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 16 & 18 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 .
