Enumeration of virus particles in aquatic or sediment samples by epifluorescence microscopy
Prepare reagents .
Collect and prepare sample according to sample type .
Prepare slide labels with critical information such as the date , sample location , and volume filtered to keep track of the samples once they have been filtered and stained .
For every set of four samples to be stained , use a permanent pen to mark the bottom of a plastic Petri plate into four labeled sections .
For each filter that is to be prepared , add a 78 - µL drop of 0.02 - µm filtered dH2O on each section of the marked Petri plates .
Thaw a 40 - µL vial of the stock SYBR solution and add 2 µL stock solution to each drop ( 78 µL ) of sterile dH2O or buffer .
Mix the stain by gently pipetting up and down .
Place the Petri plates in the dark so that the stain is not bleached .
Prepare the antifade solution in a clean , sterilized 2 - mL microcentrifuge tube .
Dilute the 10 % ( wt / vol ) stock of p - phenylenediamine 1 : 100 using glycerol / PBS as the diluent .
Keep the solution on ice and protect it from light .
For each filter that is to be prepared , add a 80 - µL drop of thawed Yo - Pro working solution on each section of the marked Petri plates .
Place a 9 - cm - diameter filter paper soaked with 3 mL aqueous NaCl solution ( 0.3 % wt / vol ) in the lid of the Petri plates to prevent evaporation of the stain .
Connect a filtration unit for 25 - mm filters to a vacuum source , ensuring the vacuum is ≤13 kPa .
Place a 0.45 - µm nitrocellulose backing filter on each filter support , and overlay it with a thin layer of dH2O .
Carefully pick up a 0.02 - µm Anodisc filter by its plastic ring and lay it over the wet backing filter , with the plastic ring facing upward .
If the sample has been preserved and frozen as described , thaw it in a 37°C water bath .
For a sample that has just been collected , fix it with 0.5 % glutaraldehyde for 15–30 min at 4°C before preparing slides .
In addition , prepare duplicate control samples by fixing 1 mL of the 0.02 - µm filtered water that was used to dilute the SYBR stain .
Because divalent cations interfere with the binding of the stain , seawater samples should be diluted to < 7 psu with 0.02 - µm filtered dH2O before filtration .
It is a good idea to make test slides ( including a control with no sample added ) to be sure an appropriate volume is filtered , that the procedure is working , and that the filters and reagents do not have viruses on or in them ( some batches of Anodiscs have been covered with bacteria and viruses ) .
For most lake and coastal seawater samples , which have viral abundances of ~ 107 mL–1 , 0.8–1.0 mL sample is added to the surface of the Anodisc filter while the vacuum is off .
Turn on the vacuum and suck the sample through the filter .
For oligotrophic or very deep ocean samples , it may be necessary to filter 4 mL or more .
Filter towers must be cleaned between samples .
Rinse the towers with 0.02 - µm filtered dH2O followed by ethanol .
Dry with lint - free paper ( e . g . , Kimwipe ) .
Once the sample is filtered , remove the Anodisc with the vacuum still on .
Allow the filter to air - dry ( typically a minute or less ) , until the surface is visibly dry .
Place the Anodisc , sample side up , on a drop of stain in the Petri dish .
Allow the filter to stain for 15 min in the dark .
Allow the filter to stain for 48 h in the dark , at room temperature .
Add a drop of dH2O on the backing filter , lay the stained Anodisc on top , and use the vacuum to remove any remaining fluid .
Some samples ( e . g . , sediments , vent fluid , and humic waters ) may require the filters to be rinsed to reduce background fluorescence .
If so , while the vacuum is still on and the filter is damp , rinse the filter twice with 1 mL of 0.02 - µm filtered dH2O .
Remove the Anodisc while the vacuum is on .
Place the Anodisc , sample - side up , on a 9 - cm filter paper or Kimwipe in the dark , and allow the filter to dry until it appears opaque .
Place 12–15 µL antifade solution on a labeled glass slide and lay the dry Anodisc on top .
Add ~ 20 µL antifade on top of the Anodisc and cover with a coverslip .
Place 12–15 µL spectrophotometric - grade glycerol on a labeled glass slide and lay the dry Anodisc on top .
Add ~ 20 µL glycerol on top of the Anodisc and cover with a coverslip .
Remove any air bubbles that are trapped under the coverslip by gently pressing on the surface .
The slides can be counted immediately or stored frozen at –20°C for at least 4 months with no decrease in estimates of viral abundance .
Count the viruses at 1000× magnification using a 100× oilemersion objective .
Begin by checking the test filters to ensure that the reagents or filters were not contaminated and the filtered volumes were appropriate .
Check each slide before counting to make sure that the filter is evenly stained and that the viruses are on a single plane of focus and not suspended in the mounting medium and are evenly distributed across the filter .
Using the ocular reticule , select an appropriate number of grid squares so that there are 10–100 stained viruses in each field .
Estimate the abundance of viruses by counting at least 20 random fields .
Keep a tally of the number of particles in each field so that the variation in abundance of particles among fields can be determined .
For each sample , record the number of particles counted in each field , the number of fields counted , the area of the field , and the volume of sample filtered .
The abundance of viruses mL–1 ( Vt ) in the sample = Vc ÷ Fc × At ÷ Af ÷ S , where Vc = total number of viruses counted , Fc = total number of fields counted , At = surface area of the filter ( µm2 ) ( see note below ) , Af = area of each field ( µm2 ) , and S = volume of sample filtered ( mL ) .
The total number of particles counted will determine the size of the 95 % confidence intervals on the estimates of viral abundance .
By assuming a Poisson distribution , the 95 % confidence intervals can be estimated using the following equations ( Suttle 1993 ) :
