James Emmett Stanek
Thesis submitted to the Faculty of the Virginia Tech in partial fulfillment of the requirements for the degree of
Masters of Science
J. O. Falkinham III, Chair
S. M. Boyle
G. H. Lacy
January 24 1997
AbstractA rapid coliphage detection assay (RCDA), based on the phage-induced release of b-galactosidase from cells of Escherichia coli (Ijzerman, M., J.O. Falkinham III and C. Hagedorn. (1993) [A liquid, colorimetric presence-absence coliphage detection method. J. Virol. Meth. 45:229-234] was modified to reduce the number of steps required to perform the assay, remove the need for specialized media and buffers, reduce the volumes required, and simplify growth and reaction conditions. Tolerances of the assay were defined at each step of the assay. The number of steps has been reduced from 12 to 7. The b-galactosidase reaction buffer was eliminated. Culture volumes were reduced from 25 ml to 5 ml and reaction volumes were reduced from 10 ml to 0.5 ml. Optimal growth conditions were 37 o C with orbital shaking at 200 rpm, a one hour subculture time and an incubation of subculture with water sample for two hours. Color development occurred at 37 o C in 30 minutes. The changes and modifications of the assay increased the ease of its performance without sacrificing the ability of the assay to detect as few as two phage particles per sample. By understanding the tolerances of the assay, technical support representatives of companies producing kits modeled after the assay will be prepared to answer questions from customers concerning possible kit failures or user error.
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