Introduction
Bacteriophages, informally known as phages, are a specific type of virus that only infect bacteria. Since their discovery in the early 1900s , researchers have began studying the various behaviors and interactions of phages within the environment (Poxleitner, Pope, Jacobs-Sera, Sivanathan, & Hatfull, 2016). Their name, bacteriophages, is derived from the idea that they are “bacteria eaters.” Due to their bacteria-destroying nature, they have been used in phage therapy in which they are used to kill bacteria that are highly resistant to antibiotics (Sadava, Hillis, Heller, & Berenbaum, 2014). Researchers analyze the genetic material of phages in order to gain a deeper understanding of them. Hence, the goal of this lab is to analyze …show more content…
It decreases the diversity of phage presence (Van Twest & Kropinski, 2008). A 50 mL conical tube was filled with 15 mL worth of soil from the environmental sample. Enrichment broth was then added to the soil sample until the mixture reached 35 mL. The mixture was then vortexed and shaken at 250 rpm for 1 hour. Next, the tube was centrifuged at 2,000 x g force for 10 minutes in order to separate the particulate matter from the liquid medium containing phage. The aggregate of particulate matter left behind is known as the pellet. The remaining liquid containing the phage is known as supernatant.The supernatant was filtered through a .22 micron filter in order to extract phages. Since bacteria and other particles tend to be bigger than .22 microns, they would not fit through the filter, thus allowing only phages through. Mycobacterium smegmatis was then added to the supernatant in the amount of .5 mL. It was allowed to incubate for 5 days. After incubating, 1.4 mL of the enriched filtrate was pipetted into a microcentrifuge tube. This was done twice using the p1000. Both tubes were then centrifuged for 1 minute to separate the pellet and the supernatant. The supernatant, 1 mL from each tube (2 mL in total), was then filtered through a .22 micron filter in order to retrieve phages. A petri dish was divided in four sections and labeled as positive control, …show more content…
This mixture was vortexed and labeled DC. Next, 90 microliters of phage buffer was pipetted into 5 tubes, labeled in order from 10-1 to 10-5. Using the p20, 10 microliters was removed from the DC tube, placed into the 10-1 tube, and then vortexed. This process was repeated aseptically, transferring 10 microliters of solution from one tube to its successive tube in order to dilute the phage. The 5 tubes were then used to infect 5 different tubes of Mycobacterium smegmatis. These infections were plated. This dilution series was repeated 3
Bacteriophages, informally known as phages, are a specific type of virus that only infect bacteria. Since their discovery in the early 1900s , researchers have began studying the various behaviors and interactions of phages within the environment (Poxleitner, Pope, Jacobs-Sera, Sivanathan, & Hatfull, 2016). Their name, bacteriophages, is derived from the idea that they are “bacteria eaters.” Due to their bacteria-destroying nature, they have been used in phage therapy in which they are used to kill bacteria that are highly resistant to antibiotics (Sadava, Hillis, Heller, & Berenbaum, 2014). Researchers analyze the genetic material of phages in order to gain a deeper understanding of them. Hence, the goal of this lab is to analyze …show more content…
It decreases the diversity of phage presence (Van Twest & Kropinski, 2008). A 50 mL conical tube was filled with 15 mL worth of soil from the environmental sample. Enrichment broth was then added to the soil sample until the mixture reached 35 mL. The mixture was then vortexed and shaken at 250 rpm for 1 hour. Next, the tube was centrifuged at 2,000 x g force for 10 minutes in order to separate the particulate matter from the liquid medium containing phage. The aggregate of particulate matter left behind is known as the pellet. The remaining liquid containing the phage is known as supernatant.The supernatant was filtered through a .22 micron filter in order to extract phages. Since bacteria and other particles tend to be bigger than .22 microns, they would not fit through the filter, thus allowing only phages through. Mycobacterium smegmatis was then added to the supernatant in the amount of .5 mL. It was allowed to incubate for 5 days. After incubating, 1.4 mL of the enriched filtrate was pipetted into a microcentrifuge tube. This was done twice using the p1000. Both tubes were then centrifuged for 1 minute to separate the pellet and the supernatant. The supernatant, 1 mL from each tube (2 mL in total), was then filtered through a .22 micron filter in order to retrieve phages. A petri dish was divided in four sections and labeled as positive control, …show more content…
This mixture was vortexed and labeled DC. Next, 90 microliters of phage buffer was pipetted into 5 tubes, labeled in order from 10-1 to 10-5. Using the p20, 10 microliters was removed from the DC tube, placed into the 10-1 tube, and then vortexed. This process was repeated aseptically, transferring 10 microliters of solution from one tube to its successive tube in order to dilute the phage. The 5 tubes were then used to infect 5 different tubes of Mycobacterium smegmatis. These infections were plated. This dilution series was repeated 3