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.
Hatfull et al. (2008), indicate the
recognition of the vast numbers of bacteriophages
in the biosphere has prompted a renewed 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 (Pope et al.,
2011). This is a very promising research area
since 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

2. recommended by Science Education Alliance
(2012).
- Sample Collection:
The first step of the experiment was the
collection of a soil sample. In this step, as part of
maintaining everything sterile, you used a pre-
packed utensil to recollect the soil sample into a
sealed and sterile test tube. After collecting the
sample, the test tube was sealed and stored at
room temperature. Data such as temperature,
climate, soil moisture, GPS site, soil depth, etc.
was recorded.
- Enrichment:
Afterwards, the second step of the
experiment was the enrichment of the soil sample
that was recollected. In this step, you added to a
sterile 50ml test tube the following: 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 also added 1ml
of the bacteria M. smegmatis. In addition, you
added 0.5g of the soil sample to the test tube with
the enrichment solution and the bacteria. Lastly,
you incubated the test tube at 37°C at 220rpm for
24 hours.
- Harvesting:
Once 24 hours passed after the enrichment
step, the test tube was centrifuged for 10 minutes.
Then, you poured the supernatant into a new
sterile 50ml test tube using sterile filtering
techniques. Once successfully filtered, the test
tube was capped and labeled. Next came the
second part of the harvesting: to plaque. The
plaque process was done on petri dishes and each
plate was divided into three sections. This step
consisted 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
(This sentence lacks clarity.). Afterwards, another
wooden stick was used to streak from section one
to section two and then, using a new wooden
stick, we steaked from section two to section
three. After the streaking was completed, 4.5ml of
top agar with 0.5ml of bacteria were added to the
plate. Once the agar solidified, the plates were
incubated at 37°C, for 24 hours, and after positive
results appeared, the plates were refrigerated.
- Plaque Purifications:
If positive results appeared. the phage(s)
that you wanted to purify were to be circled at the
bottom of plate so that you could view them
clearly. Now to a labeled tube, we added 50µl of
phage buffer. To the circled phage of the plate, we
inserted a micropipette tip and then placed it in
the tube with the phage buffer. Afterwards, a
new petri dish was labeled and the plaque
process described in the second part of the
harvesting was repeated. However, instead of
using the filtering results to plaque, we used the
tube with the mixture of the phage buffer and the
phage inserted from the plate. Once again, we
streaked section one, then from section one to
section two, and section two to section three.
Three rounds of plaque purifications were
performed.
- Second Enrichment:
The next step was to make another
harvesting. First of all, a phage was isolated with
the tip of a micropipette and then added into the
same enrichment solution as the first enrichment.
Afterwards, it was incubated for 24 hours like
in the first enrichment. Next, the test tube was
centrifuged for 10 minutes. Then, we poured the
supernatant into a new sterile 50ml test tube using
sterile filtering techniques. Once successfully
filtered, the test tube was capped and labeled.
- Medium Titer Assay:
This step consisted of creating serial
dilutions. From the filtration obtained after the
second filtration, four phage solutions were
diluted in four tubes labeled -1 to -4, add 90µl of
phage buffer. Then, we added 10µl of the
filtration to the -1 tube and centrifuge it. Next, we
added 10µl of the -1 tube to the -2 tube and
centrifuge. This process was repeated for the 4
tubes. Afterwards, we added 10µl of each tube
(filtration and -1 to -4 tubes) to a sample of 0.5ml
of bacteria. Once the solution sat for 15-30
minutes we added 4.5ml of top agar to the
bacteria solution and spread the solution on a
properly identified plaque. The plates wee

3. incubated after solidifying and checked after 24
hours. Once the plate that was successful was
identified (the one with the “web” pattern), we
add 6ml of phage buffer to it and placed it in the
refrigerator for 24 hours. Next, we extracted the
phage buffer ___the plaque, filtered, and
refrigerated it.
- High Titer Assay:
In this step, 10 plates were infected with
the bacteriophage. First of all, we labeled 10
plates and labeled a sterile 50ml test tube. To the
test tube, we added 5ml of bacteria culture and
then infected it with 10µl of the dilution that
completely lysed the bacteria. The bacteria was
incubate and shaken at 37°C for 30 minutes.
Afterwards, 45ml of top agar was addedto the test
tube. Five ml of the mixture was distributed onto
each plate and incubated at 37°C for 24 hours.
After the time has passed and the web pattern was
obtained, 6ml of phage buffer was added to each
of the 10 plates, and using sterilized utensils the
agar was broken and mixed with the buffer. Next,
place the plates were placed in the incubator at
37°C for four hours. After the time has passed,
the phage buffer was extracted from all of the
plates and placed in a sterile 50ml test tube. As
the last step, the tube was centrifuged and
filtered.
- 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 (Not
clear!). One ml of Mycobacteriophage High Titer
Phage Lysate (HTPL) was transferred to a clean
sterile microtube and centrifuged at 10,000xg for
one hour at 4°C. Afterwards, 950µl of the
supernatant was aspirated. Next, a sample buffer
was prepared by adding 25µl of Beta-
mercaptoethanol (BME) to 475µl of Laemmli
Sample Buffer (LSB) and vortexed completely.
Later, 20µl of the LSB plus BME solution was
added to the Mycobacteriophage virion coat
protein pellet. After that was done, (from here on
continue to use passive voice with past participle
verbs for the rest of the report. You are reporting
on the methodology that you used. It has to be
past tense and to make it objective the voice must
be passive.). 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
Biosafe Coomassie 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:
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. The soil sample
was taken from an urban site next to trees and
compost. In addition, the soil was dry and the
depth from where it was taken was 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

4. 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
phage #1, the pattern was on the plate with the
dilution -3. For phages #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:
Positive results for phages obtained after
the enrichment and first plating was most
probably due to 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. Once 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.
A protein gel was run once a medium was
created based on the “web” pattern. 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 were able to
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 determine if the isolated
phage is, in fact, unique. Furthermore, the
isolation of phages has applications in the field of
biomedicine. As mentioned earlier, an example of
this is the possible elimination of antibiotic
resistant bacteria using phages. In addition,
phages can also be helpful in understanding
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
%2F10.1371%2Fjournal.pone.0016329