Mycobacteriophages are viruses that infect mycobacteria. The document describes experiments conducted by students to isolate and characterize mycobacteriophages from soil samples. Soil samples were collected from various locations and depths, enriched in culture, and screened for plaques indicating the presence of phages. While proper procedures were followed, none of the samples yielded phages except for one deeper sample. The experiments helped students learn aseptic technique and other lab skills, but the primary objective of isolating a phage was not achieved, possibly due to environmental or technical factors.

1. Mycobacteriophages
By:
Anaeli Shockey
Nicolle A. Rosa
Delaine M. Zayas-Bazán
Zuleika Velázquez Ortiz
BIOL 4997
Rise Program

2. Introduction
• Mycrobateriophages are viruses that infect
bacteria belonging to the Mycibacterium genus.
• They are relatively easy to isolate and
characterize.
• They are great for studying evolution.
• Bacteriophages have two cycles: lytic cycle
(reproduction) and lysogenic cycle (integration)

3. • In our experiment we looked for
mycobacteriophages and completed the required
steps to isolate and characterize it.
• This had some implications such as:
 Learning the Aseptic Technique
 Applying other laboratory skills: plating,
centrifugation and mixing solutions among others.
 Understanding the importance of finding and
characterizing a mycobacteriophage

4. Materials Sample Collection
 Lab Notebook
 Sterile 15mL conical tube with cap
 Sterile Spoon
 GPS or Google Earth

5. Materials Enrichment Culture
 Clean Spatula
 Approximately 0.5g of the soil sample
 Sterile 50mL conical tube with cap
 Weighing pan
 Soil Samples

6. Materials Harvesting and Plaque Screening
 5mL sterile disposable pipettes
 Agar Plates
 Bacteria
 Centrifuge
 Enriched Sample
 Sterile 15mL conical tube with cap
 Sterile Filter
 Sterile Syringe
 Tips
 Top Agar

7. Methods Sample Collection
 Identify the place for soil sample collection
 Recollect the soil sample with a sterile spoon
and put it in the sterile 15mL conical tube with
cap
 Close quickly
 Record the location with a picture (place and
you) and the environmental data.

8. Methods Enrichment Culture
 Obtain ~0.5g of the soil sample
 Added it to the enrinchment solution placed in
the sterile 50mL conical tube with cap.
 Close it slightly
 Incubate at 37° C for 24 hours

9. Methods Harvesting and Preparing the
Enriched Sample
 Centrifuge the enriched sample at 3000 RPM for 10
minutes
 Prepare phage filtrate
 Obtain the supernatant of the sample
 Filter the supernatant into a sterile 15mL conical tube
with cap and close quickly

10. Method Plaque Screening
 Label the Agar Plate with the necessary information and
name the zone of the Agar Plate with number (1, 2 and 3)
 In the extracted supernatant wet the wood side of a tip.
 Make the first striate with the wet tip in the zone 1
 Use another tip to disperse the striate of the zone 1 to the
zone 2 and the same procedure with the zone 2 to the
zone 3.

11.  Add 5.0mL of Top Agar with the bacteria to the
Agar Plate from the zone with the less amount of
sample to the most amount of sample
 Let the Top Agar solidifies, invert the plate quickly.
 Incubate the inverted plate at 37°C
Observe
Phages or Not Phages?

12. Date Time Weather Location Other
Conditions GPS Information
Temperature: 28°C Site: Urban Depth: .5cm
Precipitation: 10% Proximity to: Trees This soil sample was
February 9th 2:45 PM Humidity: 65% and buildings not utilized due to the
Wind: 13km/h 18°08'44.56"N 65°49'17.55"W absence of bacteria
Sunny/Clear Humacao, Puerto Rico in the lab.
Temperature: 27.2°C Site: Urban Depth: .5cm
Precipitation: 40% Proximity to: Trees This soil sample was
February 18th 1:39 PM Humidity: 69% and buildings utilized, but it did not
Wind: 20km/h 18°08'44.56"N 65°49'17.55"W contain phages.
Cloudy Humacao, Puerto Rico
Temperature: 29°C Site: Urban Depth: .3cm
Proximity to: Beach This soil sample was
March 3rd 1:00 PM Humidity: 67% and to the road utilized, but it did not
Wind: 24km/h 18°10'9.38"N 65°44'28.82"W contain phages.
Sunny/Clear Humacao, Puerto Rico
Temperature: 31°C Site: Rural Depth: .5cm
Proximity to: Trees This soil sample was
March 3rd 4:36 PM Humidity: 49% and buildings utilized, but it did not
18°17'5.66"N 66° 0'41.57"W contain phages.
Sunny/Clear Gurabo, Puerto Rico
Delaine’s Soil Samples

13. Date Time Weather Location Other
Conditions GPS Information
Temperature: 27°C Site: Urban Depth: .5cm
Proximity to: Very close to This soil sample was
March 11th 6:36 PM buildings utilized, but it did not
18°7'5"N 66°8'43"W contain phages.
Clear/ Dark Cayey, Puerto Rico
Temperature: 29°C Site: Rural Depth: .2cm
Proximity to: Trees This soil sample was
March 17th 3:27 PM Humidity: 69% and buildings utilized, but it did not
18°17'5.55"N 66° 0'41.29"W contain phages.
Sunny/Clear Gurabo, Puerto Rico
Temperature: 29°C Site: Rural Depth: .6cm
Proximity to: Trees This soil sample was
March 17th 3:30 PM Humidity: 69% and buildings utilized, but it did not
18°17'10.43"N 66° 0'39.28"W contain phages.
Sunny/Clear Gurabo, Puerto Rico
Temperature: 29°C Site: Rural Depth: .4cm
Proximity to: Trees This soil sample was
March 17th 3:32 PM Humidity: 69% and buildings utilized, but it did not
18°17'5.79"N 66° 0'42.14"W contain phages.
Sunny/Clear Gurabo, Puerto Rico

14. Zuleika’s Soil Sample
Date Time Weather Condition Location
GPS
Other
Information
February 18 4:14 PM
Temperature: 68°
Cloudy/ Clear
Site: Rural
Proximity to: 5 minutes to
the main road, trees and my
house
18° 10'06.46"N
66° 11'30.52" W
Cidra, Puerto Rico
Depth: 1cm
This soil sample was
utilized, but it did not
contain phages.
March 3 1:19 PM
Temperature: 76°
Sunny/Clear
Site: Rural
Proximity to: 5 minutes to
the main road, trees and my
house
Cidra, Puerto Rico
Depth: 0.8cm
This soil sample was
utilized, but it did not
contain phages.
March 12 9:36 AM
Temperature: 79°
Sunny/Clear
Site: Rural
Proximity to: fruits trees
and my house
Cidra, Puerto Rico
Depth: 1.2cm
This soil sample was
utilized, but it did not
contain phages.
March 12 9:42 AM
Temperature: 79°
Sunny/Clear
Site: Rural
Proximity to: fruits trees
and my house
Cidra, Puerto Rico
Depth: 0.6 cm
This soil sample was
utilized, but it did not
contain phages.
April 8 8:18 AM
Temperature: 82°
Sunny
Site: Rural
Proximity to: 5 minutes to
the main road, trees and my
house
Cidra, Puerto Rico
Depth: 0.7cm
This soil sample was
utilized, but a problem with
the bacteria was presented.
It did not appear phages.

16. Comparison
 All of the samples were diverse
 Hours, locations, and temperatures among other determining factors
 Factors
 The municipalities
 They have varying geographic and geomorphic
 The blue represents the karst region
 The yellow represents the coastal region
 The green represents the inner mountain region

17.  Type of soil
◦ Unfortunately this information was not adequately recorded,
because the only description mentioned was dry, moist or
normal.
◦ There are three types of soil: sand, clay and silt, and each of
those contain certain characteristics that difference them.
◦ Vegetation surrounding the location
 The vegetation is diverse from municipality to
municipality.
 The depth was diverse to and might have been the
determining factor
◦ All of the negative results were superficial, while the only
positive one was at almost 6 cm.

18. Conclusion
 Although the experiments and procedures were
performed accurately, no bacteriophages were found.
 This might be for any of the reasons discussed in the
comparison or because the laboratory technicalities,
such as the availability of bacteria.
 The main objective was not accomplished, but other
objectives like practicing important laboratory skills
were completed.