The Effects of K1F bacteriophage on the
EV36 strain of E. coli
by Courtney Willard
I will be viewing the effects of K1F bacteriophage on the EV36
strain of Esherichia coli. I will be comparing this effect to those of
the E. coli that have not been infected by the bacteriophage. E. coli is
a bacteria that is commonly used in scientific studies concerning bacteria
and is one of the best understood bacterium. A K1F bacteriophage is a
virus that infects the E. coli. The bacteriophage adheres to the membrane
of the E. coli and degrades the capsule that with neuraminidase. The
bacteriophage is then able to inject its DNA into the bacteria cell. The
virus then uses the bacteria cell to replicate itself. The bacteria cell
will continue to replicate the viral DNA, and the virus itself, until the
cell becomes so full that it bursts the membrane and bacteriophage is
released into the environment. I hope to see an E. coli cell in this
ruptured state with a broken membrane. I also hope to see some
bacteriophage being released by the cell..
This project required a large amount of time outside the SEM lab.
I retained the EV36 strain of E. coli and the K1F strain of bacteriophage
from the Department of Microbiology and Immunology at the Medical Center.
The bacterium were on agar plates and the bacteriophages were in test
tubes. The E. coli needed to be grown in an LB media in a shaking water
bath with the water maintained at a constant temperature of 37 degrees
Celsius. For this, a portion of the E. coli growing on the agar plate was
removed and placed in an LB broth in a 250 mL flask. It was then placed
in a shaking water bath where it was allowed to grow overnight. Once
there was sufficient growth, I was able to place the bacteriophage in the
mixture of E. coli. I then waited for three minutes, thirteen minutes,
and twenty-three minutes before removing samples with a micro pipette to
be viewed in the SEM. I also took a sample of just plain bacteriophage so
that I could view that also.
Once I removed the samples, each one was passed through a
millipore filter, which were used to catch the bacteria and bacteriophage.
Each one of these filters were then floated in a gluteraldehyde fix of 5%
ethanol. This was gradually increased until the mixture the filters were
in was 100% ethanol. The filters were then allowed to sit overnight.
After this I took the filters and folded them over, holding them between
two U-shaped magnets so that the bacteria were not able to fall off during
the critical point drying process. The filters were then placed in the
chamber, which was sealed and then filled with liquid CO2. The pressure
of the chamber was also raised. The liquid CO2 was bled out numerous
times while the pressure remained high. Once all the ethanol was removed
from my samples, I removed them from the chamber. Next, I cut out small
pieces from each filter and placed them on the sample holders with a
carbon based glue. These were then placed in the sputterer so that they
could be coated with gold. The vacuum was placed on and argon was bled
into the vacuum. This step was repeated numerous times. The sputterer
was then turned on and the samples were coated in gold. My samples were
now ready to be viewed in the SEM
Above, E. coli cells uninfected by the bacteriophage.
After viewing the filters in the SEM, I was able to see the
effects of the K1F bacteriophage on the EV36 strain of E. coli. As I had
hoped, the bacteriophage had infected some of the E. coli and had even
caused some of the E. coli to burst releasing even more of the
bacteriophages. I also viewed E. coli cells that were not infected by the
bacteriophage. I was able to view mass amounts of the K1F bacteriophage
on the sample containing just the bacteriophage. I was unable to see any
single bacteriophages because of their minuscule size. The procedure for
this project was relatively simple. The only problem that arose was when
some of the filters sank while placed in the ethanol. I was concerned
that this would cause the bacteria and bacteriophage to fall off the
filters, but, as can be seen, they both remained attached.
Please enter any comments, criticisms, questions, etc. below.