Apr 29, 2022
An optimized protocol for flow virometry (FVM)-based detection and enumeration of T4 bacteriophage
This protocol is a draft, published without a DOI.
- 1Mel Johnson [UC Davis], Heather Bischel [UC Davis]
Protocol Citation: Hannah Safford 2022. An optimized protocol for flow virometry (FVM)-based detection and enumeration of T4 bacteriophage. protocols.io https://protocols.io/view/an-optimized-protocol-for-flow-virometry-fvm-based-b8bprsmn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: April 27, 2022
Last Modified: April 29, 2022
Protocol Integer ID: 61519
Keywords: flow cytometry, flow virometry, T4, bacteriophage
Abstract
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.
Protocol materials
Amicon Ultra-15 Centrifugal Filter UnitMerck Millipore (EMD Millipore)Catalog #UFC910024
Step 2.10
SYBR Green IThermo Fisher ScientificCatalog #S7563
Step 3.1
SYBR GoldThermo ScientificCatalog #S-11494
Step 3.1
dimethylsulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)
Step 3.1
Preparation of purified, high-titer T4 stock
Preparation of purified, high-titer T4 stock
2d 18h 45m
2d 18h 45m
Propagate T4 and host from freeze-dried specimens.
Note
The bacteriophage T4 and its host Escherichia coli (Migula) Castellani and Chalmers can be ordered from the American Type Culture Collection (ATCC; product nos. 11303-B4 and 11303, respectively) and propagated from freeze-dried specimens according to ATCC instructions (attached).
11303-B4 Product Sheet - Escherichia coli bacteriophage T4.pdf 11303 Product Sheet - Escherichia coli (Migula) Castellani and Chalmers.pdf 2d
Aliquot propagated specimens and store at -80 °C
Note
200 µL volumes are appropriate for downstream applications described in this protocol. Host should be stored as a solution containing 25 % volume glyglycerol; phage should be stored untreated.
Use propagated T4 and host to prepare purified, high-titer T4 stock for FVM.
Note
The protocol explicated here is based on Bonilla et al. (2016) [see citation below].
CITATION
Incubate propagated host Overnight in approximately 25 mL of nutrient broth (ATCC Medium 129.pdf)) at 80 rpm, 37°C .
ATCC Medium 129.pdf)) at 80 rpm, 37°C .12h
Spike approximately 20 mL of the overnight host culture into 250 mL of nutrient broth (again ATCC Medium 129). Incubate at 80 rpm, 37°C, 01:00:00 .
1h
Spike approximately 200 µL of propagated T4 into the broth solution from step 2.2. Incubate at 80 rpm, 37°C, 05:00:00 .
Note
Omit T4 spike if prepraring negative control.
Note
Following incubation, mixture may be stored overnight at 4 °C .
5h
Aliquot mixture from Step 2.3 into 5–6 sterile conical tubes. Centrifuge tubes 3200 rcf, 00:20:00 to sediment bacterial debris.
20m
Using a serological pipette and being careful not to disturb the pellet, reserve each supernatant.
Syringe-filter the supernatants at 0.2 µm into fresh sterile conical tubes.
Lyse remaining bacteria by adding 10 % volume chloroform to each tube and incubating for 00:10:00 at Room temperature .
Safety information
Chloroform is toxic if swallowed or inhaled. Use caution and appropriate technique (including glass or otherwise solvent-resistant pipette tips) while handling.
10m
Centrifuge tubes 3200 rcf, 00:05:00 to collect chloroform.
5m
Using a serological pipette and being careful to avoid the chloroform interface, reserve and combine the supernatants.
Transfer 15 mL of supernatant at a time to the upper reservoir of a 100 kDaAmicon Ultra-15 Centrifugal Filter UnitEmd MilliporeCatalog #UFC910024 . Concentrate by centrifuging 3200 rcf, 00:05:00 . Reserve the retentate.
Note
The same Amicon device may be reused to concentrate additional volumes of supernatant. When the filter begins to clog, transfer the contents of the upper reservoir into a fresh device and continue.
5m
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, 00:05:00 and reserve the retentate.
5m
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 .
Note
Before using the purified, high-titer stock for downstream applications, we recommend checking the titer through plate-based culturing, qPCR, or similar.
Prepare sample for FVM analysis.
Prepare working stock of SYBR dye. SYBR Green I (SYBR Green IThermo Fisher ScientificCatalog #S7563() is recommended if the species of interest are exclusively DNA viruses; SYBR Gold (SYBR GoldThermo ScientificCatalog #S-11494 ) is recommended otherwise. Prepare working stocks by diluting the concentrated stock in dimethylsulfoxide (DMSO)Sigma Aldrichs 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.
Note
The SYBR fluorescent dyes are photosensitive; work quickly when preparing the working stocks to minimize exposure to light.
Dilute sample in TE buffer to achieve an FCM analysis rate of about 102–103 events/sec.
Note
The dilution factor will depend on (i) the estimated titer of the sample, and (ii) the instrument-specific analysis settings used.
Add glutaraladehyde to the sample at a final concentration of 0.5 % volume and incubate at 4 °C for 00:15:00 .
Note
Diluted, glutaraldehyde-treated samples may be stored at -80 °C prior to analysis. Avoid repeated freeze-thaw cycles to avoid compromising sample integrity.
15m
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 00:01:00 .
Note
Sample should be analyzed as soon as possible after the staining is complete.
1m
Analyze sample via FVM
Analyze sample via FVM
Note
The staining protocol explicated above is designed to be compatible with sample analysis via FVM on a wide variety of flow cytometers. Below, we provide settings for sample analysis on a NovoCyte 2070V coupled with a NovoSampler Pro autosampler (Agilent). In adapting these settings to other instrumentation, the key components to retain are (1) a slow flow rate, and (2) rinses in between each sample to thoroughly flush the fluidics.
Settings for sample analysis.
Note
Collect data using the 488 nm (blue) laser as well as the FSC, SSC, and FITC (530 ± 30 nm) lasers. Analyze a 10-mL volume of each sample was using the lowest instrument flowrate (5 mL/min) and a FITC = 800 threshold. The analysis may be performed with or without instrument mixing of the sample prior to analysis.
Settings for rinses.
Note
Flush fluidics in between each sample by running 150 mL of 1x NovoClean solution (Agilent) followed by 150 mL of MQ water through the sample intake port (SIP) at the highest instrument flowrate (120 mL/min).