I'm facing difficulties while preparing double agar using the bacteria I initially used to isolate my phage. I wanted to see how different concentrations of these bacteria (measured as OD 0.1, 0.4, 0.8, and 1.5) would impact the results. To do this, I kept the concentration of the phage at a steady level (10^2 PFU/ml, as shown in the petri dishes). When I found that the double agar I made with bacteria at an OD of 0.4 was more effective, I decided to stick with that concentration. Later, I collected the zones from the double agar plates made with bacteria at both 0.8 and 0.4 OD and moved them into SM buffer. Afterward, I remade the double agar. I expected that by doing this, the PFU/ml ratio of my phage would increase, so I made dilutions of 1, 2, and 3 fold to make counting the plaques easier (the D petri dish contained the undiluted filtrate). However, despite these measures, the image shows no zones forming as I anticipated. What could be causing this absence of zone formation?
I am confused with your pictures and also having trouble seeing some of them well. For example the control plates do not look as if the bacterial lawns grew well (but maybe they did). I am surprised that you see such a difference with different OD cultures, I usually just worked with an overnight culture most of the time.
One possibility is that in your SM eluate from the plates the titer is too high, if you had a titer of 106 or higher for example you may not see individual plaques.
Michael J. Benedik
Thank you for your advice. Working with Pseudomonas, dealing with overnight cultures makes it challenging to count plaques because of the dense biofilm formation.
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