Isolation and Characterization of

Mycobacteriophages Isolated from Tropical Soils

of Puerto Rico.

Mónica Rivera-TorresCharlene Rivera-Bonet

Mentor: Dr. Michael RubinUniversity of Puerto Rico at Cayey

RISE Program

Introduction Bacteriophages: virus that infect bacteria

Structure: Head with DNA and tail

Life Cycles: Lytic and Lysogenic

Mycobacteriophages: affect Mycobacteria

Significance / Advantages

Phage therapy

Template for research and discovery of emerging bacteriophages

Novel genes

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

Materials Agar plates

Top Agar

Phage buffer

M. smegmatis culture

Centrifuge

Incubator

Phages

Methodology

Isolate Phage

Prepare Filtrate

Plaque Screening

Plaque Purification

Web Patterns

(Dilutions)

High titter Assay

MethodsSoil 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

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.

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.

Methods

Plaque Purificationo 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.

MethodsSecond Enrichment and filtration

Dilutions

High Titer Assay

ResultsPhages found in first try

o Monchar

Results1st Plaque Purification

Higher Concentration

Lower Concentration

Least Concentration

Results

2nd Plaque Purification Successful

3rd Plaque Purification Six times unsuccessful

Results A second 2nd Plaque

Purification was made but this one also came out wrong.

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.

Acknowledgements Lab Technician: Giovanni Cruz

Christopher

Dr. Michael Rubin

RISE Program

Questions?

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