Mycobacteriophage Meru: Isolation and Characterization of a Novel Mycobacteriophage

By Merit P. George
2013, Vol. 5 No. 10 | pg. 2/3 |

Results

From the initial plating of M. smegmatis enrichment cultures, plaques were identified. The plate contained plaques of both small and large sizes, ranging from a diameter of about 0.5 mm to about 3 mm. All plaques appeared significantly light and turbid, and contained smooth edges. Four consecutive rounds of purification, which included separate purification dilutions for a phage with a diameter of 0.5 mm and a phage with a diameter of 3mm, yielded a consistent picture of plaque morphology (Figure 2). Each plate that resulted from these processes showed mixed plaque sizes within a similar range (0.5 to 3.0 mm).

Figure 2

To confirm this consistency, two stick streak tests were utilized, which also yielded the same diversity in plaque sizes as well as similarities in the other characteristics of plaque morphology mentioned previously. As a result, we concluded that the mycobacteriophage isolated was of a pure population species because of persistent patterns in size, shape, turbidity, and general structure.

Empirical testing for web patterns yielded plaque morphologies that were consistent with previous purification results (mixed plaque sizes ranging from less than 1 mm to about 3 mm; high turbidity) (Figure 3).

Figure 3

The titer calculation that was made upon serial dilution and plating of M. smegmatis infected by filter-sterilized high titer phage lysate yielded a result of 4.7 x 108 pfu/mL. Subsequent to phage DNA isolation and purification, the concentration of phage DNA was found to be 86.6 ng/µL.

Because of the relatively small concentration of DNA retrieved from DNA isolation and purification methods, gel electrophoresis was only conducted with two restriction enzymes (ClaI and HaeIII). Results of the procedure (Figure 4) indicated that ClaI was not able to cut the DNA, while HaeIII was able to digest the genomic material.

Figure 4

Cross-immunity testing revealed that Meru was not immune to any of the other phages in the Fall 2011 Virginia Commonwealth University Phage Discovery course. However, there appears to be doughnut shaped accumulation in some of the spots, where lysis seems to be disrupted (Figure 5). The sequenced phages Batiatus (Cluster F) and Twister (Cluster A1) were able to lyse the Meru lysogen, indicating that Meru may be unrelated to phage from these commonly isolated clusters. Immunity testing revealed that the mycobacteriophage Tibbie, a potentially lytic phage from the course, significantly lysed the mycobacteriophage Meru (Figure 6). Finally, it is interesting to note the conversion of the Meru lysogen lawn to a more lytic condition, as observed by formation of plaques within the bacterial lawn.

Figures 5 & 6

Host range testing yielded similar plating efficiencies for the mycobacteriophage Meru on both strains of M. smegmatis (Figure 7). All plaques that appeared were turbid, especially those plated from the latter portion of the serial dilution. In terms of texture and size, the plaques were mostly smooth-edged and very consistent in diameter. However, it appears that Meru had a greater infection efficiency on ATCC type strain for the last few spottings, as evidenced by a slightly greater degree of lysis in those areas of the plate.

Figure 7

Figure 8

Lastly, electron microscopy provided a clear picture of the physical structure of Meru (Figure 8). Using a scale marker of 50 nm, the tail length of the phage was calculated to be about 204 nanometers, while the head diameter was found to be about 69 nanometers.

Suggested Reading from Inquiries Journal

If you talk to any person in the field of education today about high-profile and controversial issues in their field, the topic of high-stakes standardized testing will inevitably come up.  This issue has been around for a long time, but was really introduced into the minds of the public through the standards-based reform movement of the 1990s.  The notion of standards-based reform focuses on three main principles: state-level content standards... MORE»
Advertisement
The rationale behind the proposal is that in the event of the purchase of sexual services becoming a crime and, therefore, by encompassing a fear of the criminal label being attached to an individual, it will reduce the demand for such services and ultimately relieve pressure on human trafficking in Ireland more broadly. As current research suggests that the predominant population of buyers belong to the upper classes, it can be argued that the possibility... MORE»
Within the first ten minutes of Twilight of the Golds, it is clear that both Judaism and homosexuality play a role in the Gold family. The family is at least culturally Jewish, if not more, and the son David (Brendan Fraser), is portrayed to be gay. Yet neither “gay” nor “Jewish” is actually mentioned until much later in the film. What are the methods that the film uses to construct gayness and Jewishness? And... MORE»
Throughout the American education system, the assessment of writing skill and general academic performance through timed essay examinations has become increasingly pervasive, contributing to the determination of grades and course placements and ultimately affecting college admissions through their use in standardized tests. In March... MORE»
Submit to Inquiries Journal, Get a Decision in 10-Days

Inquiries Journal provides undergraduate and graduate students around the world a platform for the wide dissemination of academic work over a range of core disciplines.

Representing the work of students from hundreds of institutions around the globe, Inquiries Journal's large database of academic articles is completely free. Learn more | Blog | Submit

Follow SP

Latest in Biology

2016, Vol. 8 No. 08
The Ras/Raf pathway is a crucial cell signaling pathway utilized by eukaryotic cells for growth and proliferation, and it is highly conserved amongst all eukaryotic organisms. Mutations in this pathway lead to uncontrolled growth and proliferation... Read Article »
2015, Vol. 12 No. 1
Published by Discussions
Electronic cigarettes (E-cigarettes) are devices that effectively deliver vaporized liquid nicotine to the lungs and are commercially available as a nicotine replacement therapy that is safer than conventional tobacco smoking. However, there is... Read Article »
2015, Vol. 11 No. 2
Published by Discussions
To maintain sodium (Na+) homeostasis in a hypotonic environment, freshwater teleosts must constantly absorb Na+ through their gills. Teleosts in temperate climates have the extra challenge of living in an environment in which ambient temperatures... Read Article »
2015, Vol. 11 No. 2
Published by Discussions
The increasing level of obesity in the general population of industrialized nations is a major public health concern. While obesity increases morbidity and mortality, increasing body habitus also impacts the utilization and analysis of medical imaging... Read Article »
2015, Vol. 11 No. 2
Published by Discussions
With the advent of antiretroviral therapy, suppressing the HIV-1 virus and stopping the progression of the disease are now possible. Even with long-term antiretroviral therapy, HIV reservoirs remain in individuals. These individuals experience an... Read Article »
2015, Vol. 7 No. 05
There is limited data available on the safety of electronic (E)-cigarettes, despite their commercial availability and widespread use. E-cigarettes are electronic devices that deliver nicotine to the lungs but limited data is available about their... Read Article »
2014, Vol. 6 No. 09
Gene therapy has ceased to be a futuristic dream and instead become a treatment for the present. Somatic cells can be treated with gene replacements or gene fragments to express required proteins and whilst gene therapy was originally aimed at inherited... Read Article »

What are you looking for?

FROM OUR BLOG

5 Tips for Publishing Your First Academic Article
The Career Value of the Humanities & Liberal Arts
How to Select a Graduate Research Advisor