“Isolation and Characterization of Mycobacteriophages Isolated From Tropical Soils ofPuerto Rico”Michael Rubin, Giovanni Cruz, Carla Figueroa, Crystal ColónAbstract:Introduction:Bacteriophages, more commonlyknown as phages, are bacteria that attackviruses by extracting the lysis of growingbacterial cultures. Their sizes range from100 to 200 nm and they cannot replicate norpropagate outside of their host bacteria, butthey can be found inside or outside ofbacterial cells. Phages aren’t susceptible toantibiotics and as a consequence they arepervasive class of virus that has become themost abundant life-form in earth. They areused in genetics for cloning or mutations, inepidemiology they are studied because somephages can make their host bacteria moredeadly to humans, and also they can be usedas therapeutics to kill specific bacteria.Phages are susceptible life-forms to bacteria,so to obtain a pure phage requires anexcellent aseptic technique that is differentfor each step of the process. The mostcommon way to study phages is to collectsoil samples. These samples undergo aprocess that includes enrichments,harvesting, plating, purification, empiricaltests, web pattern, and a high Titer phagelysate (HTPL). At the end of this process theobtained solution is ready for DNApurification and eventually it will becharacterized. Most of the phages fromwhich their DNA’s are characterized arenamed and inserted in a genome banc wherethey are shared with the world.Materials and MethodsThe procedure started in February 18and ended in April 3. It was divided inseveral steps: soil sample collection andenvironmental data, preparing theenrichment, harvesting and preparing theenriched sample, plaque streak protocol,plaque purification (3x), empirical test andweb pattern, ten plate preparation and hightiter phage lysate, and characterization of thephage. A negative plaque was prepared withphage buffer was prepared.Soil Sample Collection and EnvironmentaldataAfter taking two photos the soil wascollected using a wrapped spoon. Theenvironmental data was recorded and alsothe date and time of sampling, location, airtemperature, depth, moisture content, andthe features of the site.
EnrichmentUsing a clean spatula, 0.5 grams ofthe sample were added to a 50 ml tube. Tothis flask it was added: 8 mL of sterile H2Ousing 2 x 25- mL pipettes, 1 mL of sterile 10x 7H9/glycerol broth, 1 mL of ADsupplement, 0.1 mL of 100 mM CaCl2, and1mL of late long/early stationary phase M.smegmatis culture to the flask. At the end itwas incubated at 37° C, shaking at 220 rpm,for 24 hours.Harvesting and Preparing the EnrichedSampleThe tube with the sample was balancedand it was spin at 3,000 rpm for 10 minutesto pellet particulate matter, including mostof the bacterial cells. The liquid from thecentrifuge sample was poured into a fresh 50mL conical tube. The enrichment samplewas filter sterilized.Plaque Streak ProtocolAfter removing a sterile woodenstick from its package and opening the tubewith the centrifuged filtered enrichment itwas gently streak across the top third of theagar plate. This step was repeated threetimes from the streaked area to the un-streaked portion. Then 4.5 mL of Top Agarwas added to 0.5 mL aliquot of M.smegmatis. At the end it was dispensed ontothe most dilute area of the streaked plate.The mixture was spread across the platefrom the most dilute point to the moreconcentrated areas by titling the plate. Afterthe Top Agar was hard, the plates were putin an incubator at 37˚ for 24 hours. If at theother day the plate shows signs of thepresence of a phage please continue with theinstructions, if not, start again collectingnew a new sample from another location.Plaque PurificationFirst the Petri dish with the plaque wasopened and touching the center of the plaquewith a sterile blue pipette tip it wastransferred to a sterile microtube with 100µL of Phage Buffer. After returning thecover to the Petri dish and using the aseptictechnique, a sterile wooden stick wasremoved from its package to perform theStreak Technique mention in the previousstep. At the end the Top Agar and thebacteria (M. smegmatis) were added to theplate and it was incubated at 37˚C. Thisprocess should be made three times.Empirical Test and Web PatternBy drawing a circle around the area,there were designated a label to the plaque.For all of the founded plaques, there wasmade plaque purification. Then, four microcentrifuge tubes were labeled from 1 to 4.To each one of them it was added; 90 µL ofPhage Buffer and 10 µL of the 10 ˚ phagesample (to the “1” tube), 10 µL of the “1”tube to the “2” tube, and successively untilthe “4” tube. At the end the Top Agar andthe bacteria (M. smegmatis) were added tothe plate and it was incubated at 37˚C. If ithas plaques proceed.Ten Plate Preparation and High TiterPhage Lysate (HTPL)With the appropriate volume anddilution of lysate, a 50 mL flask wasinfected with M. smegmatis. There were
Figure 2. Shows final results and soil properties.added 45 mL of Top Agar to the flask thatwas previously contaminated with thebacteria. After pulling 25 mL of the mixture5 mL per agar plate should be dispense. Toeach plate there were added 8 mL of PhageBuffer. After letting the plates sit for 2 to 4hours the plates were stored at 4˚C. Then thePhage Buffer was removed and transferredto a 50 mL conical tube. The tubes werecentrifuged at 2500xg for 20 minutes topellet cell debris. The supernatant wasmoved into a 50 mL filter sterilization unit,using suction the lysate was filter-sterilized.At the end the tube containing the solutionwas stored at 4˚ C.At this point the solution is ready topurify its DNA and eventually to becharacterized eventually.ResultsDuring the research eleven soilsamples were studied. Each one of them wastaken from a different location and thereforedifferent properties were observed. Thesecan be perceived in the Figure 2. Threewere from Cidra, one from Cayey, one fromBarranquitas, five from Las Piedras, and onefrom Caguas. The air temperature fluctuatedfrom 80˚ F to 91.4˚F. Most of the soilsamples were obtained during the afternoon.The earlier sample collection was at 11:16a.m. and the latest sample collection was at6:37 p.m. Most of the soil samples wereobtained from farms and under citrus trees.Any of the soilsamples showedthe presences ofphages. Most ofthe Petri Disheslooked like Figure 1, without any plaque.Some of the reasons that can explain theseresults can be: the places were the soilsamples were collected were not appropriateor that the time that the samples spend untilit was enriched was to prolonged andaffected the presence of phages.DiscusionConclusionAfter seven weeks studyingdiverse samples from the soil of differenttowns in Puerto Rico, there was any type of
plaque that indicated the presence ofbacteriophages. This shows that in thespecific places were the soil samples werepicked there is low degradation of organicmaterial. The results obtained leads us tokeep searching for more soil samples in thisspecific towns of Puerto Rico and determinewhat is affecting the growth ofbacteriophages in them. Hopefully later on,this research will be retaken.