Difference between revisions of "Phage therapy"

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== Questions and Specific Aims ==
== Questions and Specific Aims ==
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:'''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'''.