JoVE Encyclopedia of Experiments
Microbiology
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Begin with culture supernatants containing rotavirus particles, collected from infected cells at defined time intervals.
Add trypsin to cleave the viral outer capsid proteins, exposing host attachment sites.
Incubate host cells with diluted virus suspensions, shaking intermittently.
The viruses attach to the host and become internalized.
Remove the inoculum and add an agarose medium containing trypsin that supports reinfection.
The agarose solidifies, restricting viral diffusion and ensuring localized infection.
The internalized viruses replicate, leading to cell lysis and release of virions that infect nearby cells.
The virus replication causes cell lysis, forming clear zones or plaques.
Add a dye that stains the living cells, leaving the plaques unstained.
Remove the excess dye and incubate to enhance contrast.
Count the plaques and apply a mathematical model to generate the rotavirus growth curve.
The curve shows a lag phase for adaptation, an exponential growth phase, and a stationary phase when replication slows due to resource limitations.
Store the supernatant at minus 80 degrees Celsius until applying it to the plaque assay for measuring the virus titer.
When ready to perform the plaque assay, place the tubes containing the collected supernatant in a water bath at 37 degrees Celsius. Add trypsin to 1 milliliter of 10-fold diluted sample, and incubate at 37 degrees Celsius for 30 minutes. During this 30-minute incubation, carefully wash the MA104 cells in a six-well plate with 2 milliliters of 1X PBS after removing the serum-containing medium.
Serially dilute the incubated samples with serum-free medium and inoculate 1 milliliter of the diluted sample into each well. Incubate the plate for 90 minutes at 37 degrees Celsius and 5% CO2 under saturated vapor, gently shaking the plate every 15 minutes. After incubation, remove the inoculum from the six-well plate.
Add 4 micrograms per milliliter of trypsin to the prepared medium. Gently but immediately add 3 milliliters of the medium mixed with agarose gel to each well. Allow the agarose gel to solidify at room temperature for more than 10 minutes.
Then incubate the plate for two days at 37 degrees Celsius and 5% CO2 under saturated vapor. Next, add 1 milliliter of 0.015% neutral red solution diluted with 1X PBS to each well, and incubate with the same conditions. Remove the dye after 3 hours and continue to incubate for one day.
The next day, count the number of plaques in each well and calculate the pfu per milliliter. Carefully check the cell confluence before the plaque assay to assure the plaque numbers.