Kirsten Voelker

Anatomy Summer Assignment

Bacterial Identification
In this virtual lab I learned all about how a pathologist identifies
specific bacteria. First, there are some basic steps including obtaining a
sample of DNA and making copies of that specific DNA. To do this, one must
drop the bacterial colony into a micro centrifuge tube. Bacterial DNA is
extracted by dissolving the cell wall with a digestive buffer. This buffer
consists of proteolytic enzymes, which are enzymes that break down proteins
in the cell wall. However, in this lab, proteolytic enzymes were inactivated by
heating the sample in water at 100 degrees Celsius. After this has happened
the cellular debris appears as a solid after being placed in a device that spins
and separates the components by weight, known as the centrifuge. The DNA
is contained in a liquid, supernatant, that is then transferred to the PCR
(polymerase chain reaction) tube.
Once the DNA is transferred, a solution of PCR Master Mix is added to
it. At this time, negative and positive control reactions are prepared. The
positive control reaction does not contain the sample, but it contains positive
control DNA and the Master Mix. The negative control reaction also contains
the Master Mix, as well as deionized sterile water. After that has been
prepared and placed onto the PCR machine, DNA replication begins. Thirty
cycles of three steps make up this process. The three steps are, melt, anneal,

and extend. The first step (melt) separates the strands. The second step
(anneal) binds the primer to the template. The third step (extend) is the
synthesis of the new strand. One cycle (all three steps) takes less than two
minutes to complete. After each cycle each piece of the DNA has been
doubled.
There are now many copies of the 16s rDNA. At this point, a gel test is
to be run in order to make sure that the PCR reaction worked properly. The
gel should contain three lanes. One lane is for the negative control, which
will not react and form a product, unless the sterile water was contaminated.
One lane for the positive control to ensure that the PCR worked. Finally, the
last lane is for the DNA sample. The gel test is one way to purify the PCR
product. After the PCR product is in the gel, one is able to isolate the DNA
from the gel. In this lab micro filters are used inside of a micro concentrator
instead of the gel test, to filter the DNA from the PCR tube.
Now that the DNA is purified the PCR tube only contains copies of the
16S rDNA. Cycle sequencing is the next step in the identification process.
Cycle sequencing uses a thermocycler to create many copies of the DNA. At
random places the replication process can be terminated so that the copies
are all partial sequences and have different lengths. There are normal
deoxynucleotides and special dideocynucleotides, they are tagged with
fluorescent markers that differ depending on the base. They are designed to
fluoresce with different colors. The fluorescent colors of the DNA pieces of
different sizes allow one to find the sequence for complementary DNA. From

there, the original sequence can be inferred. Primers bind to specific known
sequences on one strand only to prevent problems and mistakes in the
complementary strand. This lab uses twelve primers, six for each strand of
the double-stranded DNA. Although sequencing both strands is not always
necessary, it helps reduce error. In this case, the primers bind to the
conserved regions of the 16S rDNA gene. Therefore, they should be able to
bind to the sequence no matter the bacterial source. Eventually, each DNA
strand has a primer at one end and a fluorescence-tagged terminator at the
other.
A mix of DNA pieces of varying lengths remain within the twelve tubes.
Each piece of the DNA started with the same primer, but is left with a
different nucleotide that is tagged with a fluorescent marker. The individual
DNA pieces is to be separated and the nucleotide identified. This is done by
an automatic sequencer. In this sequencer method called Gel electrophoresis
is performed on each tube. This method separates molecules based on size
differences. Later on in the sequence, a capillary tube that passes through a
laser beam shocks the fluorescent markers. Optical detectors are then able
to detect the color of the fluorescence. The DNA sequence can be
reconstructed once the sequence of nucleotides is read based on their
fluorescence.
I was given six samples each of them went through the process and I
was given the gene sequence. I then placed the sequence into the NCBI
BLAST program and was given the results. Sample A was identified as

Bartonella henselae, fluid from Lymph Node obtained from tissue and fluid
that aspirated from a lymph node of a patient who had a fever, swollen
lymph glands, and fatigue. Sample B was identified as Escherichia coli,
obtained from the stool of a patient with a normal temperature, severe
abdominal cramps and watery diarrhea. Sample C was identified as
Pseudomonas aeruginosa, obtained from urine of a patient with pain during
urination and discharge from the urethra. Sample D was identified as
Salmonella typhimurium, obtained from the blood of a patient complaining of
headaches, joint pain, a fever, constipation, abdominal pain, and loss of
appetite. Sample E was identified as Yersinia pestis, obtained from the
sputum of a patient complaining of chills, high fever, a cough with labored
breathing and sputum with flecks of blood. Sample F was identified as
Yersinia enterocolitica, was obtained from the stool of a child who has a
fever, abdominal pain, and bloody diarrhea.