Download Bacteriophage One-step Growth Curve A bacteriophage is a virus

Bacteriophage One-step Growth Curve
# of phage
A bacteriophage is a virus that infects a bacterium. The best studied are the coliphages,
viruses that infect strains of E. coli. However, many other phages are known that infect a wide
variety of other bacteria.
Most coliphages have ds DNA as their nucleic acid. In a lytic cycle, the phage infects the
cells, replicates, then lyses the cell, releasing the new virions. The steps in this process are
absorption (phage attaches to the cell), penetration (typically, the phage injects its DNA into the
cell), replication (DNA is replicated and used to make multiple copies of capsid proteins),
assembly (a spontaneous, inefficient process by which new DNA molecules and capsid proteins
come together to form new virions), and release (cell lyses, liberating new virions). The burst
size is the number of new infective phage released from one infected cell and is around 200 for
many coliphages.
In this experiment we are studying the phage called X-174 which infects a particular
strain of E. coli, E. coli C. To follow the replication of this phage, we need to determine the
concentration of the phage at the start of the experiment, then at several times later. We expect to
see a sudden increase in the number of phage, because at roughly the same time all the infected
bacterial cells will lyse and the number of phage will increase by 200 fold. This should produce a
characteristic sigmoidal curve as shown in the
accompanying figure.
In order to count the number of bacteriophage
we use a plaque assay. A sample containing the phage
is mixed with host bacteria in melted soft agar which
is used as an overlay. The agar is soft because it is at
a concentration of 0.9% instead of the usual 1.5%.
This allows the virions to more easily diffuse through
Time
the agar to infect neighboring cells. The agar is
melted so that the phage sample and host bacteria can
be easily mixed together. The host bacteria must be added because they multiply to form a lawn,
and each hole in the lawn (plaque) indicates the activity of a virus. The host cells and phage
samples are mixed together in a small volume of agar which is poured over the existing agar in a
Petri dish, thus it is called an overlay. This overlay allows the lawn and plaques to be in a thin,
easy to visualize layer. Each virus (or infected cell which acts as a source of virus) that is present
in the overlay initiates a series of cycles of infection. Each time virions are released from lysed
cells, they infect neighboring cells which are also killed, releasing new virions. The ever
widening hole produced by several cycles of infection and cell destruction eventually becomes
large enough to be seen and appears nearly clear from lack of bacteria. This hole constitutes a
plaque. As with a viable count assay like was done for the Hamburger Experiment, several
dilutions of the phage need to be measured because large numbers of phage are present and for
statistical reasons we can only use plates with between 20 and 200 plaques.
We will start the experiment with a suspension of phage (about 5 x 1011 phage/ml) and an
overnight bacterial culture. Because viruses grow better in actively growing bacteria, the
overnight culture will eventually be diluted to create a slightly turbid suspension, aiming for a
concentration of about 5 x 107 bacteria/ml. To begin the experiment, the instructor will combine
enough of the overnight bacterial culture (around 1 ml) with a dilution of phage in a
microcentrifuge tube. A ratio of phage to bacteria will be chosen such that every bacterium is
infected with at least one phage. In fact, there will be about 10 phage for every bacterium. This
ratio is referred to as the multiplicity of infection (MOI), so the MOI will be about 10. The
mixture will be allowed to stand for a couple of minutes, then the bacteria will be centrifuged,
leaving any unabsorbed phage in the supernate which will be discarded. The infected bacteria
will be resuspended with warm phage broth and placed in a shaker where the culture will be
aerated at 35C.
Since this is a “growth” study, samples will be removed at various times, and the
numbers of phage will be measured using the plaque assay. At the early time points, there will be
no phage free in the medium. Each plaque measured will arise from one infected bacterium. As
these infected bacteria begin to lyse and release phage into the medium, plaques will result both
from infected cells and from free phage. Because of the way this experiment is begun, with
unadsorbed phage being removed at the beginning of the experiment, the infection of the cells
should occur synchronously, and the number of phage released when the cells start to lyse will
increase rapidly. When all the bacteria have been infected and lysed, growth of the virus will
stop. This results in the sigmoidal growth curve. Each group of students will sign up for a
different time point in the experiment. Samples will be removed every 10 minutes from the time
phage and bacteria were combined, the last time point being at 50 minutes.
The purpose of the plaque assay is to count the phage in the culture. Just as in the
Hamburger Experiment where the bacteria grew in the hamburger and dilutions and plate
counting were done for each time point, so will the plaque assays be done to count the number of
phage present at different times. The procedure is summarized as follows: you will obtain your
phage sample from the culture and dilute it. From each of 4 of the dilutions, you will remove a
sample and combine it with some E. coli C (host bacteria for the lawn) and melted agar. After
mixing the 3 ingredients, you will rapidly pour the mixture over the agar of a pre-made plate.
The details are as follows:
Dilutions: You will need 2 red pushcap tubes (with 4.95 ml) and 2 yellow pushcap tubes (with
9.0 ml). With a 50 l micropipetor with a yellow sterile tip, very carefully remove 50 l of
infected culture and place it in the red cap tube. Mix this well. Rinse your tip up and dwon a few
times in this solution, then remove 50 l, place it in the second red cap tube, and mix well.
Using a 1.0 ml pipet, remove 1 ml from this second red cap tube and add it to the first yellow cap
tube. Mix this well, pipet up and down to rinse the pipet, then remove 1.0 ml and add it to the
second yellow cap tube and mix this well. Continue making serial dilutions in this way until all
the yellow tubes have been used. For the plaque assay, you will use the more diluted red cap
tube and the two yellow cap tubes.
Plaque assay: To perform a plaque assay on each dilution, use a micropipettor (yellow tip) to add
100 l of an E. coli C culture (host cells for lawn) to a sterile plastic tube. To this add 100 l of
a diluted phage sample. Have your agar plate ready. Finally, add 3 ml of melted soft agar to your
tube by pipetting it from a flask in the water bath. Your bacteria, phage, and melted agar must be
quickly mixed and quickly poured over the surface of a phage agar plate, quickly rocking the
plate so that the melted agar covers the entire surface of the plate. Do it quickly, because the
melted agar will gel very fast!!
As the lawn bacteria grow, the phage added to the overlay should form plaques.
Development of the plaques will be stopped (by refrigeration) as soon as the lawn is turbid
enough to provide contrast and the plaques are large enough to be counted. By taking the various
dilutions into account, we should find that some of the data collected will demonstrate the
increase in the number of virions in the infected culture over time.
Each class will perform this experiment. Within each class, you will divide up into 7
groups and each group will take one time sample and perform 3 plaque assays (each plaque assay
counts the plaques made by a different dilution of your phage sample). Your times will be 10, 20,
30, 40, and 50 minutes.
For TA use and general enlightenment:
Recipes
Phage Broth (for growth of culture, agar
plates, dilution tubes):
Tryptone 1%
NaCl 0.5%
CaCl2 2 mM
(for agar: 1.5% agar added)
Phage soft agar (for overlays):
Tryptone
1%
KCl
0.5%
agar
0.9%