プラークアッセイ:プラーク形成単位(PFU)としてウイルス定数を決定する方法
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Q1: What are bacteriophages and how do they infect bacterial cells?
Bacteriophages, or phages, are viruses that specifically infect bacteria. They attach to the bacterial cell wall and inject their genetic material into the host. The phage then hijacks the cell's biosynthetic machinery to replicate its DNA and produce numerous progeny phage particles, which are released by lysing and killing the host cell.
Q2: Why is the plaque assay considered a reliable method for determining viral titer?
The plaque assay remains one of the most reliable methods for determining viral titer because it directly visualizes the lytic activity of phages. Each plaque represents a single infective phage particle that has infected and lysed multiple neighboring bacterial cells, creating a clear, countable zone. This amplification of lytic activity enables accurate enumeration of phage particles in a sample.
Q3: What is the purpose of using bacteria in log-phase growth for the plaque assay?
Bacteria in log-phase growth are actively dividing and have the highest viability, ensuring that a large percentage of cells are capable of forming a dense, confluent lawn on the agar plate. This dense bacterial lawn is essential for the plaque assay because it provides abundant host cells for phage infection and allows clear visualization of plaques as transparent zones against the turbid bacterial background.
Q4: How does the double agar layer technique work in a plaque assay?
The double agar layer technique uses a solid, concentrated bottom agar layer and a soft, low-concentration top agar layer containing bacteria and phage. The soft overlay allows phage particles to move locally and infect neighboring cells while restricting their movement to distant areas. This creates localized zones of cell lysis, producing visible plaques that can be counted to determine phage concentration.
Q5: Why are serial dilutions necessary before performing a plaque assay?
Phage titers vary exponentially across different samples and types, so serial dilutions generate a wide range of phage concentrations to ensure countable plaque numbers. Performing dilutions in triplicate provides statistically significant and accurate data. An ideal plaque count ranges from 10 to 150 plaques per plate, making dilutions essential for obtaining reliable results.
Q6: What does PFU stand for and how is it calculated from plaque counts?
PFU stands for Plaque-Forming Units, representing the number of infective phage particles in a sample. To calculate PFU per milliliter, divide the mean plaque count by the dilution factor, then multiply by 10 to account for the additional dilution from using only 100 microliters of phage mixture during overlay preparation. The final average PFU per milliliter corresponds to the number of infective phage particles in the original sample.
Q7: How can the plaque assay be used beyond viral enumeration?
Beyond quantifying phage titer, the plaque assay can characterize plaque morphology to aid in identification of different phage types and isolate phage mutants. Additionally, the resistance or sensitivity of bacterial strains to specific phages can be used for phage typing, dividing bacterial species into subgroups based on their susceptibility to different bacteriophages.