Presentation: Isolation and Characterization of Bacteria from Tropical Soils
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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.
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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
- 10. Experimental Design http://amoya.webnode.es/foros-de-debate/ 1 gram of soil mix Label the tubes 0→ -5 water (8.5ml) NaCl (90µl) streak the plate NaCl medium plates
- 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/
- 17. Geolocation of Bacteria Alejandra Ferrer Nicole Rivera Carolina Huertas Jessica Ortiz
- 19. Soil Collection Latitude: 18° 8’ 38.6” N Longitude: 66° 14’ 45.8” W Aguas Buenas Aibonito
- 20. Results
- 21. Results Gram Staining Endospore Staining Bacteria: S15UPRCRISEJCOR30P01A Jessica Ortiz Purified bacteria Coexistence with E.coli
- 22. Bacteria: S15UPRCRISEJCOR30P01A Resistance to Tetracycline Clusters of bacteria Repetition Repetition with broth of clusters
- 23. Bacteria: S15UPRCRISEJCOR30P01A Agarose gel #1: PCR Product Agarose gel #2: Purified PCR Products
- 24. Bacteria: S15UPRCRISEJCOR30P02A Jessica Ortiz Changes in color according to incubation period at 30 C 1 day 2 days 3 days Gram Staining
- 25. Bacteria: S15UPRCRISEJCOR30P02A Resistance to Tetracycline Clusters of bacteria Repetition with broth of clusters
- 26. Bacteria: S15UPRCRISEJCOR30M02B Jessica Ortiz Purified Bacteria Gram Staining Circles of resistance against tetracycline Coexistence with M.luteus
- 27. Bacteria: S15UPRCRISEJCOR30M02B Agarose gel #1: PCR Product Agarose gel #2: Purified PCR Products
- 28. Bacteria: S15UPRCRISECHA30M01 Carolina Huertas Morphology of Bacteria Gram Stain of Bacteria Antibiotic Production of Bacteria -->
- 29. Bacteria S15UPRCRISEAFD30M01A Gram stain Colony morphology Gram stain Antibiotic production for M. Luteus
- 30. Bacteria S15UPRCRISEAFD30M01B. Alejandra Ferrer Colony morphology Gram stain Antibiotic production
- 31. Bacteria S15UPRCRISEAFD30M01A 16S_1510R and 16S_8F primers Alternative Primers
- 32. Bacteria: S15UPRCRISENRE30M01A Nicole S. Rivera EspinalAntibiotic production Gram stain Colony morphology
- 34. Bacteria: S15UPRCRISENRE30P01B Nicole S. Rivera Espinal Gram stain Colony morphology Antibiotic production
- 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