Isolation and Observation of Mycobacteriophage Bingo

Isolation and Observation of Mycobacteriophage Bingo

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Introduction

Mycobacteriophages are viral microorganisms that infect mycobacteria (Pope, Jacobs-Sera, Russell, Peebles, Al-Atrache, et al, 2011). Mycobacterium tuberculosis and Mycobacterium smegmatis are examples of mycobacterial hosts infected by mycobacteriophages (Graham et al, 2006). They are complex viruses with superinfection immune systems. Therefore, a study of their genetic dynamics and functions may provide insights essential for scientific applications, particularly in clinical medicine. Discovery of new mycobacteriophages is a constant process with many of these microorganisms potentially undiscovered (Kumar et al, 2010). In order to enrich knowledge of these viruses and encourage an increased exploration, students of the Virginia Commonwealth University took the first step of the expansion of knowledge of the mycobacteriophage diversity and the roles of mobile elements in viral evolution. They realized the potential of the knowledge of the evolution, genomics, and lifestyle of these complex microorganisms in clinical applications. It holds the key to unlocking the phenomenon of tuberculosis and other related public health problems.

This lab report describes the isolation of mycobacteriophage Bingo and discusses the results, and the implications for scientific studies that analyzes the genetics and traces the origins and future of mycobacteriophages. The isolation of mycobacteriophage Bingo uses Mycobacterium smegmatis as the host.

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Methods

A soil sample, collected in an area in Richmond, Virginia, was introduced into an enrichment culture composed of 40ml water, 40ml sterile 10×7H9/ glycerol broth, 0.5ml of 100mM Calcium Chloride, 5ml AD, and 5ml Stationary phage. This was done to isolate the mycobacteriophage. The sample was then incubated for 48 hrs at 37°C. Then, the sample was filter-sterilized at 0.22μm. Through serial dilution, introduction into liquid cultures of M. smegmatis and direct plating using top agar (7H9 broth base, 1 mM Calcium Chloride, and 0.4% agar), the sample was then tested for the mycobacteriophage. The plates were incubated for 24 hours at 37°C, after which observation for isolated plaques was conducted. The phage was confirmed pure following a series of purification. In order to understand the genetic dynamics of the phage better, I prepared a HTL (High Titer Lysate) of the phage and used it to isolate and purify the phage’s genomic DNA using nuclease mix (0.25 mg/ml RnaseA, 50% glycerol, and 150mM NaCl). The procedure followed the Promega Wizard DNA purification protocol. The next procedures were gel electrophoresis, microscopy of phage particles, lysogeny test, and immunity test.

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Results

Initially, the mycobacteriophages were not visible; they did not survive the phage infection. However, subsequent series of purification expressed clear plaques. The procedure isolated mycobacteriophage Bingo from a soil sample by use of enrichment culture. The procedure included multiple rounds of purification conducted to confirm consistency of its morphology. The plaques of Mycobacteriophage Bingo were small, but relatively clear, and with an approximate diameter range of 0.5-1.5mm.

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Discussion

The isolation and observation of Mycobacteriophage Bingo created the knowledge that there exist several unknown phages that have diverse morphology. The mycobacteriophage Bingo is unique in that it has the capacity to produce phages with differing sizes. Additional studies are necessary so as to gain broad, concrete knowledge on its evolution and potential applications in research and clinical practices. The analysis should compare the phage with other phages so as to distinguish its genomics and functional characteristics. In addition, additional research is necessary as regards the different strains of bacterial host infected by mycobacteriophages. These include Mycobacterium tuberculosis and Mycobacterium smegmatis, which are bacterial strains of public health significance.

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Tuberculosis prevails as a prime cause of mortality among the populations, even with the availability of effective drugs (Kumar et al, 2010).  Mycobacterium tuberculosis is the causative agent which is an obligate human pathogen. They survive hostile environments because of their complex cellular processes, thus posing a big challenge to medical treatment of tuberculosis. However, mycobacterial phages infect Mycobacterium tuberculosis and others effectively. This provides hope for future medical developments in medical treatment.  Science should uncover concrete knowledge as regards the evolution, classification, and the potential for application in medicine.

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References

Graham, F., et al. (2006). “Exploring the Mycobacteriophage Meta- proteome: Phage Genomics as an educational Platform”. PLOS Genetics, 2: 6.

Kumar, D, et al. (2010). “Genome-wide analysis of the host cellular network that supports survival of Mycobacterium tuberculosis” Cell, 140: 731-743.

Pope, W., Jacobs-Sera, D., Peebles, C., Russell, D., & Al-Atrache, Z. (2011) “Expanding the Diversity of Mycobacteriophages”. PLOS ONE, 6: 1.