I am working on bacteriophages. My phages worked well before and gave the results. But now they gave spots, but when I tested for plaque formation. They do not produce any plaques.
Protocol I followed for plaques assay is: serially diluted phage lysate mixed with 100ul of exponentially grown culture of bacteria and then leave for 15 to 20 min at 37C for adsorption. After incubation 3 to ml of semisolid agar is mixed and pour onto LB plates and then incubated overnight.
I don't know what's problem now.
Anyone could help please
Assuming that you are still using the same host strain for plating that you know works well, then the problem most likely is that either you aren't making the right dilutions, or your phage stock is no longer any good.
Does the lawn on your plate look good and nice and smoothly turbid? Are you doing a full range of dilutions in case the titer of your stock is very low?
May be you can do some mistake in your experiment protocol.
I will share you the protocol, please follow it
That might be the problem then. have you tried the old host to teat for plaques on it To be sure the phage stick is good?
sometimes a host will be resistant to plaque formation but if you out too many phages on a spot you might still observe killing or clearing.
EOP might be different between the hosts. Try to use higher phage titer.
The problem is either with your phage stock or your host. Be sure you have done the full range of phage dilutions to make sure you aren't just missing the right dilution. But since you changed hosts it seems as if that might be the problem. Have you ever seen plaques with the new host? If not, then I would assume that the new host is not suitable for your phage.
It would be helpful if you provided more details, what is the phage, what is the old host and the new host, etc. It is very difficult to diagnose problems with little information. Pictures of your plates might be helpful.
Do you use any chemical or antibiotic in your phage stocks? It can be that you get clear zones at high concentration due to endolysines and when you dilute you get no plaques. Just do a phage replication with stocks that give negative results for plaques. If you dont get any phage in replication protocol probably your stock is gone.
It could be due to lysis from without i.e. When the phages are very high in concentration, they kill the host by adsorbing to them and injecting holins and endolysins instead of the lytic cycle Article Lysis from without
To prevent this make serial dilution and do plaque assay of each dilution. At some dilution, you will get plaques (if the phages are still active and the host used is the same as before).
verify if your host is viable?
verify if the OD its optimum?
maybe you have small plaques not great plaques. (theres phages have great capsid and produce small plaques and viceversa.
review this points.
kind regards.
To determine whether this is an issue with your lysate you may want to make a new liquid host culture and new agar plates. Trying your plaque assay again with new materials may help. You could also incubate your lysate with your host for longer time periods and try the plaque assay again.
There could be multiple possibilities:
- If the suspension of phage titer you last stored were low, maybe the phages have become inactive.
- See if you are adding soft agar at a higher temperature, which can be tolerated by bacteria but not phages.
- Adding to William's answer, incubate phages and host for a longer time along with 5mM of CaCl2 and MgCl2.
Hope it will help.
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