1. 1
Isolation of a novel mycobacteriophage from the soils of Puerto Rico
Lizbeth Pérez Castro, and Javier Zavala Ayala
Biomedical Techniques Course, RISE Program,
Universidad de Puerto Rico, Recinto de Cayey
Bacteriophages are a class of virus. This specific class of virus infects only bacteria. The
bacteriophages cannot propagate outside their host bacteria. They’re composed of a protein
coat, which encloses its DNA or RNA, and a tail that provides anchorage. The purpose of this
investigation is to isolate a novel bacteriophage from the tropical soil of Puerto Rico. In order to
do so, there are four major steps namely: collection of the environmental sample, isolation of the
phage from the environmental sample, purification of the phage, and characterization of the
phage. Investigating phages is important because many phages can shed light not only on
viruses, but also on their host. Also the identification of the different phages can lead to
answering the question on how and why this organism keeps evolving. Understanding these two
factors can be transcendental for medical applications. Conclusions are that the purpose of the
investigation was achieved since the isolation of a novel mycobacteriophage was successfully
done from soil found in a cowshed in Gurabo, P.R.
Introduction:
Human beings often live without
thinking about all the microscopic organisms
that live among us. Bacteriophages are one of
many microorganisms, and they are a class of
virus. This specific class of virus infects only
bacteria. The bacteriophages cannot propagate
outside their host bacteria. Bacteriophages are
composed of a protein coat, which encloses
DNA or RNA, and a tail that provides
anchorage. The morphology of the
bacteriophages varies.
When a phage attaches to a bacterium,
it releases its genetic material into the host.
This genetic material combines with the hosts
genetic material and replicates. Afterwards
more phages are made and then released. There
are two types of phage cycles: lytic and
lysogenic. A key difference between the lytic
and lysogenic phage cycles is that in the lytic
phage, the viral DNA exists as a separate
molecule within the bacterial cell, and
replicates separately from the host bacterial
DNA. The location of viral DNA in the
lysogenic phage cycle is within the host DNA.
In both cases, the virus/phage replicates using
the host DNA machinery, but in the lytic phage
cycle, the phage is a free floating separate
molecule in the host DNA.
In this experiment, the bacterial hosts
used are Mycobacterium Smegmatis and
Bacillus. Mycobacteria are responsible for
many diseases and deaths worldwide (WHO
2010). It is estimated that one third of the
world's population are infected with
Mycobacterium Tuberculosis (Reyrat and
Kahnt 2001). But Mycobacterium Smegmatis is
a non-pathogenic bacterium and has a fast
growing rate, which makes them suitable for
laboratory experiments (Gordon and Smith
1953). It also acts as an aerobic organism.
Bacillus is an aerobic organism that can be
either a free-living or parasitic pathogenic
species. Bacillus and M. Smegmatis share some
common characteristics such as: rod-shaped
morphology and both are gram positive. The
hypothesis established was that new phages
will be isolated from tropical soils in Puerto
Rico.
The study of phages is important,
because they can be used as tools to move
DNA around for cloning and mutation. Genetic

2. 2
information about different phages allows
scientists to compare phages, study biodiversity
and identify new genes that may be useful for
scientific or therapeutic applications. Scientist
would like to use phages to kill specific
antibiotic-resistant bacteria that cause diseases
(Asai, 2012).
Materials and Methods:
For this experiment, soil samples were
taken in order to isolate a novel bacteriophages
from the tropical soil of Puerto Rico. The
climate of Puerto Rico influenced the
availability of bacteriophages, because phages
are mostly found in moist areas surrounded by
organic material (feces). There are four major
steps for the isolation of a bacteriophage, these
are: collection of the environmental sample,
isolation of the phage from the environmental
sample, purification of the phage, and
characterization of the phage.
For the isolation of a bacteria phage,
samples of soil were collected with the use of a
sterile spoon and sterile bag. Every sample
collection was identified by location, texture of
the soil, date, and time collected. The sample
was going to be tested with two host: Bacillus
cereus (B. cereus) and Mycobacterium
smegmatis (M. smegmatis). After three sample
collections, the soil sample located in Road
#943, Sector Los Chinos, Gurabo in the
grounds of a cowshed had phages. The
coordinates of the place are 18º 16’ 7’ N, 65º
58’ 32’ W. This last soil sample was collected
in the morning of a temperate or mild day next
to cow feces on February 24, 2014.
Approximately 0.5g of soil were weighted. To
one of the samples of 0.5 g of soil, 10 ml of the
master mix were added. To the other 0.5 g
sample of soil, 10 ml of TSB were added.
Then 1ml of B. cereus were added to the soil
containing the 10 ml of TSB, and 1 ml of M.
smegmatis were added to the soil mixed with
the 10 ml of master mix. These enrichments
were placed in the incubator at 37ºC, shaking at
220 revolutions per minute (rpm) for 24 hours.
The next day the sample was ready for
the next procedure, the isolation of the phage.
The enrichments were centrifuged for 10
minutes at 3000 rpm and 5 ml of the
supernatant was taken and filtrated with a 0.20
micrometers filter. The bacterial host was
identified. The plaque screening is done with
an isolated phage plug, and the phage
purification is completed with the use of the
streak protocol. These purification procedures
were repeated three times. Then after the third
purification, a second enrichment was done by
adding 10 ml of the master mix and 1 ml of M.
smegmatis to a phage plug of the third
purification petri dish. This enrichment was left
incubating at 37ºC, shaking at 220 rpm for a
time period of 24 hours.
The next step was to do eight dilutions
with an isolated phage plug of the third
purification. The spot test was done with these
dilutions. Then after identifying a dilution that
forms a web pattern, 80 microliters of the
identified dilution was mixed with 5 ml of M.
smegmatis. This second enrichment was then
divided equally and seeded in 9 plates. After
incubating for 24 hours at 37ºC, the web
pattern was observed. Five milliliters of phage
buffer were added to the nine plates with the
web pattern and incubated in the freezer at 2ºC.
The next day the plates were taken out and the
top agar was broken down in order to obtain all
the phages. The phages and buffer were taken
with a pipette and mixed in a 50 ml tube. This
mixture was centrifuged at 3000 rpm in the
time lapse of 10 minutes. The supernatant was
obtained and vacuum filtered. This last step
was done in order to obtain the High Titer
Phage Lysate (HTPL). With the HTPL a
second dilution was done first at -2,-4,-6,-7,-8,-
9 and the streak protocol followed. The phage
plugs done were counted per dilution. Then
proteomics were done in the workshop given
by Dr. Michael Rubin on April 25, 2014. On
this workshop 15 microliters of the HTPL and
15 microliters of Beta-mercaptoethanol (BME)
were mixed. Then 25 microliters of the mixture
were loaded on a polyacrylamide gel that went
from 12 to 1 percent. An EM grid was also
prepared in order to analyze later the phage
using Scanning Electron Microscopy. The dye

3. 3
used to stain the phage in the EM grid was 10
microliters of 1% uranyl acetate. The last
procedure done was the isolation of the phage
genomic DNA. To obtain the DNA 10ml of the
HTPL were prepared by adding 40 microliters
of Nuclease Mix and incubating it at 37ºC for
30 minutes. Then it was taken out and left for
an hour at room temperature. Four milliliters of
phage precipitant solution were added to the
mixture and left overnight at 4ºC. The next day
the mixture was centrifuge and spun at
10,000xg for 20 minutes. The supernatant was
decanted carefully without disturbing the pellet.
0.5 mL of sterile ddH2O and two milliliters of
pre-warmed (37ºC) DNA Clean Up Resin were
added and gently swirled to mix. The water-
resin-phage-genomic-DNA solution was
divided into two columns and by filtering and
centrifuging. The solutions that weren’t needed
were removed and the phage genomic DNA
was obtained and eluted together in one
column. In order to analyze the DNA an
agarose gel was prepared. The DNA was
prepared by adding 8 microliters of 1X TBE
buffer, 2 microliters of the DNA and 2
microliters of tracking dye. Ten microliters of
each solution were loaded in the gel and ran at
100V. The gel after about an hour was
observed.
Results:
After the enrichment, filtration, and
streaking of the specimen on the petri dishes,
positive results were not obtained from plates
one and two. However, the third soil sample
had positive results. The coordinates of the
location where that sample was found are 18º
16’ 7’ N, 65º 58’ 32’ W. The petri dish
displayed phage plaques on the first streak
region. The three purifications were done, and
after obtaining a phage plaque from the third
purification, the dilutions were made. The
dilution used to make the nine plates was the
dilution 10−1
. Since the top agar that day was
not enough to make the ten plates, nine were
done. After evaluating the nine petri dishes for
web patterns, the steps necessary to obtain the
HTPL were done. The HTPL had a volume of
35ml. For the last steps, new dilutions were
made to see the number of phage colonies per
liter. The results can be seen in table 2. Then
the median was calculated using the number of
phages divided by the dilution and volume
(ml). The median was 1.57 ± 0.3 x 1010
PFU/ml. On figure 4 the results of the
polyacrylamide gels can be observe.
Nitidusvenutus compared with other phages
show distinct band patterns. The results
obtained from the last procedure which was the
isolation of the DNA were negative. The
agarose gel shows two band patterns instead of
one indicating that there was contamination.
Soil Samples Information
Sample Coordinates Soil
Description
Sample
#1
18º 16’ 16’ N,
65º 58’ 10’ W
Loose, and
moist
Sample
#2
18º 6’ 58’ N,
66º 9’ 19’ W
Moist and
Chunky
Sample
#3
18º 16’ 7’ N,
65º 58’ 32’ W
Loose, and
moist next to
cow feces
Table 1. Description and location of the
different soil samples.
Number of Phage Colonies per Dilution
Dilution Number
of Phages
colonies
Photo
10−6 133
10−7 18

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10−8 1
Table 2. Presents the illustrated results of the
number of plaques obtain per dilutions. The
dilutions that are not illustrated had complete
lysis.
Figure 1. Petri dish shows positive results, this
picture shows the phage plaques found after
streaking the filtrated mixture of the third
sample of soil.
Figure 2. Shows the third purification.
Figure 3. Shows the High Titer Phage Lysate
obtained.
Figure 4. Results of the duplicate
polyacrylamide gels. Nitidusvenutus is placed
on the fifth well.

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Figure 5. Shows the results of the DNA
Agarose gel. Nitidusvenutus was loaded on the
fifth well. Two different bands appear in the
fifth well indicated that the isolation of the
phage DNA wasn’t successful.
Discussion:
Isolation of a novel bacteriophage is a task that
requires patience, time, and effort. In order to
effectively locate the phages three samples of
soil were collected. The results for the first two
samples were negative. The soil sample that
had possible results was moist and loose, and it
was near cow feces. Animals all have extensive
microbiomes, full of different species of
bacteria. It can be assumed that sampling in
areas rich in organic waste products of other
organisms can be an indicative of phages. This
is possible because maybe the host organism
was amongst this animal’s microbiota. The
phages that were in the environment now had
better possibilities of infecting the bacterium
once it was free in the feces. The phage
obtained was isolated and purified. Our
bacterial host was M. smegmatis. The dilution
that showed web pattern and was used to make
the nine plates was the dilution10−1
. Nine
plates were used to obtain the HTPL instead of
ten.
Throughout all the processes done,
aseptic technique is crucial to avoid
contamination. Our mycobacteriophage
Nitidusvenutus showed clear plaques meaning
that it probably has a lytic life cycle. The gel
obtained from the proteomic workshop showed
that Nitidusvenutus had distinct band patterns
in comparison with the other phages. Since
phages can be cluster in families by the
proteins in their capsid, the next step is to
identify the cluster that Nitidusvenutus belongs
or is related to. The results obtained from the
isolation of the DNA were negative. The
agarose gel showed two band patterns instead
of one indicating that in the process of the
preparation of the solution there was
contamination.
Many phages shed light not only on
viruses, but also on their host. The
identification of the different phages can lead to
answering the question on how and why this
organism keeps evolving. Understanding these
two factors can be transcendental for medical
applications. After performing the experiments
or procedures, the hypothesis was proven,
phages were successfully isolated from the
soils of Puerto Rico. The soils of Puerto Rico
contain novel bacteriophage that show genetic
variations as seen on the on the polyacrylamide
gel results. For future work we want to
characterize the phage structure through
Transmission Electron Microscopy, and
sequence its DNA using bioinformatics.
Acknowledgement:
Our special recognition to the
contributions of: Giovanni Cruz, Gustavo
Martínez, Edwin Alvarado, and Juan Apiz for
serving as technical assistants. Special thanks
to Dr. Eneida Díaz and Dr. Elena González, for
their roles as course professors of Biomedical
Techniques (BIOL-4997). Our gratitude to Dr.
Michael Rubin, for his role as mentor and the
RISE program and the Howard Hughes
Medical Institute, for the materials needed for
the procedures.
References:
Asai DJ, Bailey C, Barker LP, Bradley KW,
Khaja R, Lewis MF. 2012. Sea-Phages:
Resource Guide. Chevy Chase,
Maryland: Howards Hughes Medical
Institute; 168 p.
Rubin M, Vázquez E. 2012.
Microbacteriophage Proteomics: From
Genotype to Phenotype (There and Back
Again)!.Cayey, PR. Howard Hughes
Program, Department of Biology; 20 p.
Endersena L, Coffeya A, Neveb H, McAuliffec
O, Rossc RP, O'Mahony JM. 2013.

6. 6
Isolation and characterisation of six novel
mycobacteriophages and investigation of
their antimicrobial potential in milk,
International Dairy Journal Volume 28,
Issue 1, January 2013, Pages 8–14
Gordon RE, Smith MM. July 1953. Rapidly
growing, acid fast bacteria. I. Species'
descriptions of Mycobacterium phlei
Lehmann and Neumann and
Mycobacterium smegmatis (Trevisan)
Lehmann and Neumann. Journal of
bacteriology 66 (1): 41–8.
Reyrat JM, Kahn D. October 2001.
Mycobacterium smegmatis: an absurd
model for tuberculosis?. Trends in
Microbiology 9 (10): 472–473.
doi:10.1016/S0966-842X(01)02168-0
Keen EC. 2012. Phage Therapy: Concept to
Cure. Frontiers in Microbiology 3.
doi:10.3389/fmicb.2012.00238