Wet-mount Method for Enumeration of Aquatic Viruses
Place 1 mL of sample into a 1.5 mL microcentrifuge tube.
Add 1 μL Iron Chloride Solution and vortex to mix.
Centrifuge the sample at ~14K RCF for 20 minutes.
Remove supernatant using a pipette, leaving a small, undisturbed pellet of Fe oxyhydroxides behind (Figure 2).
Dissolve the pellet in 10 μL of Ascorbic-EDTA Buffer, creating a 100-fold concentration of the original sample.
Vortex and then pipette up and down to ensure complete dissolution.
Vortex and then pipette up and down to ensure complete dissolution.
Combine 10 μL sample (concentrated or unconcentrated) and 2 μL SYBR Gold Working Stock, vortex to mix, and place in dark for 15 minutes.
If sample is unconcentrated, add 1 μL of Ascorbic Acid Antifade Solution.
Add 5 μL of glycerol to stained sample and vortex to mix.
Add 2 µL Working Bead Solution to sample.
Clean glass slides and cover slips with isopropanol and Kimwipes.
Thoroughly mix the sample/bead mixture by pipetting up and down, then immediately pipette 10 μL of it onto a glass microscope slide.
Place a coverslip over the mixture and avoid trapping air under the coverslip.
Place a coverslip over the mixture and avoid trapping air under the coverslip.
View viruses under ~495 nm excitation at 1000X magnification using an epifluorescence microscope.
Count the number of viruses in one defined field of view.
Once complete, switch off the excitation and turn on the white light of the microscope to count the beads in the same field of view (Figure 3).
Repeat Step 14 & 15 by counting viruses and beads in multiple fields until at least 100 of each have been counted.
The concentration of viruses can then be determined with the following equation
Prepared samples can be stored at -20ºC either in the microcentrifuge tube (i.e., after completing Step 4) or after mounted on slides (i.e., after completing Steps 12&13) with no significant change in the calculated virus concentration.
