Characterization of Mycobacterium smegmatis bacteriophage Ravenclaw

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To start off made sure the surface was cleaned with 1% ethanol, and the Bunsen burner must be turned on. Everything performed to isolate a phage must be done in an aseptic zone, in this case it was near the Bunsen burner. 5 ml of the soil sample was gathered near WSU campus and poured into a bioreactor which already included the enriched medium and .5 ml of the M. foliorum then sat on the tube shaker for seven days.

Once the soil sample was on the shaker for a minimum of 24 hours, 1 ml of the soil mixture was transferred into a microfuge tube. The soil mixture spun in a microcentrifuge at speed for thirty seconds. Next, with a syringe with its plunger removed, a filter was attached to the top and transferred roughly half of the centrifuge mixture into it. The plunger was reinserted into the syringe, a filter the medium into a microfuge tube. The filtered medium in the microfuge tube was used as enriched lysate.

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Afterward the enriched lysate was prepared, a plaque assay was performed by adding 50 ?µl of the enriched lysate into a tube of .5 of M. foliorum and letting it incubate for ten minutes. Meanwhile the enriched lysate mix was incubated for ten minute, 20 ?µl of CaCl2 was mixed with liquid top agar. Once ten minutes had passed, add the top agar mix into the phage lysate mix and swirl. The mixture of M. foliorum, phage lysate, and a separate tub of liquid top agar, it was poured into a new l-agar plate it was then incubated at 30?°C for seven days.

With a growth of a phage on the plate, a spot test was performed in aseptic conditions. A grid was drawn on the plastic side of a new l-agar plate and labeled as a negative control. A tube of liquid top agar is added 20 ?µl of CaCl?¬?¬2 and that mixture was then mixed into a tube of M. foliorum. The mixture of CaCl2, liquid top agar, and M. foliorum was drained onto a new l-agar plate. As it becomes solid on the plate, 10 “ 25 ?µl of the soil sample was transferred onto the grids that were drawn on the bottom of the plate. The l-agar plates with the mixture topped off are places in a 30?°C incubator for a week.

As the lawn was visible, a plaque streak was performed by first choosing a plaque from the plate and touching the plaque with a sterile wooden pick that was then aseptically streaked onto a new agar plate. This process was done by using a sterile wooden stick,

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