1. Anaeli Shockey Lópeza
, Nicolle Rosa Mercadob
a
Chemistry Department, University of Puerto Rico-Cayey
b
Biology Department, Univeristy of Puerto Rico-Cayey
Isolation and characterization of Mycobacteriophages from tropical soil of Puerto Rico
Abstract:
Mycobacteriophages are viruses that infect bacteria from the genus Mycobacterium. They
are ubiquitous and are easily found in different types of soils. Phages are composed of a head,
which contains the genetic material, and a tail. Bacteriophages have two possible life cycles:
lytic or temperate. Most of them have a temperate life cycle in which they can cause immediate
lysis or enter a state of dormancy within the host. The objective of this investigation was to
isolate a new phage using soil from Puerto Rico. This is necessary because there are too many
undiscovered bacteriophages that can be of great use to mankind. The methodology for this
investigation consisted of isolating a phage using the protocols listed in the SEA-PHAGES
resource guide. Two phages were isolated from Gurabo, Puerto Rico and were taken up to the
high-titter Assay protocol. Future work would include sequencing their DNA.
Introduction:
Mycobacteriophages are viruses that infect
bacteria from the genus Mycobacterium. They are
ubiquitous and are easily found in different types
of soils. They can be isolated using a simple
procedure. Although phages are the most
abundant life-form on earth, very few of them
have been identified. Phages insert their genetic
material into the bacteria and replicate within it
provoking the lysis of its host. Bacteriophages
have two possible life cycles: lytic or temperate.
Most of them have a temperate life cycle in which
they can cause immediate lysis or enter a state of
dormancy within the host.
As said by Hatfull, et al. (2008), the
recognition of the vast numbers of bacteriophages
in the biosphere has prompted a renewal of
interest in understanding their morphological and
genetic diversity, and elucidating the evolutionary
mechanisms that give rise to them. This
investigation has several important applications
within the field of scientific research. An example
of this within the biomedical field is the possible
elimination of antibiotic resistant bacteria using
phages. Phages can also help us understand
certain aspects of the bacteria that they infect and
the effects that they might have on them. Based
on the immense diversity present in phages we
can also obtain important information on the
evolutionary line of these viruses. Some phages
are an example of how viruses can be beneficial to
humans. The objective of this investigation was to
isolate a new phage using soil from Puerto Rico.
This is necessary because there are too many
undiscovered bacteriophages that can be of great
use to mankind.
Mycobacteriophages can be found all
around the world and are the most numerous
biological entities in the biosphere, as said by
Pope, et al. (2011).This is a very promising
research due to the fact that there is still much to
be discovered concerning phages. There are yet
many important undiscovered characteristics that
may be helpful in the treatment of bacterial
diseases. Their genetic diversity provides a
promising future in research. Phages help us get a
better understanding of bacteria as well.
Materials and Methods:
As instructed by our mentor, for this
experiment, all of its materials and methods were
based on Science Education Alliance (2012).

2. - Sample Collection:
The first step of the experiment is the
collection of a soil sample. In this step, as part of
maintaining everything sterile, you must use a
pre-packed utensil to recollect the soil sample into
a sealed and sterile test tube. After collecting the
sample, the test tube must be sealed and stored at
room temperature. Data such as temperature,
climate, soil moisture, GPS site, soil depth, etc.
should be recorded.
- Enrichment:
Afterwards, the second step of the
experiment is the enrichment of the soil sample
that was recollected. In this step, you add to a
sterile 50ml test tube 8ml of sterile water, 1ml of
sterile 10x 7Hq/glycerol broth, 1ml of AD
supplement, and 0.1ml of 1000mM CaCl2. To this
enrichment solution, you add 1ml of the bacteria
M. smegmatis. In addition, you add 0.5g of the
soil sample to the test tube with the enrichment
solution and the bacteria. Lastly, you incubate the
test tube at 37°C at 220rpm for 24 hours.
- Harvesting:
Once 24 hours pass after the enrichment,
the test tube is centrifuged for 10 minutes. Then,
you pour the supernatant into a new sterile 50ml
test tube using sterile filtering techniques. Once
successfully filtered, the test tube is capped and
labeled. Afterwards comes the second part of the
harvesting: to plaque. The plaque process is done
on petri dishes and each plate should be divided in
three sections. This step consists of using a
wooden stick to streak, across the first section of
the bottom agar of the petri dish, the supernatant
that resulted after filtering. Afterwards, another
wooden stick is used to streak from section one to
section two and then, using a new wooden stick, it
used to streak from section two to section three.
After the streaking is complete, 4.5ml of top agar
with 0.5ml of bacteria are added to the plate. After
the agar solidifies, the plates must be incubated at
37°C, and, after 24 hours, if positive results
appear, the plates must be refrigerated.
- Plaque Purifications:
If positive results appear, the phage(s) that
you want to purify should be circled at the bottom
of plate so that you can view it clearly. To a
labeled tube, add 50µl of phage buffer. To the
circled phage of the plate, insert a micropipette tip
and then place it in the tube with the phage buffer.
Afterwards, label a new petri dish and repeat the
plaque process described in the second part of the
harvesting. However, instead of using the filtering
results to plaque, you use the tube with the
mixture of the phage buffer and the phage you
inserted from the plate. And with that, once again,
you streak section one, then from section one to
section two, and section two to section three.
Three rounds of plaque purifications are
performed.
- Second Enrichment:
The next step is to make another
harvesting. First of all, a phage is isolated with the
tip of a micropipette and then added into the same
enrichment solution as the first enrichment.
Afterwards, follow the same incubation
instructions as the first enrichment. Once 24 hours
pass after the enrichment, the test tube is
centrifuged for 10 minutes. Then, you pour the
supernatant into a new sterile 50ml test tube using
sterile filtering techniques. Once successfully
filtered, the test tube is capped and labeled.
-Medium Titer Assay:
This step consisted of creating serial
dilutions. From the filtration obtained from the
second filtration, four phage solutions will be
diluted. In four tubes labeled -1 to -4, add 90µl of
phage buffer. Then, add 10µl of the filtration to
the -1 tube and centrifuge it. Next, add 10µl of the
-1 tube to the -2 tube and centrifuge. Repeat this
process until -4 tube. Afterwards, add 10µl of
each tube (filtration and -1 to -4 tubes) to a
sample of 0.5ml of bacteria. Let the solution sit
for 15-30 minutes. After the time has passed, add
4.5ml of top agar to the bacteria solution and
spread the solution on a properly identified
plaque. Incubate the plates after solidifying and
check after 24 hours. Once the plate that was
successful is identified (the one with the “web”
pattern), add 6ml of phage buffer to it and place in
the refrigerator for 24 hours. After the time has

3. passed, extract the phage buffer the plaque and
filter it. Afterwards, place it in the refrigerator.
- High Titer Assay:
In this step, 10 plates will be infected with
the bacteriophage. First of all, you label 10 plates
and label a sterile 50ml test tube. To the test tube,
add 5ml of bacteria culture and then infect with
10µl of the dilution that completely lysed bacteria.
Incubate and shake at 37°C for 30 minutes.
Afterwards, pour 45ml of top agar to the test tube.
Distribute 5ml of the mixture onto each plate and
incubate at 37°C for 24 hours. After the time has
passed and the web pattern is successful, add 6ml
of phage buffer to each of the 10 plates, break the
agar using sterilized utensils and mix with the
buffer. Next, place the plates in the shaking
incubator at 37°C for four hours. After the time
has passed, extract the phage buffer from all of
the plates and place it in a sterile 50ml test tube.
As the last step, centrifuge and filter.
- Rapid Isolation, Separation, and Visualization of
Mycobacteriophages Capsid Proteins:
This step is performed with the extraction
of phage buffer from the plate with the “web”
pattern from the Medium Titer Assay
step.Transfer 1ml of Mycobacteriophage High
Titer Phage Lysate (HTPL) to a clean sterile
microtube and centrifuge at 10,000xg for one hour
at 4°C. Afterwards, aspirate 950µl of the
supernatant. Next, prepare a sample buffer by
adding 25µl of Beta-mercaptoethanol (BME) to
475µl of Laemmli Sample Buffer (LSB) and
vortex completely. Later, add 20µl of the LSB
plus BME solution to the
Mycobacteriophagevirion coat protein pellet.
After that is done, boil the sample for two
minutes, cool the protein sample for two minutes
and centrifuge it briefly. Now you prepare the gel.
This is done preparing 1x of running buffer by
adding 100ml of 10x Tris Glycine SDS buffer to
900ml of distilled water. Next, remove the gel
from the packaging, remove the tape from the
bottom of the gel, carefully remove the comb
using even pressure, and rinse the wells using
distilled water. Assemble the gel in the apparatus
and add appropriate amounts of 1x running buffer.
Afterwards, load the sample and the molecular
weight markers. Now you run the gel at 200 volts
for 30 minutes until the dye reaches,
approximately, 1cm from the bottom of the gel.
Next, strain the gel in a plastic tray using Bio-Rad
BiosafeCoomassie Blue G-250 strain. Wash the
gel in distilled water for 5 minutes and remove the
water (this step is repeated three times). Next, add
50ml of Coomassie Blue G-250 stain to the gel
and stain for one hour with gentle shaking. After
the staining is complete, rinse the gel with water
for 30 minutes. Now you’re ready to photograph
the gel on a white light box. The gel can be stored
in water (in a zip lock plastic bag) or dried and the
bands can be carefully excised using washed
gloves and clean unused razor blades and placed
in sterilized microtubes for subsequent protein
identification by mass spectroscopy.
Results:
Following all of the previous discussed
materials and methods the following data was
recorded and the following results were found in
the experiment. First of all we have the soil
recollection data. The temperature at 8:00am on
February 19, 2013 was 25.6°C and the day was
sunny and clear. The sample was taken in Gurabo,
Puerto Rico at these coordinates:
18°14'48.53"N 66° 0'6.55"W.
Thesoilsamplewastakenfrom an urban site
nexttotreesandcompost. Inaddition,
thesoilwasdryandthedepthfromwhere it
wastakenwas 5.74 inches.
After the first enrichment and harvesting,
positive phage results were found when the soil
sample was used. From the plate with positive
results, three phages were identified because of
their difference in sizes. Since they were treated
as three different phages, each of them required
three plaque purifications. The purification of the
first phage resulted in morphologically small
phages. The purification of the second and the
third phage resulted in morphologically medium
sized phages, both suspected to be the same size.
After the second enrichment, filtration and -1 to -4
dilutions, each phage yielded a “web” pattern. For

4. phage #1, the pattern was on the plate with the
dilution -3, for phage #2 and #3, the pattern was
on the plate with the dilution -4.
After extracting the phage buffer from
each of the “web” pattern plates, a medium was
created and analyzed with the SDS gel. The gel
was loaded with a marker, other phages and the
three suspected phages. After the whole procedure
was complete, the protein bands of all three
phages could be seen and the bands of phages #2
and #3 were extremely similar.
Discussion:
It is suspected that the reason why positive
results for phages after the enrichment and first
plating were present because of the sample depth
and location (next to compost). With the positive
phage results, three phages were identified
because they had different sizes. The difference in
sizes means that each phage is morphologically
different from the other. Moreover, this would
mean that each phage that is a different size would
be a different phage.
After the plaque purifications were
completed, phages #2 and #3 were suspected to be
the same phage because their sizes were relatively
the same. However, they continued to be treated
as different phages until the protein gel step was
completed to determine if they were the same
phage or not. After the dilutions were completed,
the “web” pattern of each phage was chosen based
on the arrangement of plaques in which almost all
of the bacteria was lysed. In addition, in the
“web” pattern, all of the plaques must be in
contact with each other.
After creating a medium based on the
“web” pattern, a protein gel was run. The protein
bands of phage #1, now named Shockage, were
different from the rest of the phages. The protein
bands of phages #2 and #3 were practically the
same; therefore, it is assumed that both phages are
the same. Phage #2 is now named Zombage.
Conclusion:
Through this experiment, we can isolate
Mycobacteriophages, which are viruses that infect
bacteria. From a single soil sample, taken from
Gurabo, Puerto Rico, two different phages have
been isolated. Phage #1 is named Shockage and
phage #2 is named Zombage. The next step for
each of these phages would be to sequence their
DNA in order to finish the phage characterization
and in order to determine if the isolated phage is,
in fact, unique. Furthermore, the isolation of
phages has applications in the field of
biomedicine. As mentioned before, an example of
this is the possible elimination of antibiotic
resistant bacteria using phages. In addition,
phages can also be helpful in order to understand
certain aspects of the bacteria that they infect and
the effects that they might have on them.
References:
Science Education Alliance. 2012. SEA-PHAGES
Resource Guide. Howard Hughes Medical
Institute; Chevy Chase, MA.
Hatfull G, Cresawn S, Hendrix R. 2008.
Comparative genomics of the
mycobacteriophages: insights into bacteriophage
evolution. Research in Microbiology. 159(5): 332-
339.
Pope WH, Jacobs-Sera D, Russell DA, Peebles
CL, Al-Atrache Z, et al. 2011. Expanding the
Diversity of Mycobacteriophages: Insights into
Genome Architecture and Evolution. [Internet]
[Cited 2013 May 14] PLoS ONE 6(1)
doi:10.1371/journal.pone.0016329 Available
from:
http://www.plosone.org/article/info%3Adoi%2F1
0.1371%2Fjournal.pone.0016329