Two strains of Staphylococcus aureus were mixed with soft agar and layered on bottom agar plates. Once the soft agar had solidified, 20 μL of (mitomycin C-induced) phage lysate was spotted on each plate. After the lysate was fully absorbed, plates were incubated at 30°C for 18-20 hours. While on the “Strain B” lawn usual sized (diameter 5-7 mm) zones of lysis were observed, a huge hazy zone of clearance was seen on the lawn of “Strain A”. Actually there was another concentric circle. Repeated twice and same pattern emerged.
Never seen this before. Any idea what’s going on here? Is it due to phage-mediated lysis?
Wow, never see this before. But it doesn't look like a plaque. It's like something (lysin? antolysin？) in the supernatant did this. You could treat the lysate with Millipore Amicon Ultra tube and spot the filtrate. See what happen.
Did you happen to incubate your plate with strain A in an upright position? If so, flip it for your next spot test and see if the problem is solved.
@Linhui: Thanks for the suggestion. Yes, I was hesitant to call it lysis, for, it was more like a wet/soaked circle.
@Priyadarshan: I did this twice and the second time after the lysate was absorbed the plate was inverted and incubated at 30C. The above picture is from that same spotting. I am not sure of the first time as sometimes I do incubate plates upright. Do you think it can affect the lysis/pattern? If so, I wouldn't get lazy again.
In a plaque assay, the plaques are visible to the naked eye because of the fact that the progeny phages infect and lyse the adjoining cells multiple times thereby creating a clear circular (or rather spherical) zone. The soft agar provides mobility to the progeny phage, albeit limited, because of its high water content. If the plaque assay was to be done by replacing the top agar with bottom agar, I bet you would not see plaques with your naked eyes.
Now, let us come back to your observation. It is not unusual to observe condensation on the lids of plates that had been incubated upright. This water can fall back on the top agar and mess up its water content ultimately increasing the diameter of the plaque. In my opinion, the two different concentric circles that you observe are a result of the condensate falling back. The clearer circle is because of the primary infection caused by addition of the cell lysate while the secondary faint circle is because of the condensate that fell back. The inner circle is clearer because the lysate penetrated further and the bacteria was infected much earlier in the experiment while the outer circle is hazy because either the condensate did not fully penetrate the top layer or the bacterial cells had outgrown by the time they got infected.
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