Table 1 Factors and levels included in the fractional factorial experimental design for staining optimization.
Factor | Level 1 | Level 2 | Rationale |
|---|---|---|---|
Stain type (which nucleic-acid stain was used?) | SYBR Green I | SYBR Gold | Both stains are widely used for applications of FVM to microorganisms. Huang et al. (2015) deemed SYBR Gold more effective for FVM-based analysis of waterborne viruses, while Brussaard (2004) reported better results with SYBR Green |
Diluent (what was the sample diluted in?) | Milli-Q (MQ) water | Tris-EDTA (TE) buffer | Both SYBR Green I and SYBR Gold are pH-sensitive, so using a buffer instead of MQ water as a diluent may improve results |
Dye concentration (what was the concentration of dye in the final sample?) | 5 × 10–5 times sample volume | 1 × 10–4 times sample volume | Level 1 concentration used by Brussaard (2004); Level 2 concentration used by Huang et al. (2015) |
Staining temperature (what temperature was the sample stained at?) | 25 °C | 50 °C | Huang et al. (2015) stained at room temperature (~25 °C) while Brussaard (2004) stained at 80 °C. Multiple studies have found that an elevated temperature can promote the staining reaction, but an 80 °C staining temperature may be unrealistic for applied water-treatment and -reuse scenarios. An intermediate temperature (50 °C) was selected as the “high” staining temperature for comparison with room-temperature staining |
Staining time (how long was the sample stained for?) | 1 min | 15 min | Huang et al. stained for 15 min while Brussaard (2004) stained for 10 min. Our preliminary results (not reported) suggested that a prolonged staining time may not be necessary to achieve good results. If a short staining time is workable, it would increase the potential of FVM as a real-time technique for water-quality monitoring |
Glutaraldehyde (was the sample treated with glutaraldehyde prior to staining?) | No | Yes, glutaraldehyde added at a final concentration of 0.5% | Both Huang et al. (2015) and Brussaard (2004) found that adding glutaraldehyde significantly improved the detectability of waterborne viruses by FVM. However, glutaraldehyde addition also closes off certain pathways for validating FVM results (e.g., using a flow cytometric cell sorter to separate target populations and then using culture-based methods to verify the identity of the target). This factor was assessed to determine whether glutaraldehyde addition is essential for our samples |
