I'm not entirely sure that what you are seeing is incomplete lysis, my first guess would be that the amount of phage in your spot is low enough to give individual plaques. A real spot from a lysogenic phage looks a bit different. But the resolution is not good enough in the pic for me to be sure.

However, if you want to find out, you can just streak from the center of the lysis region on a fresh plate and test individual colonies for whether they are lysogens. You can cross streak across some phage as in this picture I am attaching.

From the picture, it would be difficult to conclude that the phages are lysogenic. It could also be due to some bacterial cells evolving resistance, leading to unclear or turbid plaques. To verify this pick bacteria from the turbid area and re-streak them on a fresh plate, then perform a spot test again. If the bacteria resist phage infection, they might be phage-resistant clones.

To verify lysogenic phages:

For your phage purification:

Hi Mariel Gullian Klanian is it possible to use phosphate Buffered Saline instead as an alternative to SM buffer? Unfortunately, we only have PBS available. Also, how can we observe bacterial lysis in LB Broth? Does lesser turbidity after incubation indicate its lytic activity?

Thanks for the suggestion!

any buffer is generally fine but add 5-10mM Mg to it.

Michael J. Benedik Noted, thank you very much!

The spot test results show partial bacterial growth inhibition rather than clear lysis, suggesting the presence of lysogenic phages rather than lytic ones. To isolate and purify these potential lysogenic phages, follow this approach:

1. Isolation of Phages

• Pick a small portion from the edge of the inhibition zone using a sterile loop.
• Suspend it in SM buffer or phage buffer and vortex gently.
• Filter the suspension using a 0.22 μm syringe filter to remove bacterial debris.
• Store the filtrate at 4°C until further analysis.

2. Enrichment of Lysogenic Phages

• Mix the filtrate with a fresh culture of the same bacterial strain in early exponential phase (OD600 ~0.2-0.3).
• Incubate at 30°C or 37°C (depending on bacterial preference) for 4-6 hours with shaking.
• After incubation, filter again to remove bacterial cells.

3. Inducing Lysogenic Phages

• Treat the bacterial culture with an inducer, such as mitomycin C (0.5-1 µg/mL), to stimulate prophage induction.
• Incubate for 4-8 hours and monitor for signs of lysis.
• After incubation, filter to collect phage particles.

4. Purification and Confirmation

• Perform a double agar overlay plaque assay using the filtered lysate to check for plaque formation.
• If plaques appear, pick isolated plaques and replate to purify individual phages.
• Use PCR with specific prophage marker genes (such as integrase genes) to confirm lysogenic nature.