An Adapted Optical Density-Based Microplate Assay for Characterizing Actinobacteriophage Infection

Ultimately, we are interested in bacteriophage host interactions. This protocol enables the measurement of phage infection in slow growing bacteria, generating descriptive infection metrics. This methodology is instrumental in studying bacteriophage host co-evolution in slow-growing actinomyces bacteria.

Rapid microplate methods give excellent results for fast growing bacterial hosts, but are too brief to showcase bacteriophage infection progression in slower growing bacterial hosts such as actinomyces. Our method is adapted for slow growing bacteria, allowing bacteria and phage to be co-cultured for the multiple days required to characterize the bacteriophage infection dynamics. Our protocol allows bacteriophage host interactions to be assessed for slow growing bacteria in microplates, rather than requiring sub-sampling from a larger culture flask.

This protocol uses readily available materials to adapt for extended culture times and allows multiple replicates to be plated in a single microplate. To begin, add six milliliters of lid coating solution to the inside surface of a sterile 96 well microplate lid, holding it by the edges. Rotate the lid to ensure complete coverage of the solution.

Allow the solution to sit for 20 seconds before pouring it off. Invert the lid onto an autoclaved paper towel at an angle and let it dry for 35 to 45 minutes. Cool the melted agarose to a temperature between 50 and 60 degrees Celsius.

Then pipette 100 microliters of the 0.1%agarose solution into the spaces between the wells of the plate. Then pipette 200 microliters of agarose into the wells located in row A, row H, column one, column two, column 11, and column 12. Take a previously prepared microplate with anti-fog solution and agarose.

Pipette 75 microliters of the bacteria into columns three to 10. Next, add 75 microliters of sterile phage buffer to wells in columns three and four as a no phage positive control. Mix the solution by pipetting up and down after each addition, then add 75 microliters of the phage at varied concentrations to the columns, and mix each solution by pipetting up and down again after each addition.

Stick both short sides of the plate using 0.5 inch labeling tape, which will partially seal the plate while allowing for gas exchange. Incubate the plate on a microplate shaker and measure optical density using a microplate reader. Then with the optical density measurements, generate control and infected growth curves.

The growth curve depicts a productive phage infection shown by the reduced bacterial absorbance over time in wells with the phage added, this reduction in bacterial density will not be seen if the bacterium is outside the phage's host range.