A bioluminescence-based assay for enumeration of lytic bacteriophage

Abstract

A bioluminescence-based assay for enumeration of lytic bacteriophage was developed. The assay consists of a bioluminescent Escherichia coli as the host bacterium, the lytic bacteriophage T4 and an automated luminometer measuring luminescence over time. The assay is based on the decrease in luminescence as the bioluminescent host cells are lysed by T4. The T4 concentration, bioluminescent E. coli concentration, phage suspension medium, and temperature (25 °C and 37 °C) were varied. There was a strong negative correlation between bioluminescence intensities and T4 phage concentrations at both room temperature ( R 2 = 0.993) and 37 °C ( R 2 = 0.970). Phage was detected more rapidly at 37 °C than at 25 °C. The detection limit was also lower when the assay was performed at 37 °C with a minimum detection level of 2.4 log CFU/ml compared to 3.4 log CFU/ml for 25 °C. The assay was used to determine thermal inactivation using T4 phages heated at 70 °C for 0 to 30 min, and phage concentrations were determined using the bioluminescence assay and a standard plaque assay. There was no significant difference between the two enumeration methods ( P > 0.01). This study suggests the bioluminescence-based assay can be used as an alternative for quantitatively monitoring phage infectivity, instead of conventional standard plaque assays.