Quantifying Infectious Viruses in Water Samples Using an In Vitro Total Culturable Virus Assay

Take a virus suspension concentrated from a water sample. Inoculate mammalian kidney cells with equal inoculum volumes that may contain different numbers of infectious viruses.
Tilt the culture vessel to spread the suspension.
Incubate under agitation, allowing viruses to attach to cell surface receptors.
Add media and continue incubation.
The viruses enter the host cells and release their genomes. These genomes replicate and produce viral proteins, forming new virions.
The infected cells undergo structural changes or death, a phenomenon known as CPE.
Some cultures remain CPE-negative due to a low infectious virus dose.
Freeze and thaw the cultures to lyse the cells.
Filter the CPE-positive culture to obtain a clean virus suspension. Meanwhile, collect the suspension without filtering from CPE-negative cultures.
Inoculate fresh host cells with the suspensions and incubate.
CPE in two successive passages confirms the presence of infectious virus.
Use the number of confirmed CPE-positive cultures to estimate the infectious virus concentration in the water sample.
Inoculate 10 test vessels containing a monolayer of BGM cells for each test sample, with the volume of sub-sample one equal to the inoculum Volume, along with the total culturable virus quantal assay controls. For the Lab Fortified Blank and the Lab Fortified Sample Matrix, prepare 5, 25 and 125-fold dilutions using sub-sample three and 0.15 molar sodium phosphate, pH 7.0 to 7.5, as a diluent. In addition to the vessels inoculated with undiluted sub-sample one, inoculate 10 washed cell culture test vessels for each dilution series, using an inoculum volume on each test vessel.
Distribute the inoculum over the surface of the cell monolayers by tilting the vessels back and forth. Incubate the test vessels at room temperature for 80-120 minutes on a mechanical rocking platform at one to five oscillations per minute, or with rocking of the vessels every 15 to 20 minutes to allow any virus present to adsorb to the cells. Then, add pre-warmed maintenance medium to the test vessels before incubating them at 36.5 plus or minus one degree Celsius.
Look for the appearance of cytopathic effects in each test vessel using a microscope for the first 3 days, and then examine them every 2 to 3 days up to day 14. Transfer any test vessels that show greater than 75% cytopathic effects to a freezer set at or below minus 70 degrees Celsius. After examining the vessels on the last day, freeze all remaining cultures and the total culturable virus quantal assay controls at or below minus 70 degrees Celsius.
Then, thaw all the cultures, and filter greater than 15% of the medium from every CPE-positive test vessel through a 0.2 micron sterilizing filter. The next step is to perform a second passage of all first-passage test vessels using washed BGM kidney cell test vessels. To do this, inoculate the new test vessels with an inoculation volume that represents 10% of the thawed medium from all negative test vessels, as well as from the filtered medium of positive vessels.
Repeat the steps done for the first passage, freezing any test vessel that was negative on the first passage and positive on the second. Then, perform a third passage as described for the second passage, using only the negative assay controls and cell cultures that were negative during the first passage and positive in the second passage. Identify individual test vessels as virus positive when they show cytopathic effects in both the first and second passages, or, in the case where cytopathic effect does not occur until the second passage, in both the second and third passages.
Use the U.S.Environmental Protection Agency's Most Probable Number Calculator with the default program settings set as in the text protocol, to calculate the virus titers of all test samples. Then, use the values obtained for the Most Probable Number of infectious units per milliliter, in addition to the upper and lower confidence limits per milliliter, to calculate the Most Probable Number of infectious units per liter, and corresponding confidence limits, as described in the text protocol.