Isolation and Characterization of Mycobacteriophages Isolated from Tropical Soils of Puerto Rico.

Charlene N. Rivera-Bonet, Mónica Rivera-Torres

ABSTRACT

Phages are often studied and characterized in order to investigate their potential functions in terms of scientific or medicinal innovations. A sample was collected from the soil using aseptic techniques. Then it was enriched with an M. smegmatis culture and incubated for 24 hours at 37ºC. From the enrichment the supernatant is later filtered and used for purification. After purifying three times using an agar plate and M. smegmatis culture the plaques found in the plate(clear spots) indicate the phages location. The next steps to continuing this project are dilutions and creating a web pattern.

Introduction

Bacteriophages, better known as phages, are viruses that infect bacteria. Phages are made up of a head that contains the genetic material, and a tail, which they use to inject their DNA to the host cell. They have two types of reproduction, by which they are classified. One is the lytic life cycle, which is carried by lytic bacteriophages. They lyse the host as a normal part of their life cycle. The temperate phages carry out the Lysogenic life cycle which consists of the integration of the phage’s DNA into the host’s genome. As the bacteria divide, it divides (Kaiser 2012)..

Mycobacteriophage is a virus that only infects bacteria belonging to the Mycobacteria genus. Examples of this type of phage are tuberculosis and M. smegmatis.

Mycobacterium smegmatis has become the most important bacteria for biological studies. It is easy to be cultured and reproduces quickly. It is non-pathogenic to humans or other animals. Its basic structure and metabolism are common to other mycobacteria, including ones that can cause devastating diseases (Higa 2013).

Several advantages can be found when studying bacteriophages. By studying phages novel genes can be discovered, and new therapies against antibiotic resistant pathogenic bacteria can be developed.

Hypothesis

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

Materials and Methods

The materials used throughout this investigation included sterile spoon, test tubes and GPS for sample collection, and Agar plates, Top Agar, sterilization filters, syringes, phage buffer, M. smegmatis culture, micropipettes, pipettes, vortexer, centrifuge and incubators for the rest of the experiment.

Soil Sample Collection

Soil sample was collected using a sterile spoon and test tube. Data about the area the sample was taken from was annotated.

Enrichment

The first step after the soil sample collection was the Enrichment. For the enrichment o.5 grams of soil sample were measured and added into 50mL of solution containing 8mL sterile water, 1mL sterile 10x broth, 1mL

AD supplement,0.1mL of 100mM CaCl2.

1ml of late log/early stationary phase M. smegmatis culture (48 hour culture) were also added to the flask. Afterwards it was incubated at 37º C, shaking at 220rpm for a 24 hours period.

Harvesting

After 24 hours of incubation, the sample spun at 3,000rpm for 100 minutes to pellet particulate matter, including most of the bacterial cells. Then poured supernatant from centrifuged sample to a 50mL conical tube, filter-sterilized enrichment sample, capped and label the rube

Plaque Streak

Using a sterile wooden stick the enrichment was streaked back and forth across the top third or the agar plate without lifting it. Cover up the petri dish and discard the wooden stick.

This process was repeated using a new wooden stick; streak the adjacent un-streaked portion of the agar making sure to overlap on the first few strokes, beginning in the area streaked before.

For the remaining portion of the plate, the streaking process was repeated on the last portion of the plate.

Next 4.5mL of Top Agar were added to 0.5mL of M. smegmatis, using serological pipet to carefully dispense it onto the most dilute area of the plate and allow it to spread across the plate to the most concentrated areas. Then covered the plate and allowed the Top Agar to become hard. After it became hard it was incubated at 37ºC and to be checked the next day.

Plaque Purification:

In order to obtain pure phage, three plaque purifications were made.

After following the correct asceptic techniques, a plaque from the plate was chosen and picked with a 100uL micropipette tip. Then, it was transferred to a sterile microtube with 100uL of Phage Buffer. Using a sterile wooden stick, the solution inside the microtube into a petri dish was streaked on the plate, following the same streaking steps discussed before.

4.5mL of Top Agar were added to 0.5mL of M. smegmati,and using a serological pipet it

was carefully dispensed onto the most dilute area of the plate and allow it to spread across the plate to the most concentrated areas. The plate was covered and the Top Agar was allowed to become hard. Afterwards, it was incubated at 37ºC and checked the next day.

Results

Sample Collection

The soil sample was collected February 18, 2013 at 7:30pm. The conditions were 73°F, 1 inch deep, in a somewhat moist area, 6 inches away from cement sidewalk, 2 feet from a house and 10 feet from a large tree. The exact GPS location was 18.23657 N 66.04429 W in Caguas, Puerto Rico.

Enrichment, Harvesting and Plaque Purification

Phages were found on the first try. Since we had the opportunity to name our phage, we picked the name Monchar. After the enrichment was made, the first plaque purification came out well, as can be seen in Figure1.

Fig 1. 1st Plaque Purification.

The second and third plaque purification took more time and trials. The second plaque purification was done three times. The first time it did not come out right, because the bacteria were defective. The second time it got full of fungus and the third time, shown in Figure 2, it was successful.

Fig 2. 2nd Plaque Purification.

The third plaque purification had to be done six times. Each time something different happened. The first second and third time nothing happened. The last three times bacteria colonies formed. An example of this is shown in Figure 3.

Fig 3. 3rd Plaque Purification example.

The second plaque purification, was repeated but nothing came out as can be seen in Figure 4.

Fig 4. 2nd Plaque Purification repetition.

Conclusion

Phages were found in our soil sample collection and our hypothesis was proven correct. Due to a series of misfortunate events including defective bacteria, unwanted colonies or fungus, we only reached the second plaque purification. The soil from where the phages were collected was fertilized with compost. The type of soil may have had a lot to do with the presence of phages since compost is so rich in nutrients and allows bacteria to flourish. This organic matter that fertilized the soil was from the fallen leaves of tree located a short distance from the collection area. Another factor that could have affected the discovery was the temperature. The soil was collected during a cloudy and partially humid day.

Next steps would involve Web Patterns, or Dilutions, and High Titter Assay. Further

research can be done in order to characterize this phage and conclude its functionality in the field of medicine.

References

WiseGeek Conjecture Corporation [Internet]. [2013]. WiseGeek Conjecture Corporation; [cited 2013May14]. Available at: http://www.wisegeek.com/what-is-mycobacterium-smegmatis.htm

Doc Kaiser's Microbiology Home Page [Internet] [2012]. Doc Kaiser's Microbiology Home Page; [cited 2013May14]. Available at: http://faculty.ccbcmd.edu/courses/bio141/lecguide/unit3/viruses/lytlc.html