anammox bacteria is crucial to manipulation and management of anammox biosystems. In this study, we proposed a protocol specifically optimized for quantification of anammox bacteria abundance in anammox sludge samples using catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) and flow cytometry (FCM) in combination (Flow-CARD-FISH). We optimized the pretreatment procedures for FCM-compatibility, as well as the permeabilization, hybridization and staining protocols of the CARD-FISH. The developed method was compared with other methods for specific bacteria quantification (standard FISH, 16S rRNA sequencing and quantitative polymerase chain reaction). Anammox sludge samples could be disaggregated effectively by sonication (specific energy of 90 kJ·L^–1 with MLVSS of 3–5 g·L^–1) with the mixed ionic and nonionic dispersants Triton X-100 (5%) and sodium pyrophosphate (10 mM). Lysozyme treatment for permeabilizing bacterial cell walls and H₂O₂ incubation for completely quenching endogenous peroxidase of anammox sludges were essential to fluorescence enhancement and false positive signals control, respectively. Horseradish peroxidase molecules labeling at 20 °C for 12 h and the fluorescent tyramide labeling at 25 °C for 30 min with a fluorescent substrate concentration of 1:50 maintained the balance between increasing the signal and preventing nonspecific binding. Flow-CARD-FISH results showed that anammox bacteria absolute abundance in two different sludge samples were (2.31 ± 0.01) × 10⁷ and (1.20 ± 0.06) × 10⁷ cells·mL^–1, respectively, with the relative abundances of 36.7 ± 4.1% and 26.5 ± 3.7%, respectively, comparable with those of qPCR and 16S rRNA sequencing analysis. The enhanced fluorescence signals induced by CARD-FISH combined with the high quantitative fluorescence sensitivity of FCM provide a rapid and sensitive method that yields accurate quantification results that will be valuable in future studies of microbial community determination.