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 .
