Rapid and reproducible infectivity end-point titration of virulent phage in a microplate system.
The standard method for measuring the number of infectious phages in solution has traditionally been the plaque forming assay. An alternative method is described where the number of lytic, infectious phages is determined in an endpoint titration assay adapted for a microplate system. In this model system, susceptible Escherichia coli B6 at a density of 4 x 10(7) cells/ml, were mixed with an equal volume (100 microl) of PhiX174 diluted serially in a microtest plate. After 3h of incubation on a microplate shaker the endpoint was determined spectrophotometrically and calculated according to the method of Reed and Muench. A well was considered positive for infection if the OD630-value was < or = 10% compared to the OD630-value of the negative control of uninfected cells. ID50-titers were 2.5x higher than the PFU-titers (CV 15%) and the intra assay reproducibility revealed a CV of 9%. The method has several advantages as compared with the conventional PFU-titration. It is less time and material consuming with the possibility to assess several samples at the same time.
Fridholm
Helena
H
Department of Cell and Organism Biology, Lund University, Biology Bldg, Sölvegatan 35, SE-223 62 Lund, Sweden.
Everitt
Einar
E
D010584:Bacteriophage phi X 174 / Q000302:isolation & purification / Q000472:pathogenicity*; D004926:Escherichia coli / Q000254:growth & development* / Q000821:virology*; D008242:Lysogeny; D015203:Reproducibility of Results; D013997:Time Factors; D010948:Viral Plaque Assay; D014773:Virology / Q000295:instrumentation* / Q000379:methods*