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.
Presentation: Isolation and Characterization of Bacteria from Tropical Soils
1. Isolation and Characterization of
Bacteria from Tropical Soils
Alejandra Ferrer1
Nicole S. Rivera1
Jessica Ortiz1
Carolina Huertas1
Dr. Michael Rubin
Date: May 15, 2015
RISE Program
Biology and Chemistry Department
University of Puerto Rico at Cayey
1 This author contributed equally to this project
2. Background
• Bacteria are prokaryotic microorganisms that can be differentiated by
their peptidoglycan layer located in the cell wall.
• This aspect classifies them as gram-positive or gram-negative.
3. Antibiotic Era
• The discovery of penicillin in 1928 was by Alexander Fleming.
• 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).
• Rene Dubos- discovered and isolated an antibacterial compound
called tyrothricin.
• Recently, scientists have discovered a new antibiotic, from the soil,
which name is Teixobactin (Ling et al. 2015).
5. • Antibiotic resistance is produced when the capability of killing or
inhibiting bacterial growth is eliminated. The bacteria can grow in
the presence of the antibiotic (APUA 2014).
• In a recent investigation Walsh and colleagues (2003) isolated 412
antibiotic resistant bacteria from agricultural, urban, and pristine
soils.
6. Significance
● The importance of this study is that it attempts to find new
bacteria from tropical soils with antibacterial properties.
● This investigation responds to the need for new antibiotics that
can inhibit bacterial reproduction.
7. Problem
• Can we find bacteria in soil that have
useful properties such as antibiotic
production and antibiotic resistance?
8. Hypothesis
• It is hypothesized that different properties of the
isolated soil bacteria will be characterized such as
bacterial structure, antibiotic resistance and
antibiotic production .
• There will be more bacteria found in tropical soils
capable of antibiotic production and resistance .
(Dubisnky et al. 2010)
9. Goals
• Isolation, purification and freezing of soil bacteria
• Gram staining and colony morphology
• Test for antibiotic production and resistance
• Generation of genomic DNA, PCR, gel electrophoresis
and purification
• Sequencing and Bioinformatics Analysis
11. collect the colony
II. Purification
III. Cryogenic Freezing
10 mL
pipette
S15UPRC-RISEInitials30M01
S :Spring
15 :2015
UPRC: UPR-Cayey
RISE: RISE Program
Initials: Your Initials (MRR)
30: Incubation Temperature in Celsius
M: Media
(T = Tryptic Soy Agar; N = Nutrient Agar; L = LB Agar; M =
RDM; R = R2A; P = 1SP4)
01: Soil Sample Number
spread the
bacteria
medium
broth
80%
glycerol
micro
tube
cultured
medium plate
pipette
scratch the
plate
collect
the mix
test
tube mix
identify the tubes
http://amoya.webnode.es/foros-de-debate/
http://amoya.webnode.es/foros-de-debate/
http://amoya.webnode.es/foros-de-debate/
http://amoya.webnode.es/foros-de-debate/
12. IV. Gram staining
A drop of water in the
center of the slide.
Collect bacteria and add
them to the slide
For 1 minute and
remove it with
water
safranin
ethanol
crystal violet
bacteria
slides
water
inoculation
loop
13. V. Genomic DNA and Polymerase Chain
Reaction (PCR) centrifuge
dry bath ice bath
liquid broth
with bacteria
PCR tube
collect the
supernatant
http://amoya.webnode.es/foros-de-debate/
http://amoya.webnode.es/foros-de-
debate/
http://amoya.webnode.es/foros-
de-debate/
http://amoya.webnode.es/foros-de-debate/
15. VII. Antibiotic Production and Resistance Tests
M. Luteus
E. Coli
Grow bacteria in separate
plates
Divide the plates in two
portions
Dip the paper disk in the
bacteria culture and place it
in a side of the petri dish
Incubator
24 hourshttp://amoya.webnode.es/foros-de-debate/
http://amoya.webnode.es/foros-de-debate
16. VIII. Purification of PCR Products for Sequence
Determination
QIAquick PCR Purification kit
Calculate the
volume of the
buffer required
add calculated amount to PCR
product
Transfer into a QIAquick
column
Centrifugeadd buffer PE
Place the column in a
clean microtube
Pure water
Analyze the sample
http://amoya.webnode.es/foros-de-debate/
http://amoya.webnode.es/foros-de-
debate/
http://amoya.webnode.es/foros-de-debate/
http://amoya.webnode.es/foros-de-debate/
http://amoya.webnode.es/foros-de-
debate/
http://amoya.webnode.es/foros-de-
debate/
http://amoya.webnode.es/foros-
de-debate/
37. Conclusion
• Nine bacteria were isolated from tropical soils of Puerto
Rico and characterized.
• All bacteria are gram positive and seven of nine are
bacilli.
• Four produced antibiotics, two bacteria coexisted with
Escherichia coli or Micrococcus leteus, two bacteria
showed antibiotic resistance to tetracycline.
• Bacteria with antibiotic resistance and antibiotic
production are found in Puerto Rican (tropical) soil.
38. Future Experiments
• Test bacteria for cellulose and oil
degradation capability
• Test antibiotic production with other bacteria
• Repeat the investigation by isolating new
bacteria in order to have a better
geographical distribution
39. Acknowledgments
• RISE Program (Proposal:5R25GMO59429-16)
University of Puerto Rico at Cayey
• Dr. Michael Rubin
Department of Biology, University of Puerto Rico at Cayey
• Dra. Eneida Diaz
RISE Program, Department of Biology, University of Puerto Rico at Cayey
• Dra. Elena González
RISE Program, Department of English , University of Puerto Rico at Cayey
40. Acknowledgments
• Giovanni Cruz
RISE Program, University of Puerto Rico at Cayey
• Lizbeth Pérez
RISE Student, University of Puerto Rico at Cayey
• José Pabón
Department of Biology, University of Puerto Rico at Cayey
• Cristopher Quintanal
Howard Hughes Program, University of Puerto Rico at Cayey
• Laura Ramos
Department of Biology, University of Puerto Rico at Cayey
41. Isolation and Characterization of
Bacteria from Tropical Soils
Alejandra Ferrer1
Nicole S. Rivera1
Jessica Ortiz1
Carolina Huertas1
Dr. Michael Rubin
Date: May 15, 2015
RISE Program
Biology and Chemistry Department
University of Puerto Rico at Cayey
1 This author contributed equally to this project