The document summarizes an experiment to identify mycobacteriophages from soil samples. Soil samples were taken from different locations and enriched with bacteria to isolate any phages present. However, the testing of samples did not pass the enrichment phase due to issues with the bacteria. One sample from Gurabo was retested successfully, allowing the experiment to progress to plaque purification. The identification process is currently in the third purification step. The experiment highlights the challenges of phage identification but the potential benefits of understanding phage behavior and using phages in applications like phage therapy.

1. Bacteriophages: The Search to Find Bacteria’s Enemy
Juan E Maldonado Weng Primary Article
Gustavo A Martínez
Abstract
Mycobacteriophages are viruses that infect bacteria, specifically bacteria called mycobacteria. They exist in
two different stages: the lytic, where the phage reproduces within the bacteria and afterwards kills the cell; and the
lysogenic stage, where the phage’s DNA replicates within the bacteria. The experiment involved gathering soil; the
soil sample was to be taken from a certain point, recording factors such as: air temperature, moisture content, GPS
location, etc. Afterwards, 0.5 g of the sample is enriched with M. smegmitis in order to ensure there are phages
during the purification step and that the phages receive proper nutrition. The solution is isolated in filtrate for
placing it on agar plates that will allow the contained phage to grow. Three plaque purifications follow this step in
the process. These steps were interrupted and slowed by complications with the samples, the bacteria used, and
fungi that happened to grow on the agar plates. Despite the setbacks that have been faced, the work continues in
identifying the newly found mycobacteriophage. The identification process currently stands in the third plaque
purification step. After purifying the phage, the process would continue with the web pattern of the plaques. This
process is to continue using a a high titer lisate in order to analyze the DNA sequence of the phage to afterwards
process it and determine whether it is a new phage or previously identified one. After it is analyzed the phage’s
gene sequence is to be stored in a national genomic database specialized in bacteriophages.
Introduction
Bacteriophages are numerous and
diverse life forms which can be found in most
locations where bacteria can be found. Similar
to fingerprints, finding two of the same can be
nearly impossible. These creatures are all
unique to one another with different behaviors
and habits. The bacteriophages serve as great
examples of evolution since they have a high
rate of reproduction as they replicate as fast as
bacteria. With each new generation, they could
easily have new mutations or adapt to new
conditions.
The most efficient method to identify the
bacteriophage would be by the way they
reproduce which could be Lytic or Lysogenic.
Lytic would mean that the bacteriophage would
enter the bacteria, reproduce, and then destroy
the host. The lysogenic reproduction would
involve inserting itself into the DNA of the host
and reproduce along with it. The lifestyle the
bacteriophage could be lytic or temperate,
which means it could be both lytic and
lysogenic.
Based on Hanlon’s review, the bacteriophages
could be helpful to humans in many ways.
Phages were used to treat bacterial infection
before the discovery of penicillin. Since they

2. only affect bacteria, there is still a possibility
that bacteriophages can be used to cure diseases
and other illnesses caused by bacteria. Most of
the capabilities for phages still are very
mysterious and a good way to understand them
is by seeing their genetic code. An advantage
that modern science has is the ability to decode
their genome to understand or to at least have
them collected in an open database. A database
would help globalize the search for
understanding these creatures.
The problem is the act of collecting a
vast amount of the bacteriophages to then be
able to identify, study, and publish the
information about them for the rest of the world
to learn. Collecting samples of soil in search for
bacteriophages will help to better understand
their behavior, abilities, and function.
Materials and Methods
The process of identifying a phage is complex
yet simple. The first important step was to
collect soil to be able to extract the phages if
there were any. The soil could be from almost
anywhere. The soil could be moist, dry or
damp. The soil should have had a proper
amount of bacteriophages since there are
bacteria everywhere.
The next step was to isolate the phage.
From the soil sample, only .5 grams was used,
placed in a tube (250mL flask) and was added
several compounds. 40mL of water, 5mL of
10X 7H9/Glycerol media, 5mL of AD
supplement and 0.5mL of 100mM CaCl2 were
added to the soil sample, which enriched it.
The enrichment gave the sample bacteria to
feed the phages. The next day, the sample was
centrifuged and the phages that collected at the
bottom of the flask were collected with a
micropipette.
The step afterwards is confirming that
there is a bacteriophage and then, purifying the
phage. The extraction was spread into a plate
with top agar, which restricts the phage’s
movement. If the phage were there, in the next
day there would be spots or plaques on the agar
on the plate. From here, the phage was purified
three times. The purpose of this was to isolate
a single phage population from the potentially
mixed community from the sample. This was
the last step before time became a factor that
constrained the continuation.
The collections that were made are
indicated in the table below, with Juan’s
collection in blue and Gustavo’s collection in
red. The table puts into account the moisture
level, depth at which soil was collected, and
features of the area.
Results
Nine individual soil samples were taken
and evaluated for the project of
mycobacteriophages. Four of which were from
Luquillo, one from Cayey, and the remaining
Juan & Gustavo: Collections
# Date Time Depth Moisture Features Location
1 2/9/2013 6:06 P.M. Superficial Dry/Moist Urban Luquillo
2 2/18/2013 12:03 P.M. Superficial Dry-moist Urban Luquillo
3 3/3/2013 3:29 P.M. Superficial Dry Urban Luquillo
4 3/11/2013 6:36 P.M. Superficial Dry Urban Cayey
5 3/17/2013 1:59 P.M. Superficial Moist Urban Luquillo
6 2/18/2013 5:44 P.M. 2-2.5 cm Dry/Moist Rural Gurabo
7 2/28/2013 4:13P.M. 3cm Saturated Rural Gurabo
8 3/10/2013 6:25 P.M. 10 cm Damp Rural Gurabo
9 3/17/2013 7:16 P.M. 1 cm Moist Rural Gurabo

3. four from Gurabo. Gathering these many
samples was due not being able to find phages
after the enrichment phase of the process.
The soil samples collected from
Luquillo, a town near the north east coast of
Puerto Rico, were dry and almost humid. The
one soil sample gathered from Cayey was dry
and collected from a tall height in Gurabo,
town located around mountains. The four
samples from Gurabo were also dry.
The testing of these soil samples never
passed the enrichment phase due to technical
complications with the bacteria being used.
Under recommendation of Dr. Rubin, we re-
tested one of the samples taken in the past with
a better batch of bacteria.
Only one of the previous soil samples
was tested. This sample had come from
Gurabo, in a dry canal in a closed-access
neighborhood. The re-testing resulted in the
growth of plaques on the agar. This allowed us
to eventually move on to the plaque
purification process.
Currently, the process within this
project is still in the plaque purification
process. This is due to the fact that a few of the
previous plates had been infected with fungi, or
the solution for the third purification did not
show any phages. Continued attempts would
involve taking a plaque from the plate of the
second purification and attempting another
third purification.
Discussion
This experiment has been an experience
in laboratory work that involves: field work
such as gathering samples; hands on work in
the laboratory in the attempt of finding and
purifying the mycobacteriophages; and finally
processing the newly discovered phages to then
afterwards store it in a database. This process
has shown us to be tedious and at times
frustrating due to negative results or
complications in the growth of the phages. The
work has been interrupted and slowed down by
several complications. Currently, the process
has yet not passed the third plaque purification.
Future works would involve completing
the entire process of identifying the phage to
afterwards be sent for recording. Important
details to be comprehended are the conditions
the phage was discovered and how this can be
lead to discover more phages. As occurred in
this lab, only one soil sample had traces of
phages, which could mean various interesting
clues. In the soil samples from Luquillo, the
soil might have been exposed to coastal area
conditions. In the
The implications of utilizing phages and
understanding their unique behavior could lead
into interesting developments in the field of
medicine. There have been many recent
research articles based on phage therapy, which
could lead into new promising discoveries.
Based on Clark and March’s review paper,
many other labs have deemed phages the
potential to be transporters of vaccines and
even as alternatives to antibiotics and are
testing it.
Literature cited
Clark J., and March J. 2006. Bacteriophages
and biotechnology: vaccines, gene therapy and
antibacterials. Trends in Biotechnology
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10.1016/j.tibtech.2006.03.003 24:5 Availble at:
http://www.sciencedirect.com.uprcdb.cayey.upr

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Hanlon G. 2007. Bacteriophages: an appraisal
of their role in the treatment of bacterial
infections. International Journal of
Antimicrobial Agents [Internet] [Cited 2013
May 9] 10.1016/j.ijantimicag.2007.04.006
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(http://www.sciencedirect.com/science/article/p
ii/S0924857907002038)