Name: Microplate Preparation for Actinobacteriophage Infection
Uploaded: 2023-06-30T12:45:00
Duration: 1 min 7 s
Description: Watch this Scientific Journal Video about Microplate Preparation for Actinobacteriophage Infection at JoVE.com

microplate preparation for actinobacteriophage infection

Characterizing Actinobacteriophage Infection Using an Adapted Optical Density Assay

Bacteriophages or phages are viruses that infect bacteria. They are the most numerous biological entities on the planet1, and it is generally accepted that bacteriophages influence bacterial evolution and ecosystem processes2,3,4. Several methods exist to describe, measure, and analyze bacteriophage host range8 and infection dynamics5,6, including agar-based methods such as the double-layer agar method7 and optical density-based microplate methods8,9,10,11,12. Each method has its own advantages and disadvantages. Due to their efficiency, plating tests using the double-layer agar method are the &#34;gold standard&#34; for host range assays, but this method is time- and labor-intensive9. Rapid microplate methods, which return results in 24 h or less, give excellent results for fast-growing bacterial hosts such as Escherichia coli9,10,11,12 but are too brief to showcase bacteriophage infection progression in slower-growing bacterial hosts such as actinomycetes7,13,14,15.
An optical density-based microplate assay designed for fast-growing bacteria cannot be run for the multiple days required to characterize infection dynamics in a slow-growing host bacterium without evaporation occurring and giving artificially high bacterial densities. Thus, obtaining comparable high-throughput data on bacteriophage infection dynamics for slow-growing bacterial species requires specialized techniques adapted for these bacteria.
The microplate method presented here reduces evaporation, thus allowing slow-growing bacteria to be co-cultured with a phage for an extended 96 h period and enabling investigations into phage infection dynamics and host range. This method also showcases the Stacy-Ceballos index16, an optical density-based metric that allows for virulence comparisons among disparate host-virus systems.

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

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

Ultimately, we're 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 coevolution in slow-growing Actinomycete bacteria.Rapid microplate methods give excellent results for fast-growing bacterial hosts, but are too brief to showcase bacteria-phage infection progression in slower growing bacterial hosts, such as actinomycetes. 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.

Watch this Scientific Journal Video about Microplate Preparation for Actinobacteriophage Infection at JoVE.com

Microplate Preparation for Actinobacteriophage Infection  

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%Agaros solution into the spaces between the wells of the plate.Then pipette 200 microliters of Agaros into the wells located in row A, row H, column one, column two, column 11 and column 12.

microplate-preparation-for-actinobacteriophage-infection

Research

JoVE Journal

Biology

Bacteriophages are a key part of natural environments, and they have a powerful ability to shape bacterial populations. To understand how individual phages interact with slow-growing bacterial hosts such as actinomycetes, an easy and reliable method for quantifying long-term bacterial growth in the presence of phages is needed. Spectrophotometric microplate readers allow for high-throughput repeated measurements, but incubating a small volume for an extended time can present technical challenges. This procedure adapts a standard 96-well microplate to allow for the co-culturing of phages and bacteria without sub-sampling for 96 h, with the bacterial growth recorded every 8 h using spectrophotometric absorbance values. These optical density values are analyzed using R to yield infection metrics, including the percent growth inhibition, relative virulence, and the Stacy-Ceballos index. The methods outlined here provide an effective way to conduct and analyze extended-duration microplate growth curve experiments and includes modifications to reduce evaporation and lid condensation. These protocols facilitate microplate-based assays of interactions between slow-growing bacterial hosts and their bacteriophages.

An optical density-based microplate method is described to quantify long-term bacterial growth in the presence of bacteriophages, suitable for actinomycetes and other slow-growing bacteria. This method includes modifications to reduce evaporation and lid condensation and R code to calculate virus infection metrics, including the area under the curve, growth maximum, and relative virulence.

Bacteriophages are a key part of natural environments, and they have a powerful ability to shape bacterial populations. To understand how individual ...

Actinobacteriophage Infection and Absorbance Measurement

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.