This document summarizes a student research project that isolated and characterized mycobacteriophages from soil samples in Puerto Rico. Soil samples were collected and enriched to isolate potential phages. Plaque assays were performed to purify isolated phages. One phage, called Monchar, was successfully isolated on the first attempt. Further purification attempts of Monchar were unsuccessful. The student researchers hypothesized that phages could be isolated from the soil given its composition and location. While their hypothesis was proven correct with the initial isolation of Monchar, they were unable to fully characterize the isolated phage due to technical difficulties.

1. Isolation and Characterization
of Mycobacteriophages
Isolated from Tropical Soils of
Puerto Rico.
Mónica Rivera-Torres
Charlene Rivera-Bonet
Mentor: Dr. Michael Rubin
University of Puerto Rico at Cayey
RISE Program

2. Introduction
 Bacteriophages: virus that infect bacteria
 Structure: Head with DNA and tail
 Life Cycles: Lytic and Lysogenic
 Mycobacteriophages: affect Mycobacteria

3. Significance / Advantages
 Phage therapy
 Template for research and discovery of emerging
bacteriophages
 Novel genes

4. Hypothesis
We will be able to find bacteriophages in our soil sample
because of its condition and the location it was collected
from.

5. Materials
 Agar plates
 Top Agar
 Phage buffer
 M. smegmatis culture
 Centrifuge
 Incubator
 Phages

6. Methodology
Isolate Phage
Prepare
Filtrate
Plaque
Screening
Plaque
Purification
Web Patterns
(Dilutions)
High titter
Assay

7. Methods
 Soil Sample Collection
-Date: February 18, 2013
- Time: 7:30 pm
- Aprox. air temp: 73ºF
- 1 inch from ground surface
- Moisture: somewhat moist
- GPS: 18.23657 N 66.04429 W
- Caguas, PR, 00727
- 6inches from cement sidewalk
- 2ft from a house
- 10ft from a large tree

8. Methods
 Enrichment:
o Measure 0.5g of soil and add them into the solution
prepared in a 50mL tube. (Solution contains: sterile
water, sterile 10x broth, AD supplement, CaCl2, and
bacteria)
o Incubate at 37 °C, 220rpm for 24 hours.

9. Methods
 Harvesting:
o Centrifuge.
o Pour supernatant into new 50mL tube.
o Label, filter and cap.
o Plaque:
• Streak the filtering across the agar. 1, 1 2, 2 3
• Add top agar with bacteria and incubate.

10. Methods
 Plaque Purification
o Add 50uL of Phage Buffer to a labeled tube.
o Circle the phage you want to purify and insert
micropipette tip into the phage.
o Place it in the phage buffer.
o Repeat the plaque process.

11. Methods
 Second Enrichment and filtration
 Dilutions
 High Titer Assay

12. Results
 Phages found in first try
o Monchar

13. Results
 1st Plaque Purification
Higher
Concentration
Lower
Concentration
Least
Concentration

14. Results
2nd Plaque Purification
 Successful
3rd Plaque Purification
 Six times unsuccessful

15. Results
 A second 2nd Plaque
Purification was made
but this one also came
out wrong.

16. Conclusion
 Our hypothesis was proven correct.
 The soil from where we did our collection was fertilized
with compost
 Another factor that could have affected our discovery was
the temperature.
 Unwillingly, we only reached the second plaque
purification due to a series of misfortunate events
concerning bad bacteria, or unwanted colonies forming.
 Further research can be done in order to characterize
this phage and conclude its functionality in the field of
medicine.

17. Acknowledgements
 Lab Technician: Giovanni Cruz
 Christopher
 Dr. Michael Rubin
 RISE Program

18. Questions?
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