I have experience with Streptococcus bovis bacteriophages. However, I can write that to propagate phage from single plaque should be no problem. I made it often. I had titer above 10-9.  10 ml of Nutrient broth was inoculated with Streptococcus bovis culture. When the OD reached 0.2 single plaque isolated from double agar plate was added. You have incubated the broth overnight, it is OK. What is the best method to propagate phages? I do not know, however, I have very good experience with Streptococcus bovis bacteriophages and a high titer of bacteriophages. However, I can write that to propagate phage from single plaque is no problem usually.

This will almost certainly not be a problem, and there are several ways to success. Personally, I always preferred to pick a single plaque out of the soft agar and let the phages diffuse out over night into 1 ml saline or an appropriate buffer. You will normally have a titer some 10^8  afterwards.

Subsequently, you plate the phage on several Petri dishes, aiming at. confluent lysis. Add 5 ml of your phage buffer on the plates, wait for elution of the phage over night in the refrigerator.

Collect the supernatant, centrifuge shortly and store the phage suspension in the refrigerator. Normally it is wise to add a few drops of chloroform to avoid bacterial growth.

Good luck.

Look the work  Bradley DE. Ultrastructure of phages and bacteriocins. Bacteriol. Rev. 1967, v31,N4 p.230-314.

HI Rnaju,

Totally agree with Prof. Johannes Wöstemeyer, and we do select several plaques and incubate them in 1 ml of phage buffer in room temperature for 2-3 hours. Then, we infect the susceptible strain(s).

However, you can try select one plaque and do infection then select a new plaque (remember to test the phage each time by PCR or any other possible method to be 100% sure) three times. Finally, make a new infection and collect the entire plaques by collecting the upper layer in a 16 ml test tube + 3 ml of phage buffer, cooled centrifuge (4 C) at 4000 rpm/5 minutes then filter the supernatant by 0.2 milliporefilter. Then, hopefully, you will get higher titre.

Try and lets know if that works with you

Cheers,

Maan

Sometimes it is tough to get a great yield from a single plaque isolation. After your initial infection from the single plaque, titer your stock/lysate. Do a subsequent infection with your new stock during mid-log phase of the bacterial growth cycle at an MOI of 5-7. Let the infection go until you see good clearing (the culture should look almost like clear media, and you should see cell debris form lysis). Getting a maximum yield always take some "fiddling", be patient and good luck. 

Isolate the phage and then propogate it on the specific host bacteria.

Bacterial culture for propagation should be young. Dilute night culture, incubate for several hours, than inoculate phage from plaque, again incubate to obtain lysis and phage multiplication, than separate the phage from culture.

For studies some time enough to take phages from plaques as replicas.

Totally agree with Prof. Johannes Wöstemeyer.

I offer you a method close to the one that wrote Prof. Johannes Wöstemeyer

1. By titration of phages on your bacteria culture, please, prepare three Petri dishes as shown on the picture (plaques.jpg).

2. Pour petri dishes by nutrient medium for growing of your bacteria (about 10 ml).

3. Add 0.2 ml of culture of your bacteria in exponential growth phase on each dish.

4. Place the Petri dishes to incubate at the optimum temperature suited to your bacteria.

5. Incubate at three time intervals: 3 hours, 9 hours, 16 hours.

6. Collect the liquid in three separate tubes, add chloroform, stir and titrate the phage.

Comments.

You write: "In papers i referred the incubation time to be 8hrs for E.coli?"

This is not true. For phage T4 - 5 - 7 hours. For T7 - 3 - 5 hours.

For other phages of E.coli is totally different time.

You write: "How should i determine the termination time?"

To do this, you should investigate the development of phage in single cycle.

Good luck and success