I am working on the isolation of bacteriophages against Pseudomonas aeruginosa. From one P. aeruginosa isolate, I obtained two plaques with almost similar morphology. I performed 15 rounds of plaque purification, but was unable to separate the phages. Transmission electron microscopy (TEM) revealed the presence of a phage mixture.
To achieve separation, I am currently picking single plaques, suspending them in SM buffer, and repeating plaque assays using the double overlay method.
Are there any alternative methods available to separate phages from a mixture?
Separating phages from a mixture involves several steps, depending on the specific application and the type of phages being isolated. Here are some common methods based on recent studies:
1. **Enrichment and Isolation**
Enrichment Cultures: Phages can be enriched by incubating the mixture with a specific bacterial host. This allows phages that infect the host to multiply, making them easier to isolate.
Plaque Assays: After enrichment, phages can be separated using plaque assays, where they form clear zones (plaques) on a bacterial lawn, indicating lysis.
2. **Filtration and Centrifugation**
Filtration: The mixture can be filtered through a 0.22 µm filter to remove bacteria while allowing phages to pass through.
Centrifugation: Ultracentrifugation can be used to concentrate phages, separating them from other components in the mixture.
3. **Polyethylene Glycol (PEG) Precipitation**
PEG precipitation is a cost-effective method to concentrate phages. It involves adding PEG to the mixture, which precipitates the phages, allowing them to be separated by centrifugation.
4. **Chromatography**
Anion Exchange Chromatography: This method can purify phages by separating them based on their charge. It is particularly effective for removing contaminants like endotoxins.
Size-Exclusion Chromatography: This technique separates phages based on size and is useful for obtaining high-purity preparations.
5. **Droplet Microfluidics**
A novel method involves using water-in-oil droplets as individual chambers for phage propagation. This allows for high-throughput isolation and enumeration of phages.
6. **High-Throughput Screening**
High-throughput methods enable the rapid isolation of diverse phages from environmental samples, increasing the likelihood of finding specific phages for therapeutic or research purposes.
These methods can be combined or adapted depending on the complexity of the mixture and the desired purity of the phages.
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