Oct 22, 2024

Public workspaceHigh Throughput Flow Cytometry for Viral Enumeration of Aquatic Samples

This protocol is a draft, published without a DOI.
  • 1Department of Bioinformatics and Genomics, North Carolina Research Campus, The University of North Carolina at Charlotte, Kannapolis, North Carolina, USA;
  • 2Computational Intelligence to Predict Health and Environmental Risks (CIPHER), The University of North Carolina at Charlotte, Charlotte, North Carolina, USA;
  • 3Department of Marine Sciences and Geoscience, University of Connecticut, Storrs, Connecticut, USA
Madeline Bellanger
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Protocol Citation: Madeline Bellanger, Pieter Visscher, Richard Allen White III 2024. High Throughput Flow Cytometry for Viral Enumeration of Aquatic Samples. protocols.io https://protocols.io/view/high-throughput-flow-cytometry-for-viral-enumerati-dmtz46p6
Manuscript citation:

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: September 21, 2024
Last Modified: October 22, 2024
Protocol Integer ID: 108121
Keywords: viral enumeration, phage, flow cytometry, FCM, enumeration, aquatic
Funders Acknowledgements:
NASA Astrobiology Institute
Grant ID: NNH22ZDA001N-EXO
Abstract
Here, we describe a high throughput method in a 96-well plate format that provides a hands-free approach to viral enumeration. Our approach limits fake particles and has higher signal-to-noise ratios, all while limiting cross-sample contamination in a high throughput manner. In a standard run, 60 samples can be measured for VLPs within 2.25 hours, which is ~1 hour faster than the standard single tube approach and ~1.5 hours faster than epifluorescence microscopy (EFM).
Guidelines
This method is used to enumerate viruses in water samples collected from the environment using flow cytometry. See our protocols for EFM for microbialite viral enumeration, EPS viral enumeration, and soil viral enumeration!
Materials
Consumables
  • Whatman grade 1 qualitative filter paper
  • 0.65 µm PVDF Durapore membrane filter (hydrophilic)
  • 0.45 µm PVDF Durapore membrane filter (hydrophilic)
  • 0.22 µm PVDF Durapore membrane filter (hydrophilic)
  • 30 kDa MWCO Millipore centrifuge filters (of desired size)
  • 1.5 mL low protein binding, nuclease free microcentrifuge tubes
  • P1000 pipette tips
  • P200 pipette tips
  • P10 pipette tips
  • 96 well plate

Chemicals
  • 70% Ethanol
  • 25% EM grade glutaraldehyde
  • Chloroform
  • Benzonase
  • SYBR Gold nucleic acid stain
  • 10X TE
  • 10% Bleach
  • Molecular biology grade water

Equipment
  • P1000 pipette
  • P200 pipette
  • P10 pipette
  • Centrifuge capable of holding 50 ml conical tubes or wide neck bottles (62 mm diameter; 146 mm length, only if using Centricon-70 plus centrifuge filters)
  • Microcentrifuge
  • Vortexer
  • Heat block
  • Flow cytometer equipped with a well plate loader
Protocol materials
ReagentSYBR Gold Nucleic Acid Gel StainCatalog # S-11494
Step 26
ReagentChloroform
Step 13
ReagentBenzonase® NucleaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #E1014 SIGMA
Step 22
Safety warnings
  • Fixation with glutaraldehyde and chloroform treatment needs to be performed in a fume hood with a face shield and proper PPE.
  • Needles used to remove glutaraldehyde from serum vials need to be disposed of in a sharps container within the fume hood. Never replace the cap on a needle. Once the needle has been used, immediately deposit it into the sharps container.
Calculations and Well Plate Planning
Calculations and Well Plate Planning
Plan out your well plate. Ensure there is a well containing 10% bleach, a well containing nuclease free water, and a well containing ultrafiltrate that corresponds to the sample ran for every five sample wells. A template is provided below.
123456
A
TE Buffer
Nuclease Free Water
Ultrafiltrate
Sample
Sample
Sample
B
Sample
Sample
Sample
Sample
10% Bleach
Nuclease Free Water
C
10% Bleach
Nuclease Free Water
Ultrafiltrate
Sample
Sample
Sample
D
Sample
Sample
Sample
Sample
10% Bleach
Nuclease Free Water
E
10% Bleach
Nuclease Free Water
Ultrafiltrate
Sample
Sample
Sample
F
Sample
Sample
Sample
Sample
10% Bleach
Nuclease Free Water
G
10% Bleach
Nuclease Free Water
Ultrafiltrate
Sample
Sample
Sample
H
Sample
Sample
Sample
Sample
10% Bleach
Nuclease Free Water
789101112
A
Sample
Sample
10% Bleach
Nuclease Free Water
Ultrafiltrate
Sample
B
Ultrafiltrate
Sample
Sample
Sample
Sample
Sample
C
Sample
Sample
10% Bleach
Nuclease Free Water
Ultrafiltrate
Sample
D
Ultrafiltrate
Sample
Sample
Sample
Sample
Sample
E
Sample
Sample
10% Bleach
Nuclease Free Water
Ultrafiltrate
Sample
F
Ultrafiltrate
Sample
Sample
Sample
Sample
Sample
G
Sample
Sample
10% Bleach
Nuclease Free Water
Ultrafiltrate
Sample
H
Ultrafiltrate
Sample
Sample
Sample
Sample
Sample

Calculate the amount of sample needed based on the dilution factor.

In the template above, 60 wells are needed for the sample and 150 µL will be used per well.
60 wells * 150 µL per well = 9 mL total volume

Assume we will use a 1:100 dilution (round up if needed).
0.01 * 9 mL = 90 µL, will round up to 100 µL for easier calculations
Calculate the amount of glutaraldehyde needed.

Using 25% EM grade glutaraldehyde, a final concentration of 0.5% should be obtained:
C1 * V1 = C2 * V2
25% * V1 = 0.5% * 100 µL
V1 = 2 µL

Note
The amount of glutaraldehyde needed for the sample is often very low and difficult to pipette. More sample than what is needed can be fixed and aliquoted into separate tubes post-fixation, but prior to freezing. Multiple freeze-thaw cycles should be avoided.

Filtration and Cleaning
Filtration and Cleaning
Filter the water through a glass fiber pre filter (Whatman Grade 1 qualitative filter paper).
Collect the filtrate and filter through a 0.65 μm PVDF filter (Durapore PVDF membrane filter; hydrophilic).
Collect the filtrate again and filter two times through 0.22 μm PVDF filters (Durapore PVDF membrane filter; hydrophilic).
If only filtering samples, Go togo to step #13 . Continue through this section for optional concentration.
Collect the filtrate and add to the top portion of a 30 kDa MWCO Centricon-70 plus centrifuge filters (Millipore UFC703008).
Note
Centrifuge filters come in a range of volumes (0.5 - 70 mL). These filters can be reused for similar samples. If storing used filters for reuse, after removing concentrate add a small amount of ultrafiltrate or sterile water, enough to cover the filter surface, and store at 4ºC.

Centrifuge at Centrifigation3500 rpm, 00:12:00 (if using smaller volume filters, adjust speed and time according to manufacturer instructions).
Centrifigation
After centrifugation, collect the ultrafiltrate from the collection cup below the filter and add more 0.22 μm filtered water to the top. Continue to centrifuge as explained in step 9 until all water has passed through the filter.

After concentrating all of the water, attach the retrieval cup to the top of the filter and flip upside down. Centrifuge at Centrifigation3500 rpm, 00:05:00 .
Note
Centricon-70 plus filters come with retrieval cups, but some smaller sizes do not. Smaller sized filters can be removed and flipped upside down into a 15 or 50 mL conical tube and centrifuged to collect concentrate. Amicon ultra-15 filters do not fit into conical tubes. To collect concentrate use a pipette to remove concentrate from the filter area, careful not to puncture the filter.

Centrifigation
Collect the concentrate from the retrieval cup and pipette into a 1.5 mL low protein binding nuclease free microcentrifuge tube.
Add Amount500 µL of ReagentChloroformContributed by users to 1 mL of sample.
Safety information
A lab coat, face and eye protection, and double gloves should be worn whenever working with chloroform, in addition to being performed in a fume hood.

Note
A small glass pasteur pipette must be used when working with chloroform. The pasteur pipette’s full volume should be 500 μL.

Toxic
Centrifuge the samples at Centrifigation14.000 x g, 00:05:00 (or standard mini-fuge speed).

Centrifigation
Use glass pasteur pipette to carefully pipette the sample portion of the supernatant (top part) into a fresh 1.5 mL low protein binding nuclease free microcentrifuge tube, making sure to not get any chloroform.
Aliquot the calculated amount of the chloroformed sample from Step 2 into a fresh 1.5 mL low protein binding nuclease free microcentrifuge tube and proceed to the next section: Fixation (Go togo to step #17 ).

Fixation
Fixation
Add the calculated amount of EM grade 25% glutaraldehyde from Step 3 to the calculated amount of sample from Step 2 for a final concentration of 0.5%. Pipette to mix.
Safety information
Fixation should be done in a fume hood as it requires working with glutaraldehyde Additionally, a lab coat, face and eye protection, and gloves should be worn whenever working with glutaraldehyde.

Note
The amount of glutaraldehyde needed for the sample is often very low and difficult to pipette. More sample than what is needed can be fixed and aliquoted into separate tubes post-fixation, but prior to freezing. Multiple freeze-thaw cycles should be avoided.

Allow samples to fix in the dark at Temperature4 °C for Duration00:30:00 .

Incubation
OPTIONAL: Flash freeze samples using liquid nitrogen. If liquid nitrogen is not available, putting samples directly in a -80ºC freezer can be done. Samples can be stored in the -80ºC freezer until use.
Note
This is an optional stopping point. Freezing is not necessary and may cause a decrease in viral counts. If you do not have time to proceed with the protocol, freeze your samples until use.

Optional
Pause
Preparation for Flow Cytometry
Preparation for Flow Cytometry
Thaw fixed samples at room temperature (if applicable). During this time, start heating a heat block to Temperature37 °C . Start heating another heat block to Temperature80 °C .
Aliquot the calculated amount of sample from Step 2 into a new 1.5 mL low protein binding nuclease free microcentrifuge tube.
Add ReagentBenzonase® NucleaseMerck MilliporeSigma (Sigma-Aldrich)Catalog #E1014 SIGMA to the sample for a final concentration of 1% (250 U). Pipette to mix.
Incubate the samples in a heat block at Temperature37 °C for Duration00:30:00 .
Incubation
Dilute the samples with TE buffer working stock according to the dilution factor used in the Step 2 calculation. Ensure you end up with the total volume calculated in Step 2.
Note
  • Dilution should be at least 10-fold, but can sometimes be as high as 1000-fold.
  • If the dilution factor is unknown, testing multiple dilution factors using the tube loading setting on the flow cytometer can be used.
  • Dilution should maintain an optimal event rate of 100-500 events/sec.

Note
TE Buffer Working Stock can be prepared as follows:
  1. Dilute 10X TE Buffer with Milli-Q water (18.2 MΩ) to achieve a 1X solution.
  2. Immediately autoclave on a liquid 15 setting.
  3. Before each use, aliquot small amounts (≤50 mL) of autoclaved 1X TE buffer into a new tube.
  4. Filter the aliquoted buffer to 0.2 µm using a sterile syringe filter.

Prepare a blank of ultrafiltrate using the same dilution method in Step 24. All following steps are performed on the viral samples and the ultrafiltrate (collectively referred to as "samples").
Note
Ultrafiltrate should be from the same location as the sample.

Add Amount10 µL SYBR Gold working stock to Amount1 mL sample in the dark and pipette to mix.
Note
The stain is light sensitive so the following steps should be done in the dark. Before preparing working stock, be sure to check if there is already some prepared. Working stocks may be stored in the -20ºC freezer or one working stock at a time may be stored in the 4ºC fridge.
  1. Thaw commercial stock of ReagentSYBR Gold Nucleic Acid Gel StainContributed by usersCatalog # S-11494 at room temperature in the dark.
  2. Once the commercial stock is thawed, vortex for 10 seconds on medium-high speed, then centrifuge in a microcentrifuge for 5 minutes.
  3. Dilute the commercial stock 1:200 with autoclaved and filtered (0.22 μm PVDF membrane filters) molecular biology grade water in a fresh 5 mL Eppendorf tube.
  4. Filter the working stock through a 0.22 μm syringe filter into a fresh 5 mL Eppendorf tube.
  5. Aliquot 250 μL of the working stock into fresh black or darkened 1.5 mL low protein binding nuclease free microcentrifuge tubes.
  6. Store the working stocks at -20ºC.
  7. Working stock that is being used should be stored at 4ºC in the dark. It can work effectively for about a month, but will degrade over time (take note of when working stock is moved to 4ºC).
  8. Working stock at -20ºC can be stored indefinitely and transferred to 4ºC when ready to use. Avoid freezing and thawing multiple times.


Incubate the sample in a heat block covered in aluminum foil at Temperature80 °C for Duration00:10:00 .
Incubation
Remove samples from the heat block. Pipette samples into a 96 well plate.
Ensure samples have cooled to room temperature (≥5 minutes) and run on the flow cytometer at an optimal event rate between 100 and 500 events/sec.
Note
  • If equipped, use the violet side scatter laser configuration, like that on a Beckman Coulter Cytoflex
  • The following histograms may be beneficial in observing VLPs: FITC-A, FSC-A, SSC-A, VSSC-A
  • The following dot plots may be beneficial in observing VLPs: FITC-A vs VSSC-A, FITC-A vs SSC-A, FSC-A vs VSSC-A, FSC-A vs SSC-A

Flow Cytometry Analysis
Flow Cytometry Analysis
Gate viral populations to obtain the event count per well. Gates should not include background noise.


Note
Viral gates will appear in a different location if your flow cytometer is not equipped with a VSSC laser configuration. Viral gates will likely occur in a lower SSC-A area of the plot.


Calculate the VLP/mL for each well using the formula developed by Maltseva & Langlois, 2022:


Note
The CytExpert software can execute this formula when put into the Equation section of the Statistics Settings. The results can be exported to a CSV file to get averages across the entire sample.

Protocol references
Maltseva M, Langloi M. Flow virometry for characterizing the size, concentration, and surface antigens of viruses. Current Protocols. 2022. 2:e368. doi: 10.1002/cpz1.368