1. Isolation, Purification and
Characterization of Gêmeos and
Isinaayusferti, bacteriophages
Isolated from Tropical Soils
Amanda M. Icazatti Burtell, Fernando Pachecho Ocasio,
John Rivera Meléndez & Leyda Villagrasa Méndez
Dr. Michael Rubin
RISE Program
University of Puerto Rico at Cayey

2. Introduction
• What is a bacteriophage?
• What host bacteria are being used?
– Mycobacterium smegmatis
– Bacillus cereus
• Where can bacteriophages be found?
• What do bacteriophages need for living?
• Types of life cycles: Lytic vs. Lysogenic cycle

3. • Why are bacteriophages important?
Phages can be used in various fields of science such
as:
- Genetics - Cloning and mutation of DNA and
studies in genome
- Therapeutic uses for the creation of antibiotics
resistant to bacteria
• What is the purpose of this experiment?

4. Problem and Hypothesis
• Problem: Can novel and interesting
bacteriophages be isolated from the tropical
soils of Puerto Rico?
• Hypothesis: Both Mycobacteriphages and
Bacilophages will be found on the tropical
soils of Puerto Rico.

5. Procedure
Collection of Soil
Enrichment Centrifuge
FiltrationStreakin Process:
Purification X3

6. Dilutions
Spot test
Empirical testWeb pattern

7. Preliminary Results

8. Sample Coordinates Details Depth Temperature Date Place
Sample
#1
AMIB
Lat: 18.21561
L:on: -66.044790
Humid and
Dry soil
3’’ 25˚C 02/02/14 Caguas,
PR
Sample
# 2
AMIB
Lat: 18.234378
Lon:-66.061491
Dry soil 2’’ 25˚C 02/17/14 Caguas,
PR
Sample
#3
AMIB
Lat: 18.234604
Lon:-66.06138
Humid and
Dry soil
1’’ 27˚C 02/24/14 Caguas,
PR
Sample
#4
AMIB
Lat: 18.213103
Lat: -66.041709
Humid and
Damp soil
’
3’’ 26˚C 03/04/14 Caguas,
PR
Sample
#5
AMIB
Lat: 18.215715
Lon: -66.044879
Dry soil 1’’ 25˚C 03/11/14 Caguas,
PR

9. Sample #1
FPO
Coordinates
Lat: 18.11’15”N
L:on: 65.57’54”
Details
Dry soil
Depth
3”
Temperature
24c
Date
04/02/14
Place
San
Lorenzo,
Pr
Sample #2
FPO
Lat: 18.11’15”N
Lon: 65.57’53”W
Very dry
soil.
4” 26˚C 18/02/14 San
Lorenzo,
Pr
Sample #3
FPO
Lat: 18.7’6”N
Lon: 66.9’42”W
Humind
Soil
3” 28˚C 18/02/14 Cayey, Pr
Sample #4
FPO
Lat: 18.11’15”N
Lon: 65.57’53”W
Humid
Soil
4” 22˚C 26/02/14 San
Lorenzo,
Pr
Sample #5
FPO
Lat: 18.2’26”N
Lon: 66.35’35”
Humid
Soil
6” 30˚C 02/03/14 Ponce, Pr

10. Gêmeos (bacilophage)
• Coordenates: Lat: 23' 36" 23 S Long: 46' 41" 51 W
• Temperature: 19˚C
• Details: Dark and damp soil
• Found in a populated area near a river
• Sao Paolo. Brazil

11. Gêmeos: Preliminary Results

12. Sample Coordenates Details Depth Temperature Date Place
Sample
#1
LVM
Lat: 18.2333419
Long:
-66.0478104
Humid and
plain soil
2” 26˚C Feb/3/14 Caguas,
PR
Sample
# 2
LVM
LAT: 18.1192448
LONG:
-66.1667764
Dry and
lumpy soil
1” 25˚C Feb/16/
14
Cayey,
PR
Sample
#3
LVM
Lat: 18.2333419
Long:
-66.0478104
Humid and
plain soil
3” 27˚C Feb/23/
14
Caguas,
PR

13. Sample Coordenates Details Depth Temperatur
e
Date Place
Sample
#1
JLR
Lat: 18 Nᵒ
Long: 66 Wᵒ
Humid and
saturated
soil
1‘’ 31˚C Feb/3/14 Cidra,
PR
Sample
# 2
JLR
Lat: 18 Nᵒ
Long: 66 Wᵒ
Dry soil
under
trash
disposal
Surface
sample
32˚C Feb/21/14 Cidra,
PR

14. Isinaayusferti (mycobacteriophage)
Coordenates: Lat: 18˚ 14’ .0312 " N
Long: 66˚ 2' 52.116" W
Temperature: 27 ˚ C
Place: Caguas, Puerto Rico
Details: Humid and plain soil. Found near the
garbage in front of a house.

15. Isinaayusferti (mycobacteriophage)

16. Isinaayusferti: Preliminary Results
High Titer Phage Lisate

17. Conclusion
• The soil samples were collected and isolated
using the enrichment method.
• A bacillophage was found in Sao Paolo, Brazil
and was named Gêmeos.
• Third Purification Stock Filtrate (Gêmeos)
• A mycobacteriophage found in Caguas, Puerto
Rico was named Isinaayusferti.
• Concentrated Stock Phage (Isinaayusferti)

18. Acknoledgements
• RISE Program
• Dr. Michael Rubin
• Dr. Eneida Díaz
• Gustavo Martínez
• Giovanni Cruz
• Christopher Quintanal