TITLE: An optimized protocol for flow virometry (FVM)-based detection and enumeration of T4 bacteriophage
AUTHORS: Hannah   Safford

[DESCRIPTION]
Flow virometry can support advanced water treatment and reuse by delivering near real-time information about viral water quality. Realizing the full potential of this technique, requires rigorous sample-preparation protocols as well as objective, automated methods for expediting and improving FVM data analysis. Below, we present an optimized sample-preparation protocol for FVM-based detection and enumeration of T4 bacteriophage, an environmentally relevant viral surrogate. The protocol, which was rigorously optimized using a fractional factorial experimental design, blends and improves on existing protocols developed using a traditional “pipeline”-style optimization approach. As use of FVM for viral water-quality assessment expands, we recommend that this protocol be used to validate instrument performance prior to and alongside application of FVM on environmental samples. Widespread adoption of a consistent, optimized analytical approach will engender confidence in FVM data and will facilitate cross-laboratory comparisons.




[STEPS]
SECTION: Preparation of purified, high-titer T4 stock
1. Propagate T4 and host from freeze-dried specimens.




 
    


SECTION: Preparation of purified, high-titer T4 stock
2. Use propagated T4 and host to prepare purified, high-titer T4 stock for FVM.
 

SECTION: Preparation of purified, high-titer T4 stock
1.1. Aliquot propagated specimens and store at -80 °C 
 


SECTION: Preparation of purified, high-titer T4 stock
2.1. Incubate propagated host    in approximately 25 mL  of nutrient broth ()) at   .

SECTION: Preparation of purified, high-titer T4 stock
2.2. Spike approximately 20 mL of the overnight host culture into 250 mL of nutrient broth (again ATCC Medium 129). Incubate at  .

SECTION: Preparation of purified, high-titer T4 stock
2.3. Spike approximately 200 µL of propagated T4 into the broth solution from step 2.2. Incubate at  .
 

 

SECTION: Preparation of purified, high-titer T4 stock
2.4. Aliquot mixture from Step 2.3 into 5–6 sterile conical tubes. Centrifuge tubes 3200 rcf, 20 min to sediment bacterial debris.

SECTION: Preparation of purified, high-titer T4 stock
2.5. Using a serological pipette and being careful not to disturb the pellet, reserve each supernatant. 
SECTION: Preparation of purified, high-titer T4 stock
2.6. Syringe-filter the supernatants at   into fresh sterile conical tubes.

SECTION: Preparation of purified, high-titer T4 stock
2.7. Lyse remaining bacteria by adding 10 % volume chloroform to each tube and incubating for 10 min at Room temperature. 
 


SECTION: Preparation of purified, high-titer T4 stock
2.8. Centrifuge tubes 3200 rcf, 5 min to collect chloroform.

SECTION: Preparation of purified, high-titer T4 stock
2.9. Using a serological pipette and being careful to avoid the chloroform interface, reserve and combine the supernatants.
SECTION: Preparation of purified, high-titer T4 stock
2.10. Transfer 15 mL  of supernatant at a time to the upper reservoir of a 100 kDa . Concentrate by centrifuging 3200 rcf, 5 min. Reserve the retentate.
 



SECTION: Preparation of purified, high-titer T4 stock
2.11. Perform the wash step. Add 15 mL of Tris-EDTA (TE) buffer [1x] into the upper reservoir of the Amicon device containing the final retentate. Centrifuge 3200 rcf, 5 min and reserve the retentate.

SECTION: Preparation of purified, high-titer T4 stock
2.12. Dilute retentate 100x in TE buffer to obtain working stocks, aliquot (100 µL volumes are appropriate for downstream applications described in this protocol), and store at -80 °C. 
 


3. Prepare sample for FVM analysis.
3.2. Dilute sample in TE buffer to achieve an FCM analysis rate of about 102–103 events/sec.

 


3.1. Prepare working stock of SYBR dye. SYBR Green I (() is recommended if the species of interest are exclusively DNA viruses; SYBR Gold ( ) is recommended otherwise. Prepare working stocks by diluting the concentrated stock in s such that the final concentration of stain once added to the sample is 5 x 10-5 the sample volume.

Working stocks may be stored in aliquots at -20 °C  and thawed in the dark immediately prior to use.
 



3.3. Add  glutaraladehyde to the sample at a final concentration of 0.5 % volume and incubate at 4 °C  for 15 min.
 


3.4. Stain the sample. Add either SYBR Green I or SYBR Gold (depending on whether the species of interest include both DNA and RNA viruses) to the glutaraldehyde-treated sample at 5 x 10-5 sample volume and incubate in the dark at 50 °C for at least 1 min .
 


SECTION: Analyze sample via FVM
4. 
 

SECTION: Analyze sample via FVM
4.1. Settings for sample analysis.
 

SECTION: Analyze sample via FVM
4.2. Settings for rinses.