Protocol for SYBR Counts of Cyanophages and Bacteria
Make dilutions of the sample(s).
Record volume of samples used.
Custom has been to use 20 µl of phage stocks or 200 µl of cells diluted to 2 ml with autoclaved seawater.
If the final slides appear too sparse or dense then the volume of sample used can be adjusted accordingly.
Remove the 10% SYBR and phenylenediamine from the freezer.
Keep the stocks away from light (such as in a drawer) while they are thawing.
Connect the glass flask to the vacuum pump.
Attach the filter holder so that the grout is flat and level with the table.
Place a 0.8 µm filter on top of the grout.
Make sure it is completely flat and centered, with no air bubbles underneath (pre-wetting the grout helps).
This filter can be used many times so long as it stays intact and flat.
Place the 0.02 µm filter on top of the 0.8 µm filter. 
Again, make sure it is flat and centered with no air bubbles underneath.
Turning on the vacuum for a brief while can help achieve this.
Clamp the funnel on top of the filters.
Add the sample and turn on the pump (pressure should be ~20 kPa or 7 mmHg).
After the last liquid passes through the funnel and clamp should be removed with the vacuum still on.
Turn off the vacuum; remove 0.02 µm filter.
Keep track of which side of the filter is the top.
Blot out any seawater on the bottom or the top plastic rim with a kimwipe.
It is very important to make sure the filters are completely dry before continuing.
It is a good idea to rub the back of the filter with a kimwipe and then stick it in a dessicator (we used a makeshift box with some drying rocks) for a few minutes.
Prepare a 100 µL drop of SYBR, made fresh from 2.5 µL 10% stock + 97.5 µL 0.02 µm filtered deionized water, on the bottom of a plastic Petri dish.
Lay the 0.02 Anodisc filters sample side up on the drops of the SYBR staining solution for 15 minutes in a dark drawer or box.
While waiting it may be a good time to prepare the antifade solution.
Dry completely as in steps 7-8.
Do not touch the top of the filter.
Place the filter sample-side up on a glass slide.
Place 30 µL of antifade on a cover slip; then invert slip and place on top of the filter.
Appy pressure to ensure that the antifade fills the space underneath the square.
View with blue excitation.
Examine at least ten fields in the microscope (We usually examine 20).
Count at least 200 viruses or bacteria total for ten fields (400 for 20 fields).
Field size may be full (counting all particles in all 100 small squares) or smaller (e.g. 5 small squares), depending on the virus/bacteria concentration.
Find the average number of particles per quadrant (25 small squares).
Multiply this by a scaling factor of 1.26 x 105 to get the total number of particles on the filter.
Divide this by the volume used (e.g. 20 µL) to get the titer in particles per ml.
