I am working with Xanthomonas oryzae bacteriophages. When I try doing a plaque assay I can visualize the bacterial lawn after 2 days, but plaque formation is not seen. It takes almost 5 days for the plaque formation.
I tried streaking the plaque on bacterial lawn. Even in that I was only able to visualize the clearance after a week. How can I reduce the time taken for the plaque formation? I have attached the photo of clearance of bacterial cells by phages in streak plate method.
Using soft agar will help, also you could check the optimum growth temperature of your culture. How much of bacterial inoculum are you using for bacterial lawn
Your plaques should appear in less than 24hrs if you took care in obtaining your extract/viral suspension before inoculation into your soft agar and before the final inoculation onto bacteria cells embedded in your solid medium
I can write according to my opinion with Streptococcus bovis bacteriophages that your plaques should appear in less than 24hrs if you took care in obtaining your extract/viral suspension before inoculation into your soft agar and before the final inoculation onto bacteria cells embedded in your solid medium. I have such opinion.
Dear Fizah ,
If i am not wrong , the time taken for plaque formation also depends on the host , Incase of ur host the doubling time is less. but incase of Xahthomonas oryzae the doubling time for the pathogen is 2 hours. i am not sure whether thats the reason why i get the plaque formation very late .
I don't know if I got you right this time around. But please check the quantity of agar in your solid medium. If your solid medium is too tough, it will not allow easy assess of the viruses in the soft medium to interact with the bacteria cells in the solid medium. I guess this is the reason for the development of the lawn before the clearance.
Plaque growth is dependent on a number of factors - Steve Abedon has written a lot about this - but see chapter 17 in Bacteriophages; methods and protocols which you can down load from this web site: http://download.springer.com/static/pdf/530/bok%253A978-1-60327-164-6.pdf?auth66=1413975352_fd9fc13e89be9630703e3d0b79f4126e&ext=.pdf
Dealing with the simplest first, as mentioned above, diffusion rates are an important factor - so as a general rule, the lower the percentage agar, the faster the plaque will grow. However infection rates are also determined by (1) the expression of the receptor on the cell surface and (2) the rate of replication of the phage in the host cell. There is not much you can do to optimise (2) but given your observation that plaque formation takes many days, it is possible that the phage receptor is only expressed by your host cells in stationary phase.
It would be worth doing a simple phage cell binding assay to see when your phage binds best to the host cells. This is pretty straight forward; dilute the phage to about 10^3 pfu/ml [you need to dilute the phage to allow you to detect a drop in the phage titre] and mix with an excess of host cells (m.o.i = 0.01) harvested at the different stages of growth (you need to adjust the cell number so each sample contains about the same number of cells). Mix phage with cells and allow a time for cell binding (usually about 15-30 min), centrifuge the sample at 13,000 x g (microfuge) and then titre the number of free phage remaining in the supernatant. From the sample that gives you the largest drop in free phage titre, you should be able to identify the growth phase of your cells when your phage are best able to bind to the host. You can use this information to try and improve the method used to prepare your lawns so you speed up plaque growth
I am of course assuming you don't know what the receptor is...if you do just check when this is expressed at highest levels.
There are lots of useful methods in the book on the above link that will help you going forward.
Please, I am interested in the outcome of your research on the expression of receptors and if that was actually what was responsible for the challenge that you were faced with. Thank you.
I worked with Streptococcus bovis bacteriophages. I have experiences that I can visualize the bacterial lawn after overnight incubation in a termostat, but plaque formation is seen after next 24 hours.
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