1. Article
Abstract
Mycobacteriophages are viruses that infect a
specific type of bacteria belonging to the
mycobacteria genus. Mycobacteriophages
can be obtained by infecting mycobacterium
in a controlled environment. The two types
of mycobacterium used in the lab were
Mycobacterium smegmatis, suitable for
investigation due to its fast growing and
non-pathogenic behavior, and Bacillus
cereus, currently used as a model organism
for different live events in bacteria.
Mycobacteriophages are being studied to
comprehend bacterial pathogenesis, to
perform phage therapy and for a better
understanding of basic molecular biology.
The purpose of this investigation is to isolate
novel and interesting bacteriophages from
the tropical soils of Puerto Rico so they can
be characterized using genomic and
proteomic approaches. A
mycobacteriophage was discovered in our
seventh soil sample 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 3 times.
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 GF, et al. 2006).
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 that will be used for infection
Isolation and Characterization of Mycobacteriophage
Musamodel from Tropical Soils of Puerto Rico
Mónica C. Del Moral and Félix J. Vallés, RISE Program, Howard Hughes Medical Institute, Department of
Biology, University of Puerto Rico at Cayey

2. 2
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 its 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
bacteria; form spores and have 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 will be classified into
clusters using comparative techniques,
genomic DNA restriction digestion patterns,
polymerase chain reaction separation of
proteins using polyacrylamide gel
electrophoresis. Finally, the
mycobateriophage will be 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 GF, et al. 2010).
According to (Pope WH, 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 implications on research and
medicine. Since bacteria cause animal
diseases, and bacteriophages kill bacteria,
bacteriophages could be exploited to
function as antibiotics. Mycobacteriophages
are being studied to understand bacterial
pathogenesis, to perform phage therapy and
for a better understanding of basic molecular
biology.
Our goal is to characterize novel
bacteriophages using genomic and
proteomic approaches. However, can novel
and interesting bacteriophages be isolated
from the tropical soils of Puerto Rico? If so,
could these be characterized? We believe
that unique bacteriophages with useful
properties will be isolated and characterized
successfully.
Materials and methods
This experiments methodology is
composed of 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 is to
capture a phage to then isolate it. For the
collection it is necessary to use a sterile
spoon and deposit at least one gram of soil
into a sterile bag. For each sample, we
recorded the location (by GPS o Google
Earth), date and time of the collection, the
temperature, the depth at which the sample

3. 3
was obtained, soil description, and site
description. The sample was then taken to
the lab to extract the phage from it and
tested it with two hosts: Bacillus cereus (B.
cereus) and Mycobacterium smegmatis
(M.smegmatis). Once in the lab after
cleaning the work area aseptically, we first
added 0.500 grams into two weight boats.
Then, we labeled two 50mL conical tubes
with our initials, date, the location where the
sample was collected, sample number, and
respective host name. Afterwards, we
pipette 10mL of the enrichment mix. In the
case of the M. smegmatis labeled tube, the
enrichment mix is Master Mix, made with
H2O, 10x 7H9/glycerol broth, AD
supplement, and 100 mM CaCl2. On the
contrary, for the B. cereus labeled tube, the
enrichment mix is tryptic soy broth (TSB).
In addition, we added 1000µL of the
correspondent bacteria and the 0.500 grams
of soil to the tubes. At last, these tubes are
left incubating at 37˚C and shaking at 220
rpm for 24 hours.
The following day the enriched
sample is ready for the next step: isolation.
In this step, we first centrifuge the
enrichments at 3,000 rpm for 10 minutes to
pellet the particulate matter. Then, after
aseptically cleaning the work area, we
labeled two new 15mL conical tubes with
our initials, the date, the location where the
sample was collected, the sample number,
and the name of the corresponding bacteria
hosts. Further on, we pipette 5mL of the B.
cereus enrichment supernatant into an
assembled filtration unit, with a 0.22-µm
filter and a 5mL syringe, and added the
filtrate into the 15mL tube. This step was
repeated with the M. smegmatis enrichment.
If these filtrates were not to be used
immediately, we could store them at 4˚C.
An alternative way of “filtering” the
enrichment is by centrifugation. First, we
centrifuged the enrichments. Secondly, we
aseptically clean the work area and we
labeled two centrifugation microtubules with
our initials, the date, the location where the
sample was collected, the sample number,
and the correspondent bacteria host. Then,
we added 1,000µL of the corresponding
enrichment supernatant into each
microtubule. Thirdly, we centrifuged these
microtubules at 10,000rpm for 10 minutes.
Subsequently, we added 500µL of the
corresponding microtubules supernatant into
two new microtubules labeled as filtrate,
along with the other information.
The filtrate is then used to streak on
agar plates and assess if the sample had
phages. In order to perform the streak, we
labeled one plate prepared for B. cereus with
tryptic soy agar (TSA) and another plate
prepared for M. smegmatis prepared with
luria base agar (LB) with our initials, the
date, the location where the sample was
collected, the sample number, and the
respective bacteria host name. Once we
performed the streak in three quadrants with
both filtrates, we mixed 4.5mL TSA top
agar with .5mL of B. cereus, and deposited
it on the correspondent plate from the most
diluted region. This is repeated with the M.
smegmatis bacteria, but with the LB top
agar. We waited about 30 minutes for the
top agar to harden and incubated the plates
inverted at 37˚C for 24 hours.
The day after, we assessed if any of
the plates had phage plaques. If the plates

4. 4
did not have any plaques, we needed to
collect a new soil sample and repeat the
procedure until a phage is found. When one
of the plates had phage plaques, we
continued to the next step of the experiment:
the purification of the phage. First, we
labeled a centrifugation microtubule with
our initials, the date, and original phage
plug. Secondly, we added 50µL of phage
buffer to the microtubule. Then, using a
1,000µL micropipette, we extracted a plaque
plug and deposited it in the phage buffer.
After waiting a few minutes for it to
dissolve, we labeled a M. smegamtis plate,
because that is our phages host, as the first
purification, along with the usual
information. Additionally, we performed a
streak in three regions using the phage plug
and buffer mix we prepared. We then
pipette the 4.5mL of LB top agar and .5mL
of M. smegmatis mix to the plate, allowed it
to harden, and incubated it 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, we had to
repeat that same purification.
Once we had our three purifications,
we did a second enrichment, but using a
phage plug from the third purification. First,
we labeled one 50mL conical tube with our
initials, the phages name, the date, and
“second enrichment”. Secondly, we pipette
10mL of Master Mix, 1,000µL of M.
smegmatis using a micropipette, and added a
phage plug from the third purification.
Then, we left this tube incubating at 37˚C
and shaking at 220rpm for 24 hours.
This is as far as we have reached in
the methodology. Later on, we will do a
Spot test to see at which dilution we should
obtain a web pattern with the phages. Then,
we will do an Empirical test to see and
confirm our web pattern. Subsequently, we
will prepare a medium titer phage lysate
(MTPL) using the plate that shows the most
clear web pattern. This MTPL will enable
us to replicate the dilution and prepare the
high titer phage lysate (HTPL). With this
step we would conclude our purification
segment and move on to the characterization
portion of our work. First, we will observe
our phages using a scanning electron
microscope. Afterwards we will isolate the
DNA, amplify it with PCR, run a
polyacrylamide gel electrophoresis, send our
phage DNA for sequencing, and at last, use
bioinformatics to characterize the phages
genome.
Results
Neither 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 and we saw phage plaques. 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, we tried doing the first
purification with another phage plug, with
which we obtained positive results. 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, which is not

5. 5
supposed to occur. This might have been
due to adding the top agar and bacteria mix
from one of the less dilute regions or
moving the plate before the top agar solidify
by mistake. 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, that instead of the third region
being the most dilute it was the second.
Discussion
After six unsuccessful attempts to
find a mycobacteriophage, we obtained the
wanted results with our seventh soil sample.
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. We believe that this sample
had phages because the soil was more fertile
there. In addition, we know that this phage
uses M. smegmatis as bacterial host to
reproduce. Once we had confirmed that we
found a phage, we named it Musamodel.
The name is made up of “Musa”, meaning
inspiration, and “model” because of the
name of the researcher who found it and as
the model to be studied.
Up to this point, we believe that our
phage, Musamodel, is virulent and has a
lytic life cycle because of the plaques on the
latest purification. These plaques seem to be
perfectly circular and completely clear,
characteristic of a virulent phage. However,
we need to continue with the steps of the
research in order to know for certain the
characteristics of our phage. Therefore, our
future plans include doing the empirical test,
the ten plate preparation, the phage analysis,
and the bioinformatics/sequencing portion of
the characterization.
Ultimately, we can say that the part
of our hypothesis that stated that we would
find a phage was accepted. Although, we
have not been able to characterize
Musamodel yet, we are currently working
towards achieving that goal. Still, we have
confirmed that phages can be isolated from
the tropical soils of Puerto Rico because of
the richness of the soils.
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

6. 6
Size. Journal of Molecular Biology
397(1): e119-43
Appendices
 Appendix 1: Soil sample
collection data
 Appendix 2: Seventh soil sample
plate
 Appendix 3: First purification
 Appendix 4: Second purification
 Appendix 5: Third purification
 Appendix 6: Third purification-
second attempt
Appendix 1. Soil sample collection data
Appendix 3. First purification Appendix 2. Seventh soil sample plate

7. 7
Appendix 4. Second purification Appendix 5. Third purification
Appendix 6. Third purification- second attempt