During screening of microbial extracts for antibacterial activity I keep seeing an unusual effect on the growth of P. aeruginosa (PA), which I hope you can see in the images attached. Basically I am doing disk diffusion assays, so plates are inoculated with bacterial solution matching 0.5 McFarland standard. Whenever I do this with PA I see what looks like viral plaques. I have tried preparing the inoculum using both the overnight growth and direct suspension methods, but still see the same effect. The same thing happens with two different strains of PA, and on two types of media (MHA and BHA). Interestingly, the technician in the lab has tried to replicate this several times without success.
Also, there seems to be a strange effect where there is a halo of these plaque looking things around some of the disks, yet the bacteria are growing within the zone.
If anyone has come across this before, or has any idea, I would love to hear from you!
It looks a lot like phage plaques, your bacteria stock might be contaminated with a phage. If you want to test if it is the case, why not pick up one of these plaques (using a pipet tip), ressuspend it in media (50-100µl), do a serial dilution and plate each dilution as drops? If it is a virus, and you do like -1 to -10, you will see a nice dilution effect and can even titre it.
That is indeed an interesting effect!
Did your technician try to replicate it with your bacterial stock or with her own? It is possible that you have a contamination in only one stock.
Also, do you have the same effect with other bacteria?
If the bacteria still grows within the halos, I do not see how there will be any phage there. I see this all the time when Pseudomonas aeruginosa grows on media containing substrates they can hydrolyze without proper dye to highlight the clear zones. I am certain that a particular substrate in the medium is effectively hydrolyzed. You could follow it and find out what.
Thanks all, been scratching my head over this one for a while now!
Phage induction was the first thought when we observed the effects, so the technician was concerned that it may be contaminated. But no, the bacteria I am working with come from her own stock so the bacteria she used to try and replicate the effects were the same, but she could not do so. And again, it happens with two different strains of PA, but not with any other bacteria I have worked with.
As for the hydrolysis of substrates in the medium, I wonder if this would produce a more consistent pattern over the plate (like the third picture), rather than the random scattering seen in the other two images. Just a thought..
Can the plaques be replicated by sampling a plaque and inoculating a plate seeded with your Pseudomonas strain? If so I would be almost certain that it is phage. It looks absolutely typical. It has probably picked up the phage after your technician donated the strain, explaining why her stock does not show the effect.
Hi. I hope the following website could help you:
https://www.sciencedirect.com/topics/medicine-and-dentistry/pseudomonas-aeruginosa
So the lab tech mentioned to me that she had a fresh plate of P. aeruginosa that she had made for some minor research students. This had come from her stocks again and some of the students had been doing disk diffusion tests, none of which produced this effect. That being the case, we assumed that there is no viral contamination. However, I decided to experiment and prepared a couple of swab plates from her sample and again I managed to get the same effect as before (the plaque looking effect). Again this was done by making a standardised suspension of PA in MHB and then swabbing onto both MHA and BHA. I know it definitely looks like viral plaques, and if I get time will do a plaque assay in future, but it just seems unlikely that I am somehow contaminating it myself every time.
I know I should just move on, but my curiosity gets the better of me!
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