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1. Isolation and Characterization of Bacteria from Tropical Soils
Nicole Rivera- Espinal1, Alejandra Ferrer-Díaz1
1Department of Biology, University of Puerto Rico at Cayey. Both authors contributed equally to
this project.
Abstract
The purpose of this experiment is to encounter bacteria from tropical soils that have the
capability of producing antibiotic. In order to do so, it was necessary to isolate the bacteria,
purify it five times and freeze it at a temperature of -80˚C. After purification, gram staining was
performed to classify the bacteria as gram positive or negative. The bacteria were characterized
by doing purification of genomic DNA and then, a Polymerase Chain Reaction (PCR) with the
primers 16sRNA, 16S_1510R and 16S_8F. An electrophoresis was conducted to see if the PCR
process went well. The PCR product was purified and analyzed by using Bioinformatics. Finally,
both bacteria’s S15UPRCRISENRE30M01 and S15UPRCRISEAFD30M01A had positive results
as antibiotic producers for M.luteus.
Introduction:
In the beginning of Earth history, the
first thing considered as organisms were
bacteria. Even though the first ones found
were photosynthetic they functioned as a
fundamental base to the understanding of
life and evolution (Evert and Eichhorn
2013). Bacteria are prokaryotic
microorganisms that can be found
practically everywhere, for example, in the
soil, water and even in the human body.
These can have different types of
morphology, and they are classified as:
cocci, bacillus, spirillum and spirochetes.
Also, bacteria can be differentiated by their
peptidoglycan layer located in the cell wall
that classifies them as gram-positive or
gram-negative. The gram-positive ones are
characterized by the thickness of their wall.
On the contrary, gram-negative bacteria
possess a thinner wall (Beveridge 2001).
Due to the properties of these
microorganisms, scientists were currently
searching for beneficial ways in which
bacteria can contribute to human welfare.
The discovery of penicillin in 1928
by Alexander Fleming indicated that a new
transcendental and significant era was about
to start (ACS 2015). Since 1940’s scientists,
using Fleming’s research as a source, have
been studying and developing antibiotics in
order to reduce illness and death from
infectious diseases (CDC 2014). For
example, Rene Dubos of the Rockefeller
Institute for Medical Research, in the late
1930’s, discovered and isolated an
antibacterial compound—tyrothricin, from
the soil microbe Bacillus brevis—capable of
destroying Gram-positive bacteria. This
discovery revived the stalled interest in

2. penicillin and launched the era of antibiotics
(Van Epps 2006). All these contributions
have led to a substantial control of powerful
diseases that plays an adverse role in human
health. Despite all the important facts
discovered about antibiotics, it is essential to
know that right now these types of
medications are playing an ineffective role
on bacteria due to bacterial antibiotics
resistance.
Antibiotic resistance is produced
when the capability of killing or inhibiting
bacterial growth is vanished; in other words,
the bacteria are strong enough in comparison
to the antibiotic and, as a consequence, they
continue to multiply in the presence of
therapeutic levels of an antibiotic (APUA
2014). Antibiotic resistance is a natural
incident. When an antibiotic is used, bacteria
that can confront that antibiotic have a
higher chance of survival than those that are
"susceptible." Although this effect is natural,
the accelerated and worldwide spread of
antimicrobial-resistant organisms in recent
years has been unparalleled (Vasoo et al.
2014). As a result, the scientific community
has been looking for an effective alternative
that can counteract bacterial resistance in
order to control infectious diseases and its
spread. Recent studies have indicated that it
is possible to diminish bacterial resistance
by manipulating the environment in which
the bacteria has been cultured. For example,
scientists discovered a new antibiotic which
named Teixobactin that can disrupt
peptidoglycans located in the cell wall of
gram-positive bacteria’s (Ling et al. 2015).
The importance of this experiment is the
way by which the scientists discovered the
antibiotic. They used an apparatus named
ichip whose function is to isolate uncultured
bacteria by in vivo techniques. This
discovery was a major breakthrough because
it is virtually certain to be effective for the
multiresistant strains that are now all but
impossible to treat (The scientist 2015).
Since their discovery, bacteria have
had an important economic and bio
sustainable role. These microorganisms are
used in some industries for the production of
tobacco, tanning leather, hides, cheese,
yogurt, buttermilk, vinegar, and sauerkraut.
Also, they are used in the development of
antibiotics. Given that bacteria are evolving
and creating resistance to many common
antibiotics used today, people are suffering
strongly from infectious diseases. In this
study, the main purpose is to find different
types of soil bacteria and characterize them,
in order to see if they are capable of
producing antibiotics that battle the new
strains of infections that are affecting us.
Studies have showed that tropical soils are
more likely to contain major nutrient
availability than other types of soils (Xenos
2014). Due to that fact, we have formulated
the following problem: Is it possible to find
bacteria with antibiotic properties in the soil
of Puerto Rico? Our hypothesis is: Bacteria
from tropical soils are capable of developing
properties such as antibiotic production. Our
hypothesis is sustained by several studies
such as the one developed by Dubinsky et
al.2010. This investigation attempts to prove
how soils in Puerto Rico can provide
abundance and activity of microbes varied
across the landscape. The significance of
this study is that it is determined to

3. encounter bacteria with antibacterial
properties, in order to counteract antibiotic
resistance. Finally, this investigation
attempts to respond an antibiotic worldwide
concern by using in vitro techniques.
Materials and Methods
Isolation and purification of the Bacteria:
Soil samples were collected from the
tropical soils of Puerto Rico in order to
obtain a range of bacteria. Important data of
the soil and the place were noted such as
temperature, soil composition, texture, and
living organisms, percent of humidity, and
GPS coordinates from the place. Brief
description of the surroundings of the soil
was included. After collecting a significant
amount of soil, just one gram diluted. The
soil was added to a test tube with 8.5 mL of
sterile H2O and 90µL of NaCl. The pH of
the sample was measured with a strip that
has a qualitative and quantitative range that
specifies the acidity of the soil. Ninety
microliters of Sodium Chloride were added
to six micro tubes that were already labeled
with numbers 0 to -5. Ten microliters of the
soil mix was added to the micro tube marked
as zero. In order to finish the dilution
process, 30µL of the zero micro tube were
collected and poured into the -1 micro tube;
this step was followed in a chain action until
the -5 micro tubes. Different medium plates
were selected to cultivate bacteria from the 0
and -5 micro tubes; these were Rhizobium
Medium (RDM) and ISP4. A drop of 30µL
was added to the center of the medium
plates and spread all over it in the form of a
cross. The plates were stored in the
incubator at 37˚C.
To obtain one specific colony of
bacteria, it is necessary to perform the
purification process that consists of the
recollection of isolated bacteria and their
cultivation in the mediums that facilitated
their reproduction. This process is repeated
at least three times. The streak plates with
the bacteria were saved in the incubator at
37˚C for a period of 24 hours.
Observational data at the macro level, a
plate of bacterial culture was taken and
observed under the dissecting light
microscope to analyze its morphology. After
bacteria were completely purified, a
cryogenic freezing was completed to store
them in the fridge at minimum temperatures.
Bacteria Characterization:
In order to assure that the isolated
bacteria were not contaminated and
additionally to comprehend the bacteria, the
gram staining technique was performed. In
the Gram stain, crystal violet, Iodine,
alcohol and safranin were used for bacterial
classification.
Bacteria were tested with E. coli and M.
luteus to see if they could serve as an
antibiotic agent. E. coli and M. luteus were
grown in dish plates that were already
divided into two portions, to analyze the
antibiotic properties of the follow bacteria
S15UPRCRISENRE30M01,
S15UPRCRISENRE30P01,
S15UPRCRISEAFD30M01A, and
S15UPRCRISEAFD30M01B. Then, two
paper disks were immersed in the micro tube

4. that contained the supernatant of the bacteria
found in tropical soils. After that, the paper
disks were placed into each of the portions
of the dish plates. This process was
performed in a period of 24 hours to see if
the bacteria inhibited E.coli and M.luteus.
Isolation of DNA:
For a large concentration of isolated
bacteria, it is necessary to complete a
bacterial enrichment. This step consists in
the mixture of 4.0 ml of RDM broth for the
bacteria that are cultured in rhizobium
medium and TSB broth for the bacteria that
grew in the ISP4 medium. This mixture is
saved in a semi-opened test tube for 24
hours in a shaker incubator at a temperature
of 37°.
The Polymerase Chain Reaction
(PCR) is a process commonly used in
Genetics and Molecular Biology that
consists of amplifying specific DNA and
producing several copies of it. Before
performing this process, it is required to
purify the genomic DNA. In order to do so,
300µL of the mixture, that was previously
prepared, were transferred to a micro
centrifuge tube. Then, the sample was
heated at 100˚C for 10 minutes followed by
an ice bath for another 10 minutes. To
separate the DNA supernatant from the
pellet, the micro centrifuge tubes were
placed in the centrifuge at 10K RPM for 10
minutes, Then, 150µl of the DNA
supernatant was added to a micro tube. Only
6µl of the DNA supernatant was transferred
to a PCR tube. Twenty five microliters of
master mix, 2µl of forward primer and
reverse primer and 15µl of nuclease free
water were also added to the PCR tube. The
PCR tubes were put in a thermal cycler
machine in order to start the PCR process.
The first step was denaturalization at 95˚C
in order to break the DNA double helix. The
second step was annealing at 48˚C that
consists of primer attaching. The last one
was extension at 72˚C. The first and third
step took one minute; the second one is 30
seconds. To test the samples of the PCR
process, the agarose electrophoresis was run.
Once the PCR process yielded positive
results, DNA purification process was
carried out by using a “QIAquick PCR
Purification Kit”.
Based on the amount of PCR product
used the volume of buffer needed was
calculated. To start this protocol, the
calculated amount of Buffer PB was added
to 1 volume of the PCR reaction; this
concoction was mixed in the centrifuge until
it turned yellow. It was then centrifuged to
allow for the separation of the products by
passing the samples through the column.
The flow-through was discarded and the
column was placed in the same tube. Next,
750µl of Buffer PE was added to wash the
QIAquick column. After that, QIAquick was
centrifuged one more time to remove the
residual wash buffer. In order to elute the
DNA, pure H2O (pH 7.0-8.5) was added to
the center of the QIAquick membrane and it
was centrifuged for 1 minute. Because the
purified DNA was going to be analyzed on a
gel, 1 volume of Loading Dye was added to
5 volumes of purified DNA.
Results:

5. A. Gram staining
Upon the completion of this process
it was noticed that all four of the bacteria
stained purple, indicating they were gram
positive. Likewise, it was noted that two of
the bacteria namely,
S15UPRCRISENRE30M01(figure 3) and
S15UPRCRISENRE30P01(figure 4), were
shaped in the form of bacillus. Moreover,
S15UPRCRISEAFD30M01A (figure 2) had
the form of a cocci bacteria while
S15UPRCRISEAFD30M01B (figure 1) was
shaped in the form of a streptobacillus.
Fig 1. Gram stain of bacteria S15UPRCRISEAFD30M01B
Fig 2. Gram stain of S15UPRCRISEAFD30M01A
Fig 3. Gram stain of bacteria: S15UPRCRISENRE30M01
Fig 4. Gram stain of bacteria: S15UPRCRISENRE30P01
Antibiotic producer with M. luteus and E.
Coli:
As shown in figure 5 Bacteria
S15UPRCRISENRE30M01 had a positive
result as an antibiotic producer with M.
luteus. A circular edge was underlying the
disk plate, which means that the bacteria
inhibited the reproduction of M. luteus. The
width of the circular edge observed was
broad, but that does not determine the
antibiotic strength of
S15UPRCRISENRE30M01. This bacterium
had negative results when it was tested with
E. coli because no edge was observed
around the disk plate.
Both bacteria
S15UPRCRISEAFD30M01B and
S15UPRCRISENRE30P01 presented
negative results with bacteria E.coli and
M.luteus. As visible in Fig. 5, bacteria
S15UPRCRISEAFD30M01A presented an
edge around the disk plate giving a positive
result as an antibiotic producer for M.
Luteus. When bacteria
S15UPRCRISEAFD30M01A was tested
with E.coli, inhibition was not observed
signifying it is not an antibiotic producer for
this bacterium. This information is
summarized in table 3.
Fig 5. Positive result for
bacteriaS15UPRCRISENRE30M01

6. Fig 6. Minimal positive result for bacteria
S15UPRCRISEAFD30M01A
PCR product:
After completing the PCR process
for the first time with the primer 16sRNA,
the results were negative. As shown in
figure 8, it was not possible to appreciate the
DNA amplification bands in comparison to
the KB ladder rail. After repeating the
procedure with alternative primers, the DNA
amplification appeared positive in bacteria
S15UPRCRISENRE30P01 and
S15UPRCRISEAFD30M01A. Conversely,
it was not possible to observe the DNA
amplification bands in bacteria
S15UPRCRISEAFD30 and
S15UPRCRISENRE30M01M01B, The
results from these bacteria were considered
negative. These results are shown in table 3.
Fig 7. Rail #1 and #4 represents the
amplification of bacteria’s S15UPRCRISENRE30P01
and S15UPRCRISEAFD30M01A, respectively.
Fig 8. Negative results for bacterial DNA
amplification with primer 16sRNA
Table 1: Soil collection
Date of
Collection
Site of Collection Air
temperature
Moisture Coordinates pH of
soil
January 28,
2015
Aguas Buenas
Caverns
82˚F Moist 18° 25' 86" N66° 01' 32''
W
5.5
February 1,
2015
Aguas Buenas
Caverns
82˚F Moist 18° 25' 86" N66° 01' 32''
W
5.5
February , 2015 Aibonito shooting
club
75° F Moist Latitude: 18° 8’ 38.6” N
Longitude: 66° 14’ 45.8”
W
6.0
February 1,
2015
Aibonito shooting
club
75° F Moist Latitude: 18° 8’ 38.6” N
Longitude: 66° 14’ 45.8”
W
6.0

7. Table 2: Bacteria Isolated
Bacterial Designator Soil Number of
purificatio
ns
Form Surfac
e
Elevatio
n
Color
S15UPRCRISEAFD30M
01A
“dots”
Aguas Buenas 5 Circular Smoot
h
Raised White
S15UPRCRISEAFD30
M01B
Aguas Buenas 5 lines with
projectio
ns
Rough Flat White
S15UPRCRISENRE30M
01
Aibonito shooti
ng club
5 Punctifor
m
Rough Flat Cream with
a cover of
dense white
S15UPRCRISENRE30P0
1
Aibonito shooting
club
5 Circular Smoot
h
Raised Cream and
yellowish
Table 3: Results to multiple testes
Bacterial Designator Gram Staining Antibiotic
Resistance
Antibiotic
Production
PCR Product
S15UPRCRISEAFD30M01A Cocci, Gram Positive Not determined Positive,
M.luteus
Positive
S15UPRCRISEAFD30
M01B
Streptobacillus, Gram
Positive
Not determined Negative Negative
S15UPRCRISENRE30M01 Bacillus, Gram
Positive
Not determined Positive,
M.luteus
First time:
negative
Positive
S15UPRCRISENRE30P01 Bacillus, Gram
Positive
Not determined Negative Negative
2nd
time:
negative

8. Discussion:
Due to the results obtained during the
completion of this research it is concluded that
the hypothesis established was correct. The
hypothesis was proven because the bacteria
obtained from tropical soils had antibacterial
properties. From the four bacteria encountered
from different environments, two developed
antibacterial properties. These two represent
50% of the bacteria examined. It is important
to emphasize that the bacteria that did not
developed antibiotic properties, for either
M.luteus or E.coli, are not discarded from
developing these properties with another
bacteria. Also, 50% of the bacteria had
positive results on the PCR product which
means that bacteria from tropical soils can be
characterized and tested in Bioinformatics.
For further studies, bacteria are going
to be tested with cellulose to determine if they
have the potential to break it down into
monomers and ingest it. Also, an endospore
stain can be performed to observe if bacteria
exhibit a prolonged dormancy stage. This is
relevant to the study because it implies that the
bacteria encountered can live and reproduce in
a hostile environment.
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Acknowledgments
RISE Program
University of Puerto Rico at Cayey
Dr. Michael Rubin
Department of Biology, University of Puerto
Rico at Cayey
Lizbeth Pérez
RISE Student, University of Puerto Rico at
Cayey
Mr. Giovanni Cruz
RISE lab technician
Dr. Eneida Díaz
Department of Biology, University of Puerto
Rico at Cayey
Dr. Elena González
Department of English, University of Puerto
Rico at Cayey