1. Article
Abstract
Mycobacteriophages are being studied to
comprehend bacterial pathogenesis, to
perform phage therapy, and for a better
understanding of basic molecular
biology. The research question of this
experiment is whether or not novel and
interesting mycobacteriophages can be
isolated from the tropical soils of Puerto
Rico. It is hypothesized that unique
mycobacteriophages with useful properties
will be isolated and characterized
successfully. The purpose of this
investigation is to isolate
mycobacteriophages from soil samples so
they can be characterized using genomic and
proteomic approaches. A
mycobacteriophage was discovered in the
seventh soil sample obtained from Caguas
P.R. The sample was then enriched and
filtrated. The filtrate was then streaked on a
Petri dish and incubated. After obtaining the
mycobacteriophages, plaques were extracted
and purified 4 times. The first purification
had to be repeated with a different phage
plug since no plaques were present on the
plate. The first two purifications showed a
greater concentration of plaques on the
second and third streak regions when
compared to the first region, unlike the third
purification. The third purification had to be
repeated as well due to contamination. The
isolated phage, named Musamodel, seems to
have a lytic life cycle since the plaques are
circular and completely clear, characteristic
of a virulent phage. This investigation
proves that mycobacteriophages can be
effectively isolated from the soils of Puerto
Rico. Since the investigation was not
finished, some techniques and procedures
still remain to be fulfilled. These procedures
include the spot test, making phage stocks,
the empirical test and the 10 plate
preparation, and finally the analysis and
bioinformatics/ DNA sequencing of the
mycobacteriophage.
Introduction
Mycobacteriophages are viruses that
infect a specific type of bacteria belonging
to the mycobacteria genus. “These
bacteriophages are the most ample life forms
in the biosphere and possess genomes
characterized by highly diverse genetic
designs” (Hatfull et al. 2006). The goal is to
characterize novel mycobacteriophages
using genomic and proteomic approaches,
therefore the following research questions
will be answered: Can novel and interesting
mycobacteriophages be isolated from the
tropical soils of Puerto Rico? If so, could
these be characterized? It is hypothesized
that unique mycobacteriophages with useful
Isolation and Characterization of Mycobacteriophage
Musamodel from Tropical Soils of Puerto Rico
Mónica C. Del Moral, Félix J. Vallés, Dr. Michael Rubin, RISE Program, Department of Biology, University of
Puerto Rico at Cayey

2. 2
properties will be isolated and characterized
successfully.
Mycobacteriophages could be
identified as virulent or lysogenic. Virulent
phages follow a lytic life cycle by lysing all
bacteria they infect. On the other hand,
temperate phages follow a lysogenic life
cycle in which they either enter a dormant
state by incorporating their genetic material
into the DNA of the host bacteria, or
replicate and lyse the host bacteria like
virulent phages. Mycobacteriophages
consist of a capsid, which contains the
genetic material, the genetic material or
DNA, and a tail, which serves to attach to
bacteria and functions as a passageway for
DNA from tail to bacterium.
Mycobacterium can be used for
hosting or infection in labs to obtain these
mycobacteriophages for later
characterization, annotation and
classification. In this case, the two
mycobacteria used for infection are
Mycobacterium smegmatis (M. smegmatis)
and Bacillus cereus (B. cereus). M.
smegmatis, an aerobic organism usually
found in the soil, water, and plants. They are
commonly used in research due to their fast
growing and non-pathogenic behavior. B.
cereus, on the other hand, can be obligate
aerobes or facultative anaerobes, commonly
found in the soil. They are one of the best-
understood prokaryotes and are currently
used as a model for differentiation, gene
regulation, and cell cycle events in bacteria.
Both bacteria are Gram-positive, acid-fast,
and spore forming, with a rod-shaped
morphology and a strepto-cell arrangement.
If the infection of the mycobacterium
is successful, a mycobacteriophage should
be obtained from the soil sample. The
mycobacteriophage discovered will be
characterized using morphologic, genomic,
and proteomics techniques. Genomic
sequences from the mycobacteriophages will
be identified, annotated using
Bioinformatics tools, and then submitted
into an international DNA database. The
mycobacteriophage are usually classified
into clusters using comparative techniques,
genomic DNA restriction digestion patterns,
polymerase chain reaction and separation of
proteins using polyacrylamide gel
electrophoresis. Finally, once discovered
the mycobacteriophages are added to the
other 1000 mycobacteriophages that have
been sequenced, annotated and analyzed by
the scientific community. “Genome
clustering facilitates the identification of
genes that are more likely to genetically
mutate and to have been exchanged in recent
evolutionary times” (Hatfull et al. 2010).
According to Pope et al. 2011,
“mycobacteriophages provide extremely
useful tools for the study and manipulation
of their host”, hence, bacteria could be better
understood by the study of
mycobacteriophages, and could be used for
different research and medicine application.
Since bacteria cause animal diseases, and
bacteriophages kill bacteria, bacteriophages
could be used in substitution of antibiotics.
Mycobacteriophages are being studied to
understand bacterial pathogenesis, to
perform phage therapy, and for a better
understanding of basic molecular biology.

3. 3
Materials and methods
The methodology used has four
major steps: collection of the environmental
sample, isolation of the phage from the
environmental sample, purification of the
phage, and characterization of the phage.
During the sample collection phase, the
objective was to capture a phage to then
isolate it. For the collection, it was
necessary to use a sterile spoon and deposit
at least one gram of soil into a sterile bag.
For each sample, the location (by GPS o
Google Earth), date and time of the
collection, the temperature, the depth at
which the sample was obtained, soil
description, and site description was
recorded. The sample was then taken to the
lab to extract the phage from it and tested on
two hosts: Bacillus cereus (B. cereus) and
Mycobacterium smegmatis (M. smegmatis).
First, 0.500 grams were added into two
weight boats. Then, two 50mL conical
tubes were labeled with the initials, date, the
collection location, sample number, and
respective host name. Afterwards, 10mL of
the enrichment mix were pipetted. In the
case of the M. smegmatis labeled tube, the
enrichment mix was Master Mix, made with
H2O, 10x 7H9/glycerol broth, AD
supplement, and 100 mM CaCl2. On the
other hand, for the B. cereus labeled tube,
the enrichment mix was tryptic soy broth
(TSB). In addition, 1000µL of the
correspondent bacteria and the 0.500 grams
of soil to the tubes were added. At last,
these tubes were left incubating at 37˚C and
shaken at 220 rpm for 24 hours.
The following day the enriched
sample was ready for isolation. In this step,
first, the enrichments were centrifuged at
3,000 rpm for 10 minutes to pellet the
particulate matter. Then, two new 15mL
conical tubes were labeled with the initials,
the date, the collection, the sample number,
and the name of the corresponding bacteria
hosts. Further on, 5mL of the B. cereus
enrichment supernatant were pipette into an
assembled filtration unit, with a 0.22-µm
filter and a 5mL syringe, and the filtrate was
added into the 15mL tube. This step was
repeated with the M. smegmatis enrichment.
If these filtrates were not to be used
immediately, we stored them at 4˚C.
An alternative way of “filtering” the
enrichment was by centrifugation. First, the
enrichments were centrifuged. Secondly,
two centrifugation microtubules were
labeled with the sample information. Then,
1,000µL of the corresponding enrichment
supernatant were added into each
microtubule. Thirdly, these microtubules
were centrifuged at 10,000rpm for 10
minutes. Subsequently, 500µL of the
corresponding microtubules supernatant
were added into two new microtubules
labeled as filtrate, along with the other
information.
The filtrate was then used to streak
on agar plates and assess if the sample had
phages. In order to perform the streak, one
plate prepared for B. cereus was labeled
with tryptic soy agar (TSA) and another
plate prepared for M. smegmatis prepared
with Luria base agar (LB) with the sample
information. Once the streak was performed
with both filtrates, 4.5mL TSA top agar was
mixed with .5mL of B. cereus, and deposited
it on the correspondent plate from the most
diluted region. This was repeated with the
M. smegmatis bacteria, but with the LB top

4. 4
Table 1. The Seven Soil Samples Collection Data
agar. After about 30 minutes of wait for the
top agar to harden, the inverted plates were
incubated at 37˚C for 24 hours.
The day after, the plates were
assessed to see if they had phage plaques. If
the plates did not have any plaques, a new
soil sample needed to be collected and
repeat the procedure until a phage was
found. When one of the plates had phage
plaques, the purification stage of the
experiment was begun. First, a
centrifugation microtubule was labeled.
Secondly, 50µL of phage
buffer was added to the
microtubule. Then, using a
1,000µL micropipette, a plaque
plug was extracted and
deposited it in the phage
buffer. After waiting a few
minutes for it to dissolve, a M.
smegmatis plate was labeled,
because that was our phages
host, as the first purification, along with the
usual information. Additionally, a streak
was performed using the mix we prepared.
Then 4.5mL of LB top agar and .5mL of M.
smegmatis mix were pipetted to the plate, it
was allowed to harden, and it was incubated
inverted at 37˚C for 24 hours. Further on,
two more purifications were done, each
made with a phage plug from the previous
purification plate. If at any of the steps the
purification did not result with any plaques,
the same purification had to be repeated.
Once the three purifications were
obtained, a second enrichment was done, but
using a phage plug from the third
purification. First, one 50mL conical tube
was labeled with our initials, the phages
name, the date, and “second enrichment”.
Secondly, 10mL of Master Mix, 1,000µL of
M. smegmatis were added using a
micropipette, and a phage plug from the
third purification was added. Then, this tube
was left incubating at 37˚C and shaking at
220rpm for 24 hours.
Results
None of the first six soil samples that
were enriched, filtered and streaked on the
Petri dishes had positive results. It was not
until the seventh soil sample that a phage
was found. The Petri dish displayed almost
no plaques on the first streak region, a large
concentration of plaques on the second
region, and less concentration of plaques on
the third region. The first purification
attempt had negative results since no
plaques were present on the plate. Therefore,
the first purification was done with another
phage plug, and positive results were
obtained.
Figure 1. The Seventh Soil Sample Phage Plate

5. 5
The first and second purification
plates showed a greater concentration of
plaques on the second and third streak
regions when compared to the first region.
This is not supposed to occur. This might
have been due to adding the top agar and
bacteria mix from one of the less diluted
regions or moving the plate by mistake
before the top agar solidified. The third
purification had to be repeated since it had
gotten contaminated.
The repeated third purification had positive
results.
However, it had the same pattern of phages
concentrations as the first two purifications.
The second region was more diluted than the
third region.
Discussion
After six unsuccessful attempts to
find a mycobacteriophage, successful results
were obtained with the seventh soil sample
(Table 1). Interestingly, this sample was
collected right next to the roots of a plantain
plant. This was the only sample that was
collected that close to a plant. The sample
probably had phages, because the soil was
more fertile there and it uses M. smegmatis
as bacterial host to reproduce. Once the
presence of a phage was confirmed it was
named Musamodel. The name comes from
the word “Musa”, meaning inspiration, and
the word “model” in honor of one of the
researchers and due to its function in the
study.
The novel phage, Musamodel,
appears to be virulent and to have a lytic life
cycle based on the plaques found on the
latest purification (Figure 5). These plaques
seem to be perfectly circular and completely
clear, characteristic of a virulent phage.
However, in order to know for certain the
characteristics of our phage. Other steps
Figure 2. First Phage Purification Plate
Figure 3. Second Phage Purification Plate
Figure4.Third Phage Purification Plate (First Attempt)
Figure 5. Third Phage Purification Plate
(Second Attempt)

6. 6
need to be carried out. Therefore, future
plans include doing the spot test, phage
stocks, empirical test, the ten-plate
preparation, the phage analysis, and the
bioinformatics/sequencing portion of the
characterization.
Ultimately, the hypothesis unique
bacteriophages with useful properties will be
isolated and characterized successfully was
partially proven. Musamodel was isolated,
but not characterized. Currently work is
being done towards achieving that goal. It is
inferred that phages can be isolated from the
tropical soils of Puerto Rico, because of the
richness of the soils. There is no doubt that
a lot needs to be learned from these phages
and that there are many yet to be discovered.
Acknowledgements
 Dr. Michael Rubin- Howard Hughes
Program director at the University of
Puerto Rico at Cayey and mentor
 Eduardo Correa- teaching assistant and
mentor
 Giovanni Cruz- laboratory technician
 Gustavo Martínez- teaching assistant
Literature cited
 Hatfull GF, Pedulla ML, Jacobs-Sera D,
Cichon PM, Foley A, et al. (2006)
Exploring the Mycobacteriophage
Metaproteome: Phage Genomics as an
Educational Platform. PLoS Genet 2(6):
e92.
 Pope WH, Ferreira CM, Jacobs-Sera D,
Benjamin RC, Davis AJ, et al. (2011)
Cluster K Mycobacteriophages: Insights
into the Evolutionary Origins of
Mycobacteriophage TM4. PLoS ONE
6(10): e26750.
 Hatfull GF, Jacobs-Sera D, Lawrence
JG, Pope WH, Russell DA, et al. (2010)
Comparative Genomic Analysis of 60
Mycobacteriophage Genomes: Genome
Clustering, Gene Acquisition, and Gene
Size. Journal of Molecular Biology
397(1): e119-43