I have isolated two phages from two urine samples from a UTI patient. For isolation, I mixed equal amounts of LB broth and urine and added 1 ml of e.coli overnight. After filter sterilization, the filtrate was used for spot assay.Picture shows spots that are formed from urine samples.
But when I chose a plaque for purification, they gave me no plaques.
I'm confused, if this is urine that has antibacterial activity? If there are phages then why didn't they produce plaques as I purified them.
Any advice or help on how can I confirm that antibacterial activity is due to urine or phages?
First you can isolate and grow the bacteria from urine by following cetrifugation of urine, culture the bacteria then check the activity of your isolated phages against thats specific strain of bacteria. In phage spot assay use the patient urine sample is a control to check if it has any antibacterial effect. Wish you good results ahead
thank you Fazal Mehmood Khan for your kind suggestions i did follow the same procedure. i have added the overnight bacterial culture, urine sample and lb broth. then next day i added chloroform then centrifuged it and then did spot assay. it gave the clear spot as shown in picture but when i pick small plaque from this spot for further purification it did not produce any spot. plus i do not have urine sample now to use as control. can you suggest me how to confirm now
Did you dialyze the phage after chloroform treatment? The plaque observed could be as a result of the chloroform.
I think you can first perform the experiment without chloroform treatment to ensure that the phages are active.
And also use a small amount of buffer to resuspend the plaque, like 500ul then if it clears the whole plate, you can dilute the phage lysate to obtain single plaques for purification. Wish you good luck
In general when you dont get plaques after the second round of phage purification than probably the phage is lysogenic, temperate. You should pick up a single plaque and try to replicate in liquid media. After this you have to spot the second enrichment. In general a lysogenic phage with produce single plaques or nothing at all. ( do you know if this patients used antibiotic recently?)
In case that patient have a chronic bacterial infection then use urine to srpead on a plate (bottom layer) and look if you get paques.
Why don't you get a urine sample from a healthy individual not on antibiotics and use that as a control?
But if you think there are bacteriophages, then just do a dilution series of the urine sample and and see if you get individual plaques and at what titer.
@Hira Niaz in this case the clearing is due to antibiotic taken by patient. Just do several step of enrichment lowering the sample each step. In 2-3 round of enrichment you will avoid antibiotics and see if you had phages in urine.
well,
It must be known whether the patient is taking antibiotics or not. Then you can capture a single plaque and make a double-layer method. Such plaque is often caused by an antibiotic.
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