Hello, I encountered a problem isolating a phage for Pseudomonas aeruginosa bacteria. When performing the spot test, lytic zones appeared. but when trying to do the plaque assay test I didn't observe any lytic zone. the growth of bacteria was homogenous as no phage filtrate was added. Still, I observed only one thing different: in my isolate of Pseudomonas the pigment of the isolate disappeared when treated with phage filtrate compared to control one -the same observation was taken in the enrichment step the treated test tubes showed no pigment compared to control one- and still disappeared until dilution no. 5. The pigment return appeared as in the pictures attached. I re-ran this test several times and it shows the same result without any lytic zone in the plaque assay test. what is a possible Explanation of this result? is it related to Pseudomonas especially, phage type, or maybe a technical error? Any advice to do this test better? thank you for your answer
Hi G. Hamdi Ismail
This could be a result of numerous reasons. One of the most commonly observed is associated with instability of phage particles in the buffer/environment of your choice. This results in the loss of phage infectivity while phage lysins reales in the media would still have activity in spot assay although you would not observe anything in plaque assay.
Try to optimize the phage storing conditions (buffer composition, temperature, etc.) or duration when phage is still infectious.
Good luck
Povilas Kavaliauskas thank you for replying.. any recommendations for the best buffer I can use for better results??
For that, you have to try several. We routinely use Tris-based buffer with BSA or gelatin.
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