University of Puerto Rico at Cayey
Department of Biology
Bacteria Isolation and Characterization of Soil Bacteria in Aguas Buenas, Puerto Rico
Sara Díaz
Patricia Rodríguez
Some bacterium are pathogenic while others are harmless. The discovery of new bacterium is important because they are
ecologically and medicinally beneficial. A research was carried out to isolate and characterize new soil bacteria with
beneficial properties, namely antibiotic production and resistance. A soil sample from Aguas Buenas was collected and the
bacterias were isolated, purified, and characterized. In order to prove the hypothesis, a series of tests were carried out. Results
demonstrated that the bacteria has some resistance against antibiotic Rifampin.
Prokaryotes have been present for approximately
directly from soil organisms. This investigation shares
three fourths of planet Earth’s history. Throughout this time
similar objectives to our research but utilizes a different
they have developed the ability to adapt to almost all
approach. Studies suggest that the widespread use of
ecological habitats. They possess diverse metabolic
antimicrobial chemicals may be causing antibiotics to lose
capabilities that allow them to use a variety of organic or
their effectiveness. Using antimicrobial chemicals frequently
inorganic compound as a food source. The concept of
enough may have an adverse effect, by destroying some
bacteria, tends to be directly related to diseases due to their
bacteria and allowing the resistance subset to proliferate. Due
pathogenic characteristics. However, these microscopic
to the overuse of antibiotics, bacterium have become
single celled organisms are essential to our existence because
resistant to them. This has led to a major public health crisis.
our exposure to them is an important part of the development
Certainly, it’s clear that new antibiotics are needed in order
of our immune systems. Some bacteria have symbiotic
to confront resistance, and their need will be even more
relationships with plants and invertebrates where they carry
pressing in order to combat emergent diseases in the future.
out the processes of nitrogen fixation and cellulose
There are a vast amount of bacterial species in the world and
degradation in order to sustain these higher life forms.
most of them are still unknown. Discovering new bacterium
Additionally, bacterium play an indispensable role in the
in our environment could have great future benefits. By
cycling of chemical elements necessary for human life, soil
broadening our knowledge on this field we can determine the
fertilization and provision of nutrients. Some bacterium are
properties and characteristics of various bacterium and later
useful in the preparation of foods, chemicals, and antibiotics.
develop innovative ways to counteract their negative effects.
Previous studies have been made in order to discover and
This is important because many bacterium possess properties
characterize new bacterium with the purpose of evaluating
beneficial to society’s overall health. Some of these
their specific characteristics, for medical and ecological
beneficial properties are the ability to degrade oil, produce
benefits and applications. As an outside reference for this
cellulose activity, and produce or resist antibiotics. The
investigation, a research found in NCBI Pub Med done by
hypothesis for this research is that we will discover a new
MacNeil IA et.al titled Expression and Isolation of
soil bacteria with at least two beneficial properties, namely
Antimicrobial Small Molecules from Soil DNA Libraries was
antibiotic production and oil degradation. The purpose of this
used. They carried out a research with the mission of finding
research is to discover and catalogue new soil bacteria, while
new uncultured bacteria species that would provide an
incorporating practical lab techniques. The future
undiscovered gene pool. This gene pool could then be used
implications of this study are that finding bacteria to degrade
to produce natural products for clinical purposes. They were
oil will help clean up oil spills all around the world, and also
able to support the idea that technology has the potential to
produce cellulose activity. Although cellulose is commonly
provide new natural products that surge from microbial
produced in plants it is also produced by bacteria. In addition
diversity and its importance for new tool drug discovery. The
one of the main goals is to find bacteria that produce or resist
technology they used was a bacteria artificial chromosome
bacteria in order to develop new antibiotics.
library containing genomic fragments of DNA isolated
the original bacterial purifications for each medium type.
Methodology
Both were observed under the microscope and their cellular
Soil was collected from Aguas Buenas in a labeled zip lock
bag using a sterile spoon. Tools like a GPS, a camera and a
ruler were used to obtain the location’s coordinates,
photographs and soil measurements. In the laboratory, the
plates for the first enrichment were appropriately labeled. 1.0
g of soil was placed into a beaker and 9ml of distilled water
were added, creating the 100 solution. The 10−2 𝑡𝑜 10−5
serial dilutions were made from the 100 soil suspension and
deposited in six labeled test tubes. 50µl from the 10^0
solution were placed on the center of the respective medium
plate. An L shaped streaking loop was used to streak using
the appropriate techniques. The previous step was repeated
using the 10−5 solution. The plates were left for incubation
at 30⁰C for 48-72 hours. Afterwards, an RDM and a TSA
plate were used to incubate the bacterial colonies. The swab
streak technique was carried out using an inoculating loop
and the plates were incubated at 300C. In order to be able to
prepare samples for cryogenic freezing, a liquid culture of
each isolate was prepared by transferring 3mL of broth into
the appropriate media. The purified bacterium were
incubated at
300C
and left overnight. The next day, two
cryogenic tubes were labeled for each isolate, a working
stock and a permanent stock. 1ml of 80% glycerol was added
to the liquid cultures. The tubes were left overnight in the
freezer at -800C. After 24 hours, the cultures were taken out
of the freezer. A viability test of the cell stocks was
performed by streaking the cultures using an inoculating
loop. Lastly they were incubated at 300C. The genomic DNA
purification was performed by transferring 500µl of the
morphology was documented. A drop of water was placed on
a microscope slide. A small amount of a single colony was
picked up with a sterile loop and the cells were suspended by
rubbing the loop in the water drop. The suspension was
spread out, air dried, and heat fixed in the iron. The heat
fixed smear was flooded with crystal violet for 1 minute and
then rinsed gently in flowing tap water. Remaining water was
washed off with Iodine solution for 1 minute. The smear was
covered with a few drops of ethanol and then drained off and
rinsed off with tap water. Additional water was washed off
with the safranin solution for 1 minute. The excess safranin
was washed off and the slide was blotted between sheets of
paper towel to dry. The smear was placed under the
microscope and examined with oil immersed lens.
Depending on the visible color, the gram type of the bacteria
was identified.The test for antibiotic production was carried
out by using an overnight broth for each bacteria
respectively. Using the broth, each bacteria was patched onto
appropriate plates of M. luteus and E.coli. The plates were
incubated at 370C for 48 hours. The next step was inspecting
the plates and checking for any evidence of clearing. The test
for antibiotic resistance was carried out by dipping a sterile
cotton swab into the bacterial culture tube and swabbing it on
the entire surface of the agar plate. The plate was left to dry
for 15 minutes. The selected antibiotic discs were placed on
the culture-painted plate which were incubated for 48 hours.
Data analysis took place to check for inhibition growth
zones. Aseptic techniques were used throughout all the
previous procedures.
liquid culture broth into a micro centrifuge tube. The tubes
were placed in each station: 1000C water, ice, and the micro
centrifuge at 14K RPM for 10 minutes. Finally, 300µl from
the supernatant were transferred into clean tubes and kept in
ice. To set up the PCR 6µl of DNA were added to the PCR
tube. The gel electrophoresis was prepared by having 1
molecular gel marker of 1.5K base band. The PCR products
were purified. The gel electrophoresis amplification was
performed by placing the PCR samples into the gel. The
colonies were examined and characterized by using one of
Results and Discussion
A total of two bacteria were isolated successfully and
characterized. The soil samples were collected from Aguas
Buenas, Puerto Rico. The temperature was moderate at 270C.
The bacterial names are: S15UPRC-RISEPRM30M01A and
S15UPRC-RISESCDM30T01B.
The
first
sample
was
collected from "La Charca" in Aguas Buenas. The shape of the
bacteria was circular with a curled margin and a raised/flat
elevation. The texture was smooth and its appearance was
gram stain was gram positive and the bacteria’s cells were
glistening. Its optical property was opaque. The result of the
identified as bacillus. Observation 2.2 shows a photograph of
gram stain was gram positive and the bacteria was identified
the gram stain slide. Observation 2.3 shows a photograph of
as bacillus. The results of the antibiotic resistance were all
the antibiotic resistance plate against the antibiotics
negative. The test for antibiotic production showed that the
Vancomycin, Rifampin, Tetracyclin, Streptomycin, and
bacteria did not produce antibiotics against E. coli or M.
Kanamycin. Observation 2.4 shows a graph of each antibiotic
luteus. It showed that they could coexist because it did not
and their respective diameter of bacterial clearing. It
inhibit bacterial growth. The
second isolation was also
demonstrates that the bacteria was most effective in resisting
collected from "La Charca" in Aguas Buenas. The shape of the
Rifampin and less efective in resisting Streptomycin. The
bacterial colony was rhizoid with a undulated margin and a
bacteria was not able to inhibit M. luteus or E. Coli growth,
flat elevation. The texture was smooth and its appearance was
giving negative results in the antibiotic production tests.
dull. Its optical property was transparent. Observation 2.1
Future experiments include the performance of oil and
shows a photograph of its bacterial colony. The result of the
cellulose assays.
References:
1) Macneil, I.A, CL Tiong, C. Minor, PR August, TH Grossman, and KA Loaicono. "Expression and Isolation of
Antimicrobial Small Molecules from Soil DNA Libraries." NCBI PubMed. 3 Apr. 2001. Web.
Annex:
Observation 2.1
Observation 2.4
3.33
6.25
7.14
5.88
7.69
ANTIBIOTIC RESISTANCE
DIAMETER OF CLEARING (CM^-1)
Observation 2.2
Observation 2.3
V A N C O M Y C I N R I F A M P I N T E T R A C Y C L SI NT R E P T O M Y C I KNA N A M Y C I N
ANTIBIOTIC