In my laboratory, where we study viruses, we
grow lots and lots of cells, you can grow
cells in two different ways. On the left side
of this slide is a photograph of what we call
a spinner culture. These are suspension cultures
of cells. The flask, these glass flasks that
you see here have a magnet suspending in them
and that is turned by a magnet in the structure
underneath them and so these are constantly
spinning and the cells are growing in a liquid
medium. On the right we can also grow cells
in what we call monolayer cultures. Here they're
growing in plastic dishes or flasks, covered
with mediu. And I think you can see at the back
of this incubator me actually taking this
photograph. This is a true selfie, CELLFIE,
all the other ones are fake. When you grow
viruses in cells, it can be very easy to detect
the growth, because many viruses kill the
cells that they replicated in. And this is
called cytopathic effects. And this is a series
of four photographs where we were tracking
the cytopathic effects of poliovirus in an
infected culture. On the upper left is a monolayer
of uninfected cells, these cells were then
infected with poliovirus. And then four hours
later, the picture on the upper right shows
you the effects of the virus very early in
replication, the cells are becoming rounded
and detaching from the monolayer. At eight
hours post infection, the lower left-hand part
of the slide, you can see most of the cells
are now detached. And by 12 hours, on the lower
right, all of the cells are detached and many
of them have broken up. So this is called
cytopathic effects. It is an easy way to tell
that your virus is replicating or growing
Another important aspect of studying viruses
is to detect the virus in the sample, to know
how much virus is present. And I want to tell
you two different ways that we do this; one
is we can measure infectivity of the viruses,
we can do that by a plaque assay which is
shown here and we can also measure physical
virus particles, or their components. This
photograph is an electron micrograph of virus
particles and you could count them for example,
and know how many particles are in a preparation.
Infectivity assays actually tell you how many
infectious viruses are present, physical particle
measurements only tell you the particles,
because not all virus particles are infectious.
Let's explore the plaque assay a bit. In this
assay, we take a virus stock and we assume
there's a lot of virus in this virus stock.
And then we have to make tenfold serial dilutions
of the stock in order to obtain a dilution
which has a reasonable number of particles
in it. So here we're making tenfold serial
dilutions and we end up with -1, 2, 3, -4, etc
dilutions of the virus. We then take a small
amount of each dilution and we add it to a
monolayer of cells in a plastic dish, we cover
the monolayer with an agar overlay and we
incubate it. The viruses will infect cells
and they will destroy the cells in a small
area. They are restrained in their diffusion
by the agar overlay and they form what we
call plaques. We can then stain the plaques
and count them. So earlier I said we need
to make dilutions because we need to find
a suitable number of viruses and I think the
three plates shown here illustrate that. The
one on the left part of the slide, there are
too many plaques, we can't count these with
any accuracy. The one in the middle has 17
plaques, which is a reasonable number. Anything
between 10 and 1 or 200, depending on the
size of the dish will work. And then on the
far right, only two plaques. This does not
provide a statistically rigorous number. From
the 17 plaques, we know the dilution that was
used to produce that plate, we can calculate
the titer of this virus stock at 1,7Ã—10 to
the eighth plaque forming units per milliliter.
It's very important, this is plaque forming
units, we can't say virus particles because
we don't know for this particular virus how
many viruses form a plaque. So that's an example
of a plaque assay, this is used in many laboratories,
it was developed in the 50s and it is still
used today because it's incredibly powerful.
In fact in my laboratory we do them all the
time and I've built in my office what I call
the wall of polio. This is a stack of plates
used to do plaque essays. I've glued them
all together myself. This is what I do for
relaxation and when people visit my lab, I
take their picture in front of the wall of
polio, so if you come to visit me, please
ask and I will take your picture as well.
Physical measurements of viral particles can be done in a variety of ways.
We can use a technique called hemagglutination.
Some viruses bind red blood cells and we can use that to make a physical measurement of particles.
We can look at the virus particles by electron microscope as I showed you earlier.
This is not so convenient because not everyone has an electron microscope.
We can measure viral enzymes in the virus particle.
Many viruses package into the particle and enzyme,
and many assays have been devised to take advantage of that.
We can use serologic techniques to measure virus particles and their components.
We can use antibodies against viruses and their proteins to measure how much of each one is there.
And we can also measure nucleic acids now by a variety of techniques.