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 ino