1. Isolation and Characterization of Mycobacteriophages
Isolated from Tropical Soils of Puerto Rico.
Charlene N. Rivera-Bonet, Mónica Rivera-Torres
ABSTRACT
Phages are often studied and characterized in order to investigate their potential functions in
terms of scientific or medicinal innovations. A sample was collected from the soil using aseptic
techniques. Then it was enriched with an M. smegmatis culture and incubated for 24 hours at
37ºC. From the enrichment the supernatant is later filtered and used for purification. After
purifying three times using an agar plate and M. smegmatis culture the plaques found in the plate
(clear spots) indicate the phages location. The next steps to continuing this project are dilutions
and creating a web pattern.
Introduction
Bacteriophages, better known as phages, are
viruses that infect bacteria. Phages are made
up of a head that contains the genetic
material, and a tail, which they use to inject
their DNA to the host cell. They have two
types of reproduction, by which they are
classified. One is the lytic life cycle, which
is carried by lytic bacteriophages. They lyse
the host as a normal part of their life cycle.
The temperate phages carry out the
Lysogenic life cycle which consists of the
integration of the phage’s DNA into the
host’s genome. As the bacteria divide, it
divides (Kaiser 2012)..
Mycobacteriophage is a virus that only
infects bacteria belonging to the
Mycobacteria genus. Examples of this type
of phage are tuberculosis and M. smegmatis.
Mycobacterium smegmatis has become the
most important bacteria for biological
studies. It is easy to be cultured and
reproduces quickly. It is non-pathogenic to
humans or other animals. Its basic structure
and metabolism are common to other
mycobacteria, including ones that can cause
devastating diseases (Higa 2013).
Several advantages can be found when
studying bacteriophages. By studying
phages novel genes can be discovered, and
new therapies against antibiotic resistant
pathogenic bacteria can be developed.
Hypothesis
We will be able to find bacteriophages in
our soil sample because of its condition and
the location it was collected from.

2. Materials and Methods
The materials used throughout this
investigation included sterile spoon, test
tubes and GPS for sample collection, and
Agar plates, Top Agar, sterilization filters,
syringes, phage buffer, M. smegmatis
culture, micropipettes, pipettes, vortexer,
centrifuge and incubators for the rest of the
experiment.
Soil Sample Collection
Soil sample was collected using a sterile
spoon and test tube. Data about the area the
sample was taken from was annotated.
Enrichment
The first step after the soil sample collection
was the Enrichment. For the enrichment o.5
grams of soil sample were measured and
added into 50mL of solution containing 8mL
sterile water, 1mL sterile 10x broth, 1mL
AD supplement,0.1mL of 100mM CaCl2.
1ml of late log/early stationary phase M.
smegmatis culture (48 hour culture) were
also added to the flask. Afterwards it was
incubated at 37º C, shaking at 220rpm for a
24 hours period.
Harvesting
After 24 hours of incubation, the sample
spun at 3,000rpm for 100 minutes to pellet
particulate matter, including most of the
bacterial cells. Then poured supernatant
from centrifuged sample to a 50mL conical
tube, filter-sterilized enrichment sample,
capped and label the rube
Plaque Streak
Using a sterile wooden stick the enrichment
was streaked back and forth across the top
third or the agar plate without lifting it.
Cover up the petri dish and discard the
wooden stick.
This process was repeated using a new
wooden stick; streak the adjacent un-
streaked portion of the agar making sure to
overlap on the first few strokes, beginning in
the area streaked before.
For the remaining portion of the plate, the
streaking process was repeated on the last
portion of the plate.
Next 4.5mL of Top Agar were added to
0.5mL of M. smegmatis, using serological
pipet to carefully dispense it onto the most
dilute area of the plate and allow it to spread
across the plate to the most concentrated
areas. Then covered the plate and allowed
the Top Agar to become hard. After it
became hard it was incubated at 37ºC and to
be checked the next day.
Plaque Purification:
In order to obtain pure phage, three plaque
purifications were made.
After following the correct asceptic
techniques, a plaque from the plate was
chosen and picked with a 100uL
micropipette tip. Then, it was transferred to
a sterile microtube with 100uL of Phage
Buffer. Using a sterile wooden stick, the
solution inside the microtube into a petri
dish was streaked on the plate, following the
same streaking steps discussed before.
4.5mL of Top Agar were added to 0.5mL of
M. smegmati,and using a serological pipet it

3. was carefully dispensed onto the most dilute
area of the plate and allow it to spread
across the plate to the most concentrated
areas. The plate was covered and the Top
Agar was allowed to become hard.
Afterwards, it was incubated at 37ºC and
checked the next day.
Results
Sample Collection
The soil sample was collected February 18,
2013 at 7:30pm. The conditions were 73°F,
1 inch deep, in a somewhat moist area, 6
inches away from cement sidewalk, 2 feet
from a house and 10 feet from a large tree.
The exact GPS location was 18.23657 N
66.04429 W in Caguas, Puerto Rico.
Enrichment, Harvesting and Plaque
Purification
Phages were found on the first try. Since we
had the opportunity to name our phage, we
picked the name Monchar. After the
enrichment was made, the first plaque
purification came out well, as can be seen in
Figure1.
Fig 1. 1st
Plaque Purification.
The second and third plaque purification
took more time and trials. The second
plaque purification was done three times.
The first time it did not come out right,
because the bacteria were defective. The
second time it got full of fungus and the
third time, shown in Figure 2, it was
successful.
Fig 2. 2nd
Plaque Purification.
The third plaque purification had to be done
six times. Each time something different
happened. The first second and third time
nothing happened. The last three times
bacteria colonies formed. An example of
this is shown in Figure 3.
Fig 3. 3rd
Plaque Purification example.

4. The second plaque purification, was
repeated but nothing came out as can be
seen in Figure 4.
Fig 4. 2nd
Plaque Purification repetition.
Conclusion
Phages were found in our soil sample
collection and our hypothesis was proven
correct. Due to a series of misfortunate
events including defective bacteria,
unwanted colonies or fungus, we only
reached the second plaque purification. The
soil from where the phages were collected
was fertilized with compost. The type of soil
may have had a lot to do with the presence
of phages since compost is so rich in
nutrients and allows bacteria to flourish.
This organic matter that fertilized the soil
was from the fallen leaves of tree located a
short distance from the collection area.
Another factor that could have affected the
discovery was the temperature. The soil was
collected during a cloudy and partially
humid day.
Next steps would involve Web Patterns, or
Dilutions, and High Titter Assay. Further
research can be done in order to characterize
this phage and conclude its functionality in
the field of medicine.
References
WiseGeek Conjecture Corporation
[Internet]. [2013]. WiseGeek Conjecture
Corporation; [cited 2013May14]. Available
at: http://www.wisegeek.com/what-is-
mycobacterium-smegmatis.htm
Doc Kaiser's Microbiology Home Page
[Internet] [2012]. Doc Kaiser's
Microbiology Home Page; [cited
2013May14]. Available at:
http://faculty.ccbcmd.edu/courses/bio141/lec
guide/unit3/viruses/lytlc.html