Lily Hewitt 02/21/2022 Lab Report Draft Assignment I. Introduction There is a plentiful amount of phage on earth, and an insane amount of diversity within the phage (Díaz-Muñoz, Koskella 2014). Phage was discovered roughly in the early 20th century and have researched to be promising for future biotechnology. However, less than approximately three thousand phages have been discovered and distinguished through scientific research. Therefore, identifying a phage out of the estimate of 1031 possible phage biological particles will be a vital discovery to the scientific community (Datta, S. et al. 2017). There is a lot of potential for the use of phages in science such as in the agriculture industry, petroleum industry, for vaccines, therapeutics, and especially to test for pathogenic bacterial strains (Haq, Irshad Ul et al. 2012). An important mark in history for phage, was when it was first used as therapeutics for humans in 1919, that eventually was applauded for the use of phage against E. Coli in mice (Virol J. 2012). Phage needs a host to infect in order to replicate and can do that either through the lytic cycle or lysogenic cycle. Therefore, phage is present in the environment infecting host all around such as bacteria and can take charge of ecosystem responsibilities and host functions. Microbacterium foliorum is a perfect host that has been secluded from grass in Germany. The strain used as a host in this specific experiment is called M. foliorum SEA B-24224, that has been provided by the Agricultural Research Service of USDA (Microbacterium foliorum. 2017). As of today, there is still not much knowledge of phages inour worlds, so discovering more phage and how they infect host will strengthen the knowledge of certain hosts and how phage can help in the scientific realm. So, this experiment isn’t for nothing, it will bring the world closer to future biotechnology and treatment discoveries. In finding phage that uses the host Microbacterium foliorum, there can be advancements made on the relationship between phage and microbacteria hosts. To begin this procedure a soil sample was needed to be used for the infection of M. foliorum. The soil sample was mixed with M. foliorum in a bioreactor to shake in a tube shaker for 48 hours to isolate phage from the soil sample. Then the enrichment medium was transferred into a microfuge tube to prepare for enriched lysate. The enriched lysate was then tested using plaque assay and the spot test protocol. For the plaque assay protocol, the enriched lysate was mixed with M. foliorum, and a separate mixture was made (Top Agar and CaCl2) and mixed with M. foliorum and the phage lysate. This was poured on an L-Afar plate to solidify and store in the 30 degrees Celsius incubator for 24 hours. For the spot test protocol, the same mixture above was made to pour on to the L-agar plate that had a negative control and experimental sample. Once this was solidified, it was important to spot 10-15 ul of the phage lysate into the negative control and experimental sample circles. The plate was then also placed in the 30 degrees Celsius incubator for at least 24 hours. If the plaque assay test and spot test protocols were successful, we did the plaque streak plates protocol. The plaque on the plate could have contained a mixed population of phages, therefore the plaque streak test created successfully isolated plaques that contained the one type of phage. It’s important to notice that the streak test was performed for several weeks over the course of lab to ensure the phage created a pure phage population. Each week a new streak is made fromthe plaque picked form the previous steak. So, the plaque has a well isolated single phage (Phage Lab Manual. 2018). II. Materials and methods i. Isolate a novel phage from the environment The most important part of this lab was the soil collection. A soil sample was obtained from around WSU campus into a 10 ml tube. The samples were then used in the lab to be infected by Microbacterium foliorum to begin the protocols of the lab for the isolation of the new bacteriophage. ii. Enriched Isolations The soil sample was then transferred into the enrichment medium of the bioreactor and was mixed with M. foliorum. Then the bioreactor was placed in a tube shaker to shake for 48 hours or more for preparation of filtering. The filtering process was to create the enriched lysate. 1 ml of the enrichment medium was transferred via pipet into a microfuge tube and was spun for 1 minute to transfer yet again via syringe into a microfuge tube, which became the enriched lysate. This was then used to start the spot test protocols and the plaque assay for finding phage. iii. The Spot test and the plaque Assay With the spot test, two individual circles were drawn on the bottom of the L-agar plate to define the Negative control and the Experimental sample. Then, 20 𝜇𝑙 of 𝐶𝑎𝐶𝑙2 was mixed with M. foliorum to pour onto the L-agar plate. Then it was left to solidify for the 10-15 𝜇𝑙samples of the enriched lysate was able to be transferred into the circles via pipet. The L-Agar plate was then left in the 30-degree Celsius incubator for approximately 24 hours. The plaque assay was a better protocol to test for phage than the spot test. With the plaque assay the phage lysate was mixed well with 0.5 mL of M. foliorum and sat for 10 minutes to incubate. Top agar and 20 𝜇𝑙 of 𝐶𝑎𝐶𝑙2 was mixed and poured into the phage lysate and M. foliorum mixture and poured onto an L-Agar Plate. Just like the spot test, this was placed in the 30-degree incubator for at least 24 hours. iv. Purifying the Phage: The Plaque Streak Protocol During the experiment after 5 trials phage was never found through all the enrichments a plaque assays. Therefore, a phage was adopted from another lab group to continue with the lab and begin the next few steps. With the plaque streak the plate was borrowed from another grouped and picked by a sterile wooden applicator and then streaked out onto a new agar plate. The streaks were done carefully 3 times and did not overlap on each other. The third streak was marked with an X to indicate where to pour the mixture of 20 𝜇𝑙 of 𝐶𝑎𝐶𝑙2, top agar, and M. foliorum. Then the plate was left to solidify and was transferred into the 30-degree Celsius incubator for approximately 24 hours. v. Pure Phage Enrichment (not performed yet)III. Results i. For the
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