Prophage Induction with mitomycin C (1ug/ml) gave me reduction in bacterial turbidity measured at OD 600 nm. A spot test of the lysate had given a noticeable clearance. But there no plaque formation after trying with different combinations of host & lysate. Flow cytometry analysis showed the presence of viral population after PEG precipitation. Has anyone experienced the same ? How to propagate prophage after induction with mitomycin C? How do I scale up my phage lysate ? Would TEM analysis of PEG precipitated lysate give me clear results of prophage induction? Should I go for different means of concentrating the sample
Before answering your question, it would be interested to know what kind of prophage you are working on and in which bacterial species it propages. Do you know if the phages produce big clear plaques or very tiny and turbid plaques (as might be expected with a lysogenic phage).
You can try to titerate the concentration of Mitomycin C. You want to increase the phage yield, so you will need to let the cells replicate more before they start inducing. You can plot the concentration vs. phage yield to test the dose-response. Alternatively, you can try other antibiotics like Ciprofloxacin to induce the prophage.
Normally, when I induce my prophage, I add an appropriate concentration of inducing agent (generally 1ug/mL of Mitomycin C for the E. coli phages I typically worked with). Following induction, if you spot this mixture after induction, you will probably see clearing on a lawn of bacteria as a result of the residual mitomycin C. This is why before I continue on to the next step, I always verify my titer via tittering on a sensitive strain. If you failed to get a good yield here, there is no point in continuing onwards. Something is wrong with your induction step and you are not making enough viral particles. I've had this problem occur myself from time to time.
There are a few possible causes in my experience. Occasionally, our group noted that you can get some sort of contamination that affects the health of the bacteria. When the bacteria are growing poorly, the phage yield from induction is poor. We've had thoughts that it might be some sort of filamentous phage or something comparable but the jury is still out on that one. When this problem is occurring we noted the following observations: (1.) the lawns of bacteria look thinner than normal after we poor them and (2.) overnight cultures look thinner than expected. Regardless of the cause, when this happens we throw out all old media, clean all the lab surfaces with 10% SDS and try again with fresh media. This usually fixes the problem.
If this is a phage for which you are just beginning to work with and you are unsure of the protocol to obtain the best titer, you might need to do some trial runs to optimize your induction. What works well for one phage might not work for another. Some phages also require certain additives to help them form visible plaques on a lawn like calcium, magnesium or even tryptophan. For these phages the additives help them to adsorb and without them while you can induce the lysogen, they don't appear to form plaques well.
Finally, this sounds silly but how thick is your agar in your plates? Thinner plates less robust looking plaques. I like to put at least 40 mL of media agar solution in a standard sized petri dish when I'm doing tittering plates. I've also heard of a colleague who had trouble propagating lambda phage even though they had done it many times previously. It turned out that something was off in their LB stock. When they obtained a new lot of LB, their inductions starting working again.
In any case, if you have any additional details regarding what you are working with, I could provide some more insight, but based on my past experience working with phages, I think this is a good starting point.
Thank you everyone for your valuable suggestions.
I have not got any plaque formation and its obvious because of the superinfection exclusion after numerous trials. The only way i could prove its a phage is by concentrating the lysate and do a TEM.
Ma'am Nichole : Even I have experienced the same lawn formed is thinner after induction. Induction with 1ug/ml gives me prominent decrease in OD and spot test gave me clearance. I feel there very less viral particles released after induction.
Its just a preliminary work for isolation of phage. I would like more insight on this.
Please could you suggest certain standard protocols you have followed. It would be helpful for me as a beginner.
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