1. Mycobacteriophage isolation from tropical soil
sample: Serotinus
Alejandra M. De Jesús-Soto1
, Kenny J. Colón-Colón2
1
Department of Mathematics, University of Puerto Rico at Cayey, Puerto Rico
2
Department of Biology, University of Puerto Rico at Cayey, Puerto Rico
A B S T R A C T
Mycobacteriophages are viruses that infect a specific type of bacteria. This abundant
microorganism can be easily obtained from soil samples. Using Mycobacterium smegmatis as a
host, a mycobacteriophage was isolated and purified from a soil sample. The goal is to obtain a
pure phage population in order to analyze the information and then include it in the
Mycobacteriophages Database. The first step was to collect a soil sample in order to make the
enrichment and filtrate. Three plaque purifications followed this step in the process. A pure
phage population was isolated from a tropical soil in Puerto Rico and named Serotinus. Future
work includes performing the spot test, a process to identify the phage’s web pattern. Knowing
the phages genetic information allows the identification of characteristics that may lead to
important discoveries in modern medicine. Phages therapy seems to be an alternative medical
treatment.
1. Introduction
Mycobacteriophages are DNA
viruses that infect a specific type of bacteria
belonging to the mycobacteria genus. These
are the most abundant type of
microorganism in the biological universe.
Mycobacteriophage infections may or may
not lead to the death of the bacterium. This
depends on its life style, which can be lytic
or lysogenic. Lytic cycles results in the
destruction of the infected bacteria and its
membrane. It is used by temperate or
virulent phages to achieve reproduction. The
virulent phage infects bacteria with genetic
material in order to undergo cell lysis. On
the other hand, temperate phages undergo
lysogenic cycle, in which the phage DNA
first integrates into the bacterial
chromosome to produce the prophage and
make the host a lysogen. When the
bacterium reproduces, the prophage is also
copied. The prophage can be copied or
activated, a process in which the prophage
exits the chromosomal material to enter the
lytic cycle.
The findings of existent phage
research have traversed many arenas of
genetics and have even instigated the
conception of new therapies and medical
treatments (George 2013). A critical
problem in heath arose due to the emergence
of pathogen bacteria resistant to most
currently available antimicrobial agents. The
development of alternate anti-infection
modalities is one of the highest priorities of
modern medicine. Therefore, phage therapy
seems to be a solution for this problem.
Mycobacteriophages have been
isolated from a variety of environments
around the world. They can be easily
obtained from soil samples. Using

2. Mycobacterium smegmatis as a host, we
sought to determine the presence of
mycobacteriophages in a soil sample.
Therefore, we hypothesized that we can
isolate a mycobacteriophage population
from a tropical soil sample in Puerto
Rico. The objectives of this research are to
isolate, purify, and harvest a novel phage
population while the ultimate goal is to
identify the characteristics of the phage that
may serve modern medicine.
2. Materials and Methods
Figure 1.Flow chart of the experimental procedure. Steps to isolate, purify and characterize a mycobacteriophage population
from a soil sample.
Each of the stages of the experiment was
carried out by using the Sea-phages
Resource Guide (2012) of the Science
Education Alliance. There were some
adjustments on procedures that had to be
executed throughout the process to ensure
the isolation and harvest of the
bacteriophage. For this reason, alterations
made during the procedure are included.
Note that all materials used underwent a
sterilization process, either by chemical
methods using ethanol at 70% and Lysol®
or by physical method using autoclave in
aseptic technique during their use. It is
recommended not to talk while doing any of
the procedures to further avoid
contamination.

3. 2.1. Sample Collection
Soil sample was collected from optional
locations such as in wet places or where
there was decomposition. After digging a
few centimeters the collected samples were
maintained in a sterile container. Data
regarding localization coordinates,
environmental temperature, excavation
depth and moisture content was recorded.
2.2. Enrichment
In a 50 mL conical tube, 0.500 g of the soil
sample was mixed with Master Mix,
substance that contained 8 mL of sterilized
water, 1 mL of 10x 7H9/glycerol broth and
1 mL of AD supplement and 0.1 mL of CaCl
(Science Education Alliance. 2012). Finally,
1 mL of Mycobacterium smegmatis was
added and the solution was incubated at
37°C and aerated at 220 rpm for 16-24 hours
(Science Education Alliance 2012).
2.3. Filtration
After completion of the incubation period,
solution was centrifuged at 3,000 rpm for
15-20 minutes. Afterwards, supernatant was
filter-sterilized and poured into a 15mL
conical tube. Both the pellet and the
supernatant were stored.
2.3.1 Centrifuge
Another option to obtain a purer sample
from the enrichment protocol after
centrifuging the conical tube of the
enrichment protocol was to take 1 mL of the
supernatant from the conical tube and
transfer it to a microtube. The microtube
was centrifuged at 10,000 rpm for 10
minutes. Then, 500 µL of the supernatant of
the centrifuged microtube was transferred to
another microtube to obtain the filtrate.
2.4 Plate Streaking
A sterile utensil (wooden stick) was
moistened with the filter-sterilized
supernatant once. It was then used to gently
swipe streaks on the Luria medium agar
plate in quadrants. Figure 2 shows the
correct way of streaking a plate. The
segment labeled as (1) was first streaked,
and then, another sterile swab was used to
drag part of sample segment (1) to segment
(2). The process of taking a sample from
segment (2) and dragging it to segment (3)
was repeated. Then, 4.5 mL of ~55.0o
C Top
Agar and 0.5 mL of the Mycobacterium
smegmatis were poured into the plate from
quadrant 3 to quadrant 1. The plate was
closed by placing the cap on top. Finally,
after agar solidification the plate was placed
in an incubator at 37°C for ~24 hours
(Science Education Alliance 2012). After
this part of the procedure, the presence of
mycobacteriophages in the collected sample
was finally determined.
2.5. Plaque purification
Figure 2. Streaking Technique. Available source:
http://faculty.mc3.edu/jearl/ML/streak1.gif
After confirming the presence of
mycobacteriophages, the next step consisted
in the extraction of an isolated plaque using
the wooden tip of a sterile swab. Part of the

4. plaque was transferred to a sterile microtube
that contained 25 µL (microliters) of Phage
Buffer (PB) which would help with the
dilution and sustain the mycobacteriophages
on the plaque. The plaque sample was
streaked in a “smeg plate” (2.4 plate
streaking protocol) and was left to rest from
16-24 hours until the next day. This plaque
extraction procedure was progressively
repeated with each plate until the third (3)
plate was obtained. This was done to assure
the pureness of a single type of phage from
each plaque sample. The plaque purification
samples of each three repetitions were
placed in an environment temperature of
4°C (Science Education Alliance. 2012).
2.6 Phage-Titer Assay
The third plaque purification sample went
through another enrichment protocol (2.2).
This was made for supplementing the
purified mycobacteriophage. Eight (8)
microtubes were labeled from 1 to 8. A
volume of 90 µL (microliters) of PB was
added to each microtube. In the microtube
labeled (1), 10 µL (microliters) of the
supernatant of the enrichment of the third
plaque purification sample was added. Later,
10 µL (microliters) were extracted from
microtube (1) to microtube (2). The same
process was done going from the microtube
(2) to microtube (3). This step was repeated
successively until all 8 microtubes were
diluted.
2.7 Empirical Test
A smeg plate was prepared after labeling it
and drawing eight (8) square divisions on
the bottom of it with a number
corresponding to the division on an edge for
better plaque view. Before going to the
important part of the empirical test, 4.5 mL
of TA and 0.5 mL of Mycobacterium
smegmatis were mixed and spread on top of
the Luria medium Agar. Next, 10 µL of the
sample of microtube (1) from the phage-titer
dilutions was added to the square segment
labeled with the same number. The process
was repeated for each microtube sample on
the corresponding numbered segment. The
plate is then placed in an incubator at 37°C
for 16-24 hours (Science Education Alliance
2012).
2.8 Mycobacteriophage Proteomics with
Electrophoresis
The Medium Titer Phage Lysate (MTPL)
was used for conducting the proteomics part
of the experiment. 1 ml of the MTPL was
transferred to a sterile microtube and
centrifuged at 1x104
rpm for 1 hour. Then,
950 μl were aspirated from the supernatant.
Next, 50 μl of a mix of 25 μl of BME with
475 μl of LSE buffer was added to the pellet
of the microtube. The microtube was placed
on a heat-block for 2 minutes of heat
exposure and then was moved to a cooler
with ice for at least 2 minutes. For the
electrophoresis procedure, the gel used was
a precast gel. The running buffer was
prepared by adding 100 ml of 10X TGS
buffer to 900 ml of distilled water. The gel
was rinsed with distilled water and placed
on the electrophoresis device, following that
the running buffer was poured in the wells.
The sample was added in one of wells of the
gel and it was run at 200 volts for about 30
minutes. After the 30 minutes had passed,
the gel was transferred to a rocker where it
was washed with distilled water for 5
minutes, later stained with Bio-Rad BioSafe
Coomassie Blue G-250 Stain and left
shaking for 1 hour. After finishing, the gel
was washed with water for 30 minutes. The
gel was taken for subsequent protein
identification by mass spectroscopy after

5. excising the bands and placing them in
microtubes.
2.9 Preparing phage sample for Electron
Microscopy
A plastic-faced paper was placed over a
counter for working in a clean area. Parafilm
was placed in a petri dish lid. A double-
sided tape was pasted in the parafilm of the
petri dish lid. After removing the liner from
the tape, using tweezers, a new grid (shiny
side up) was placed along the edge of the
double-sided tape. 10 μl of the phage
preparation was added to the grid without
spilling it outside the edge, passed that, it
was left during 2 minutes for that the phage
could retain to the grid. The excess of phage
substance was absorbed (edge of the grid)
using the tip of a filter paper. The grid was
washed two times pipetting 10 μl of sterile
water on top of the grid without touching the
surface of it or spilling outside the edge.
After waiting 2 minutes for the water to
wash the grid, the excess was absorbed
using the tip of another clean filter paper.
Finally, the same process was repeated
adding 10 μl of 1% uranyl acetate to the
grid. The grid is left to air-dry. The samples
were kept by the mentor, Dr. Rubin, for
future analysis using the electron
microscope of the Central and Caribbean
University at Bayamón, Puerto Rico.
3. Results
Twenty individual soil samples were
collected and analyzed (Table 1). Positive
results were obtained from sample #18
which was collected from an urban area in
the city of Cayey. Location coordinates
were: 18.11524 North, 66.137 West. The
sample was collected 2.3 centimeters
beneath the surface in an environment
whose temperature skirted 25.0º Celsius.
After collecting and enriching the
sample, the presence of mycobacteriophages
was confirmed. Then, after three plaques
purifications, a phage with clear plaques was
isolated (Figure 2). From these plaques was
obtained the MTPL, Medium Titter Phage
Lysate. The next step performed was a spot
test in order to identify the
mycobacteriophage’s web pattern to do ten
plates from the dilutions but time made it
difficult to continue so the electrophoresis
assay phage sample was done with the
MTPL and without doing the ten plate
cultivation. The result of the electrophoresis
assay showed that the protein bands were
similar to other previous isolated phage
which means that it is probable that we had
been working with a previously sequenced
mycobacteriophage, but either way, the
bands were excised for a spectroscopy test
for measuring the absorbency of the phage
solution. As for the analysis of the phage
sample using electron microscope, the grid
prepared was left with the mentor to be
taken for a visual evaluation later on.
4. Discussion
This research on the collection and
analysis of a soil sample for the isolation of
a phage has been a worthwhile laboratory
experience. Although the process was
somewhat tedious and frustrating at the
Figure 3: Clear plaques obtained after third
purification

6. beginning, due to all the negative results it
was a learning experience that taught us
patience, dedication and perseverance.
Finally, after analyzing 20 samples, a
mycobacteriophage was isolated and named
Serotinus. After three purifications, the
phage formed clear plaques on his host
Mycobacterium Smegmatis, indicating that
Serotinus is most likely a lytic phage.
Future work would include
completing the entire process of identifying
and characterizing the phage in order to
record and send the information to the
Mycobactoriophages Database. The
implications of isolating phages could lead
to new and interesting developments in
modern medicine. Currently, much research
is being done related to phage therapy, and
the study of novel phage populations could
lead to promising discoveries.
5. Acknowledgments:
This work was funded by the Howard
Hughes Program and the RISE Program at
the University of Puerto Rico at Cayey. The
authors would like to thank Dr. Michael
Rubin, Dr. Edwin Vazquez, Mr. Joseph
Perez, Mr. Giovanni Cruz, Mr. Gustavo
Martínez and Mr. Christopher Quintanal for
their support during the entire process.
6. Literature Cited
Alvarado EJ, Cruz-Arzón JA. 2013.
Mycobacteriophage isolation from tropical
soil sample: Mikriplithari and
Ususindagari. Department of Mathematics-
Physics, University of Puerto Rico at Cayey,
Puerto RicoDepartment of Biology,
University of Puerto Rico at Cayey, Puerto
Rico.
George MP. 2013. Mycobacteriophage Meru:
Isolation and Characterization of a Novel
Mycobacteriophage. [Internet]. [cited 2014
May 26], 5(10):1-3. Available from
http://www.studentpulse.com/articles/770/m
ycobacteriophage-meru-isolation-and-
characterization-of-a-novel-
mycobacteriophage
Rubin M, Vázquez E. 2012. Mycobacteriophage
Proteomics: From Genotype to Phenotype
(There and Back Again)!.Howard Hughes
Program, Department of Biology, University
of Puerto Rico at Cayey.
Science Education Alliance. 2012. SEA-PHAGES
Resource Guide. Howard Hughes Medical
Institute. Chevy Chase, Maryland

7. Table1. Soil samples tested in order to verify the presence of mycobacteriophage population. Table includes sample information: date
of sampling, coordinates, ambient temperature, depth, moisture content, area and proximity.
Sample Date Coordinates Temperature
(ºC)
Depth
(cm)
Moisture
content
Area Proximity
1
AMDS
Feb / 4 / 14 Lat. 18.119793
Long. -66.15790
23.33 2.54 Moist Urban
UPR Cayey
Campus
Tree
Dead Leafs
2
KJC
Feb / 4 / 14 Lat. 18.119506
Long. -
66.157878
23.33 4.4 Dry Urban
UPR Cayey Campus
Dead tree
Trunk
3
AMDS
Feb / 18 /
14
Lat. 18.11801
Long. -66.13711
26.11 3.3 Dry Urban.
Coca Navas Street Cayey, P.R. 00736
Palm leaf
House
Cement
4
KJC
Feb / 18 /
14
Lat. 18.187409
Long. -
66.140466
19.44 2.54 Dry Rural
Urb. Campo Lago, Cidra, P.R. 00739
Sewage
5
AMDS
Feb / 23 /
14
Lat. 18.11797
Long. -66.13706
27.78 5.3 Saturated Urban
Coca Navas Street Cayey, P.R. 00736
Under plant
Roots
6
KJC
Feb / 23 /
14
Lat. 18.119251
Long. -
66.161560
27.22 2.5 Dry Urban
UPR Cayey Campus
Mango Tree
7
AMDS
Feb / 24 /
14
Lat. 18.11524
Long. -66.16313
25.7 2.0 Moist Urban
Antonio R. Barceló Street Cayey, P.R.
00736
Underneath a decaying
fruit
Fruit – tree
8
KJC
Feb / 24 /
14
Lat.18.115079
Long. -
66.155562
24.44 3.8 Moist Urban
Los Veteranos Avenue, Cayey, P.R.
00739
Garbage
Dump
9
AMDS
March / 9 /
14
Lat. 18.11529
Long. -66.14004
27.06 3.1 Dry Urban
Ciaprian Ortiz Rodriguez Avenue
Cayey, P.R. 00736
Underneath sheep
excrement
Tree

8. 10
AMDS
Mar / 9 / 14 Lat. 18.12745
Long. -66.12056
26.2 5.2 Saturated Rural
Vegas Cayey, P.R. 00736
Dairy
Cows excrement
Cows
11
KJC
Mar / 9 / 14 Lat. 18.187424
Long. -
66.140468
17.78 4.4 Saturated Urban
Urb. Campo
Lago, Cidra, P.R. 00739
Water
Drainage
12
KJC
Mar / 9 / 14 Lat. 18.187581
Long. -
66.140479
25.56 3.17 Dry Urban
Urb. Campo
Lago, Cidra, P.R. 00739
Cement
Drainage
13
AMDS
Mar / 16 /
14
Lat. 18.11402
Long. -66.16907
28.33 2.5 Saturated José De Diego Avenue Cayey, P.R.
00736
Septic tank
Plantain trees
14
AMDS
Mar / 16 /
14
Lat. 18.11746
Long. -66.17972
28.61 2.6 Moist Rural
“Buena Vista Sur” Cayey, P.R. 00736
Stagnant water
Plants
15
AMDS
Mar / 16 /
14
Lat. 18.1174
Long. -66.17993
28.33 2.3 Moist Rural
“Buena Vista Sur”
Cayey, P.R. 00736
Under a plantain tree
16
KJC
Mar / 16 /
14
Lat. 18.187940
Long. -
66.140350
23.33 3.04 Moist Rural
Lake shore, Cidra, P.R. 00739
Plantain
Crop
17
AMDS
Mar / 20 /
14
Lat. 18.1179
Long. -66.137
26.11 1.5 Dry Urban.
Coca Navas Street, Cayey 00736
Coffee plant
Cement
House
*** 18
AMDS
Mar / 20 /
14
Lat. 18.11524
Long. -
66.16313
25.0 2.3 Moist Urban
Antonio R. Barceló Avenue, Cayey,
P.R. 00736
Underneath a decaying
fruit
Fruit – tree
19
KJC
Mar / 20 /
14
Lat. 18.119730
Long. -
66.157776
28.89 5.71 Dry Urban
UPR Cayey Campus
Under plant
Roots
20
KJC
Mar / 20 /
14
Lat. 18.119564
Long. -
66.158065
28.33 5.01 Dry Urban
UPR Cayey Campus
Under Rotten
Fruit