Table 1.
Methods for detecting newly isolated bacteriophages.
| Method | Description | Advantages | Limitations * | Example References |
|---|---|---|---|---|
| Spot testing | A plate is inoculated with host bacteria to form a lawn, then small drops of phage filtrate are placed on the surface. After incubation a zone of lysis indicates presence of phage. | Simple. Allows testing of multiple phage filtrates on the same plate. |
Host must grow to confluence on solid media. Prone to false positives due to bacterial killing by media components or phage binding that does not lead to a productive infection. |
[76,77] |
| Plaque testing | Increasing dilutions of phage filtrate are mixed with bacteria and placed on plate surface by spreading or soft agar overlay. After incubation plate is checked for the appearance of plaques. | Demonstrates productive phage growth. Plaque appearance can suggest lytic vs. temperate life cycle. Plaque size may suggest phage size due to diffusion effects (larger phages diffuse more slowly, etc.) |
Host must grow to confluence on solid media. Not all phages are capable of forming plaques even on productive hosts due to limited diffusion in agar or low productivity. |
[78,79,80] |
| Culture lysis | Phage filtrate added to broth culture of bacteria and incubated. Monitored for cell lysis as indicated by loss of culture turbidity. Metabolic dyes can be used instead of turbidity to assess bacterial metabolic activity [81]. |
Useful for bacteria that will not grow to confluence on solid media as well as any bacteria that grows well in broth. Can be adapted to automation using spectrophotometry to measure turbidity either in single tube or multiple well plates. |
As with spot testing, can have false positives due to non-productive lysis. Cell debris from cells lysed during early infections may bind to and inactivate free phages, interfering with later infections. Hosts that rapidly evolve phage resistant mutants will cause false negatives as mutants maintain turbidity. |
[25,82] |
| Routine test dilution (RTD) | Phage lysates are diluted to the point of producing just less than confluent lysis on a plate. | Useful with phage that do not form distinct plaques or very small indistinct plaques. | Prone to false positives when media components are not highly diluted. | [83,84,85] † |
* These limitations can also be viewed as a screening method when isolating phages for phage therapy. That is, phages that cannot form plaques or whose host rapidly evolves resistance may be poor candidates as therapeutic phages. † All of these references used RTD for host range and other characterization, not detection of newly isolated phages, but in principle RTD could be used with a high titer, poorly plaquing novel phage.