Skip to 0 minutes and 10 secondsEarlier in the course, you watched Harriet isolate bacteria from a soil sample. Now I want to find out if there are any viruses in the soil sample. To see if there are any viruses that infect bacteria, called bacteriophages, I have asked another colleague, Jordan, to demonstrate the plaque assay technique for us.
Skip to 0 minutes and 37 secondsThe soil suspension that Harriet made is centrifuge to pellet the soil particles and cellular microbes, including bacteria, to the bottom of the tube. Viruses are much smaller than bacteria and are not heavy enough to be pelleted to the bottom of the tube at the speed Jordan used. So they should remain in the liquid at the top, called the super supernatant. Jordan takes up some of the supernatant into a sterile syringe and filters it into a new tube to remove any remaining bacteria from the sample as they would contaminate our experiment.
Skip to 1 minute and 23 secondsTo set up the plaque assay, we need a small amount of molten soft agar, which Jordan takes out of the water bath. He adds a small amount to the supernatant, which we just filtered from our soil sample. Jordan uses aseptic technique to prevent contamination of the experiment. Then we also need to add a small amount of bacterial culture to the molten agar. This was prepared by placing a single colony in liquid growth media and growing the bacteria in a shaking incubator overnight. The molten soft agar, our filtered soil solution, and this host bacteria, are now poured onto the surface of an agar plate and allowed to cool so that the agar solidifies.
Skip to 2 minutes and 12 secondsThe plate can then be incubated for 24 to 48 hours. If they're only bacteriophages specific to the host bacteria that we used, we would then expect to see zones on the plate called plaques.
Skip to 2 minutes and 31 secondsAs you can see, most of the plate looks cloudy. This is due to the growth of a large number of bacteria in the soft agar, so many that the colonies have all merged into a single thick mat. We call this a lawn of bacteria, plaques are small clear circles in the lawn where the bacteria have been destroyed by the bacteriophages. You can see there are hundreds of plaques on this plate. So it looks like there are plenty of viruses in the soil sample, even though we only tested one host.
Skip to 3 minutes and 12 secondsI have asked another colleague, Sinead, to demonstrate how to culture animal viruses in our tissue culture laboratory. Work is carried out in a safety cabinet, as this provides a more sterile environment than working next to a Bunsen. It is important to set up a control to check whether the cells we are using are healthy and not already contaminated with the virus. So we will be setting up two flasks for this experiment. Firstly, we add a culture medium to both flasks.
Skip to 3 minutes and 48 secondsNext, we add a specific insect cell line called SF9 also to both flasks.
Skip to 3 minutes and 59 secondsSinead then takes the sample containing the virus and inoculates that test flask. This is not added to the control flask, so it can remain as a comparison for us to review any changes. The flasks must then be incubated for several days to allow the cells to grow.
Skip to 4 minutes and 24 secondsBoth flasks look identical to the human eye. So let's take a look under the microscope. In the control flask, the cells look healthy. They have attached to the bottom of the flask in a dense layer. In the test flask that was inoculated with the filtered supernatant from the soil, the cells look quite unhealthy. Some have become detached from the flask and are much larger. Some are filled with dense particles and look very sick. So it looks like there were insect viruses in the soil sample. But we would need to purify them further, as there may be more than one type at this stage.
How to isolate viruses in a lab
Viruses are acellular microbes. If you want to culture them in the lab you need to provide them with host cells in which to replicate. Viruses are usually only able to replicate in one or a few species of host organism. When they infect multicellular organisms like humans, they usually only infect a few types of cell. There are many different types of immortalised cell lines available to virologists who study viruses that infect animals including humans.
In this video, my colleague Jordan demonstrates how to isolate bacteriophage (viruses that infect bacteria) from the soil sample used in Step 2.5. Sinead then demonstrates the basic techniques used when culturing a low bio-safety hazard virus.
© University of Reading