環境試料中のバクテリオファージの検出
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Q1: What is the structure of a bacteriophage?
Bacteriophages are viruses consisting of a nucleic acid genome, either DNA or RNA, surrounded by a protein coat called a capsid. Some phages have an additional lipid envelope and spike-like surface appendages or tails. These ultramicroscopic particles are too small to view with a light microscope and can only be seen using an electron microscope.
Q2: How do bacteriophages infect and reproduce in bacterial cells?
Bacteriophages attach to the bacterial cell surface and inject their genetic material into the host. The viral genome is replicated and capsid proteins are produced using the host cell's biochemical machinery. Once phage particles assemble, they are released by lysing the host cell, often bursting out and killing the bacterium in the process.
Q3: What is a plaque assay and how does it measure phage concentration?
A plaque assay mixes phage with bacteria and soft agar overlaid on regular agar plates. Bacteria form a continuous lawn, and when phages infect and lyse bacteria, plaques form as clear zones. Counting discrete plaques and calculating plaque-forming units (PFUs) per milliliter determines the original phage concentration in the sample.
Q4: Why are coliphages useful indicators of water contamination?
Coliphages infect E. coli, a common gut bacterium indicating fecal contamination. They are reliable surrogate indicators for disease-causing enteroviruses that are also fecally-transmitted but difficult to assay. Quick and inexpensive enumeration methods make coliphages attractive tools for assessing fecal contamination in environmental water samples.
Q5: What conditions are necessary for preparing bacteria in a plaque assay?
Bacteria must be in the log phase of growth to ensure every bacterium is alive and allows phage to produce progeny. Three hours before the assay, inoculate overnight culture into fresh broth and incubate at 35-37°C with shaking. This timing ensures bacteria reach optimal metabolic activity for supporting phage replication.
Q6: How can plaque assays be adapted to detect viruses other than bacteriophages?
Plaque assays can be performed with mammalian viruses like influenza by growing mammalian cells as monolayers in tissue culture dishes. Virus-containing media are added to allow infection, then cells are overlaid with immobilizing medium such as agarose. After incubation lasting up to two weeks, infected cells are fixed and stained to visualize and count plaques.
Q7: How are individual phage isolates obtained from environmental samples using plaque assays?
Phages are collected from environmental samples like soil by filtration, then used to infect susceptible bacteria in a plaque assay. Individual plaques are picked and streaked onto new agar plates overlaid with bacteria-containing top agar. Phage concentration decreases along the streak, allowing discrete plaques from single phage types to be isolated and further analyzed.