I prepared a bacterial lawn by spreading 100 µL of log-phase Salmonella (~10⁹ CFU/mL) on LB agar. Then, I spotted 10 µL from a series of 10-fold serial dilutions of my enriched phage sample (10⁻¹ to 10⁻⁸) and incubated the plate overnight at 37 °C.
The results showed a clear reduction in the number of visible dots across the dilution series, consistent with a typical phage pattern. However, the individual dots appear raised and opaque, resembling bacterial colonies rather than flat, clear phage plaques. At the lowest dilutions (10⁻¹ and 10⁻²), there was extensive clearing or disruption of the lawn, while at higher dilutions (10⁻⁴ to 10⁻⁶), discrete white dots were visible. This makes it difficult to distinguish between:
- True phage plaques indicating active lytic phages, or
- Bacterial carryover/contamination, where live bacteria from the enrichment grew on the lawn.
I have attached an image of the plate for your reference.
Before moving forward, I plan to troubleshoot by:
- Filtering the lysate through a 0.22 µm filter to remove bacteria and re-testing.
- Comparing filtered vs. unfiltered samples to confirm whether the dots disappear.
- Possibly using a heat-treated control or liquid enrichment test to differentiate phage activity from other factors.
I would greatly appreciate your opinion on how to interpret these results and whether you have additional suggestions for controls or troubleshooting steps.
Thank you so much for your time and guidance.
The 'dots' are colonies of the bacteria. Plaques occur when the continuous growth (lawn) of bacteria (colonies growing together) have an area of the lawn where the phage has killed the bacteria. A 'hole' or clear area in the lawn of bacteria growth represents a plaque created by the actions of the phage. Please do a google search for 'phage plaques' to see examples.
I'm not sure what the top two plates are, but it looks like some sort of contamination, possibly in the top agar?
In the bottom plate those white dots look like colonies, I agree with Robert John Wolff on that. Most likely your phage stock is not sterile. You can either filter sterilize it through a 0.22 micron filter or else sterilize it with chloroform, add a few drops and vortex for 30 seconds. Most phages are tolerant to chloroform (but there are few that are not).
From what I can see in your photos, those look like bacterial colonies rather than phage plaques. This usually points to some kind of contamination.
This suggests that some bacteria may have passed through during the filtration step and survived. I’d recommend centrifuging your phage prep and then filtering it through a 0.22 µm filter before spotting again, as this should help remove any remaining bacteria.
I agree with Michael J. Benedik and Robert John Wolff
Thanks for your valuable comments I am new in this area, and your comments were helpful for me. I filtered them, but they are still contaminated with the bacteria so I will sterilize with chloroform as well and try again. Thank you so much Michael J. Benedik Robert John Wolff I. S. Gómez-Cano
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