Difference between revisions of "Phage therapy"
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→Questions and Specific Aims
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== Questions and Specific Aims == | == Questions and Specific Aims == | ||
:'''Pilot Project Aims''' | :'''Pilot Project Aims''' | ||
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:'''Isolate ARNG-derived bacteriophage from 10 patients'''. | :'''Isolate ARNG-derived bacteriophage from 10 patients'''. | ||
:''Bacteriophage Isolation''. Bacteriophage will be isolated from 10 clinical samples as previously described (REF). Briefly, isolated colonies will be selected and cultured in 5 mL TPY broth (trypticase, phytone, and yeast extract) and cultued for 24 hours at 37oC in 5% CO2. | :''Bacteriophage Isolation''. Bacteriophage will be isolated from 10 clinical samples as previously described (REF). Briefly, isolated colonies will be selected and cultured in 5 mL TPY broth (trypticase, phytone, and yeast extract) and cultued for 24 hours at 37oC in 5% CO2. | ||
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:After culture, 1500 µL of broth will be transferred to Trypticase Soy Agar (TSA) plates in triplicate (500uL each). One plate will be used for harvesting bacteriophage, a second plate will be used for propagating bacteriophage, and a third plate used for storing cultures. The remaining culture (3500uL) will be centrifuged for 10min at 2500 RPM. The supernatant will be removed and filtered through a 0.22 µm syringe filter. The syringe filters removes all cellular debris resulting in a pure bacteriophage culture. Bacteriopahge cultures will then be added to a bacterial lawns using 10-fold titrations to determine titers. Positive titers will result in plaque formation. | :After culture, 1500 µL of broth will be transferred to Trypticase Soy Agar (TSA) plates in triplicate (500uL each). One plate will be used for harvesting bacteriophage, a second plate will be used for propagating bacteriophage, and a third plate used for storing cultures. The remaining culture (3500uL) will be centrifuged for 10min at 2500 RPM. The supernatant will be removed and filtered through a 0.22 µm syringe filter. The syringe filters removes all cellular debris resulting in a pure bacteriophage culture. Bacteriopahge cultures will then be added to a bacterial lawns using 10-fold titrations to determine titers. Positive titers will result in plaque formation. | ||
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:'''Characterize ARNG-derived bacteriophage from 10 patients'''. | :'''Characterize ARNG-derived bacteriophage from 10 patients'''. | ||