Purification and isolation of Bricole phageBy Nicole Northam and Briana McLaren and Nazir Barekzi, PhD

The purpose of this was to isolate and purify a phage from acollected soil sample according to the procedures outlined by theSEA PHAGES manual protocol. The soil sample was collected inVirginia Beach, VA at 36°51’21.0”N, 76°03’10.8”W on September2nd, 2014 from a garden. The researchers began by enriching the soilsample and then performed spot tests and streak tests. Theresearchers further purified the phage samples with methods suchas phage titer assays, additional streak tests, a medium titer lysate,empirical assays, titer dilutions, and high titer lysates to isolate onetype of phage from the sample. To date, the researchers support theidea that one type of phage has been isolated through manymethods of purification and that it is lytic in nature.

The strain of bacteria used was MC2155. The researchers useda mixture of 40mL of sterile H2O, 5mL of sterile 10x7H9/glycerol,5mL of AD supplement, 0.5mL of 100mM CaCl2, and 5mL of latelog/early stationary phase of M. smegmatis to 1g of soil.

The researchers performed enrichment and direct plating ofthe soil, plaque screening with serial dilutions, spot tests, streaktests, phage titer assay, medium titer lysate, empirical assay, hightiter lysate, analyzing phage under an electron microscope, stainingphage with 1% uranyl acetate, and DNA isolation procedures.

The researchers conducted about 23 purifications using themethods listed above to isolate one phage type.

Reference:Phage Laboratory Manual. 2014. Howard Hughes Medical Institute.R Adamia. (2012, March 8). Applied Aspects of Bacteriophages. Retrievedfrom https://www.youtube.com/watch?v=sjH6m5VuR6I.Mayer, G. (2010). Bacteriophage. Microbiology and Immunology. Retrievedfrom http://pathmicro.med.sc.edu/mayer/phage.htm.

Funding:Old Dominion UniversityHoward Hughes Medical Institute - Science Education Alliance

Acknowledgements:ORCA, BSSF, Dr. Gauthier, Janis, Mary Hayward, friends of ORCA

In the early stages of research, it was believed that theresearchers had isolated a lysogenic phage based upon themorphology of the plaque forming units which appeared cloudy.Upon later purification via another medium titer lysate whichproduced web plates, those plaque forming units were smaller in sizeand appeared clearer than earlier plaques, indicating the presence oflytic phage. These results could indicate purification was successfulas the researchers may have previously had a mixture of lysogenicand lytic phage. 23 purifications occurred through a variety ofexperimental methods. Some methods such as stick streaks and spottests were performed multiple times and the medium titer lysate andhigh titer lysate were performed twice to isolate the phage.

The findings of this research infer that through the purifications,one type of phage has been found from the sample and it appearslytic in nature.

The researchers learned proper aseptic technique and theimportance of consistently using sterile methods. All of theexperimental methods used in this research were new to theresearchers and in particular, the researchers learned how to operatean electron microscope (EM), and how to prepare a sample for theEM. The researchers can continue to improve on aseptic techniqueand continuing to be as careful and precise in conductingexperiments.

Bacteriophages are viruses that infect bacteria. They have twodifferent lifecycles, lytic and lysogenic. Lytic lifecycles occur whenthe bacteriophage inject their own DNA into the bacteria’s cell,where it then replicates and produces even more bacteriophageinside the bacteria. Eventually, the host cell bursts due to theactivity of the virus. Lysogenic lifecycles involve the virus’s DNA toalso be injected but does not burst. Instead, it replicates along withthe bacterial DNA. However, lysogenic lifecycles can turn into lyticlifecycles (Mayer, 2010).

Bacteriophage are important because they have a high medicaltreatment potential. Phage are specific to their host; specific phagecan be used to treat respective bacterial diseases. Bacteriophagecan combat sickness in animals caused by different pathogens,illnesses in aquaculture that could lead to large environmental andeconomical repercussions, and diseases found in plant life thatwould negatively impact agriculture. Unlike antibiotics, phage donot develop resistance, which make them valuable to research andtreatment (Adamia, 2012).

ABSTRACT

INTRODUCTION

METHODS AND MATERIALS

RESULTS DISCUSSION

References, Funding, and Acknowledgments

Figure 1. Location ofsoil sample collection36°51’21.0”N, 76°03’10.8”W Figure 2. Morphology

of plaque forming units

Figure 3. Gel electrophoresis of phage. Bricole phage is highlighted with ladder on far left.

Figure 4. EM of phage,Tails about 300nm in length

The next steps for the phage is continuing DNA analysis. Theresearchers will perform DNA digestion via the use of restrictionenzymes and then run a gel electrophoresis to analyze the DNA ofthe phage sample. These procedures will assess the DNA quality,quantity and size. In the next semester, genomic sequencing of thephage will be conducted and one sequence from the class be sent offto be analyzed. As a class, each phage will also be uploaded to thepage database.

FUTURE DIRECTIONS