Flow cytometric quantification of viruses in activated sludge

Area of Science:

• Environmental microbiology
• Wastewater treatment technologies
• Virology

Background:

• Viruses are crucial to activated sludge microbial dynamics but difficult to quantify.
• Accurate virus quantification is essential for understanding microbial ecology in wastewater systems.
• Current methods for virus enumeration are often time-consuming and labor-intensive.

Purpose of the Study:

• To optimize and validate a flow cytometry protocol for determining virus abundance in activated sludge.
• To assess the feasibility of using flow cytometry for routine virus quantification in wastewater treatment plants.
• To compare flow cytometry counts with traditional methods like transmission electron microscopy.

Main Methods:

• Optimization of sample preparation involving Tween 80, sodium pyrophosphate, and Tris-EDTA.
• Staining of viruses with SYBR Green II for detection via flow cytometry.
• Validation of the protocol using transmission electron microscopy for comparative analysis.

Main Results:

• An optimized flow cytometry protocol was established, yielding the highest virus counts.
• Average viral concentrations in 25 activated sludge plants were determined to be 2.35 × 10⁹ mL⁻¹.
• Flow cytometry counts showed a strong correlation with transmission electron microscopy counts (r² = 0.77).

Conclusions:

• Flow cytometry provides a rapid, reproducible, and efficient method for quantifying viruses in activated sludge.
• This technique is suitable for routine monitoring and large-scale studies of viral populations in wastewater systems.
• The optimized protocol facilitates research into phage-host dynamics within activated sludge environments.