I believe that the double layer method is the most accepted one and you have already had it, right?

I had the similar issue before: The first round of incubation, I got the clear plaque. While for the second round of incubation, no clear plaque appeared. I do not know the exact reason of that. One possible reason might be that you added too many phages in one plate and finally there was no clear plaque.

Did you dilute the phage titer before the second round of incubation? If so, by how many times? 

I suggest that you just repeat the experiment and see the result. If it still does not work, just move on and focus on other phages that can always give clear plaques. 

thank u sir ,I will repeat the experiment and I tried for that.

thank u for ur time and concedaration

Some strains of S. aureus may be resistant to bacteriophage infection probably due to the presence of prophages. Adittion of mitomycin should be useful for this case. I also suggest you to test several S. aureus isolates, strains or genotypes (if available) or even a mix of them for the primary isolation of bacteriophages and further isolation in the individual strains. Also, be aware that rain may dilute phages in the sampling sites. Try not to collect samples in the rainy season.

thank u sir for ur kind information , now i got potential phages with spot test with phages isolated from sewage ,but now taken Pseudomonas aeroginosa . i got very clear plaques ,but now which method have to follow for the isolation of phages from plaque, i read in one article ,just addition of SM buffer  and incubation for 8 hr and centrifugation at 6000rpm for 10 min at 4oc.this method is best or follow any new method for the isolation of phages from plaques . please tell method suitable for isolation of phages from plaques. i attached  plaques of MDR pseudomonas aeroginosa

Remember that P. aeruginosa produces swarming in certain culture medium. This may enmask your plates. Be sure that your culture medium does not promote swarming.

ok sir ,thank you for your advice.

Actually, I am working on the  isolation of phages for staphylococcus aureus from soil. For the same, I can't able to find any straight forward protocol. Please suggest something.

Phage enrichment method was use to isolation of phages. U take any protocol from net apply for isolation of phages..

Thanks.

Bacteriophage isolation

Hi Abhishek,

i am following this protocol for isolation of phages. any how, phage isolation for S.aureus some what difficult when compared with P.aeruginosa and E.coli.

this is my personal experience. 

Sewage samples collected at sewage canal at RIMs hospital, Kadapa, Andhra Pradesh to isolate bacteriophage filtrates against MDR-S.aureus. Sewage water was centrifuged at 8000 rpm for 10 min to remove sewage debris. Filtrate was filtered through 0.45 μM syringe driven filters (Millipore filters, Himedia, Mumbai).  The filtrate was added to a fresh MDR- S.aureus bacterial culture in LB broth. The mixture was incubated at 37 °C for 15 h at 100 rpm, then centrifuged at 10,000 rpm per 10 min and filtered through a 0.45μm membrane. The isolated phage filtrates were screened for the presence of host specific phages by the spot test. Supernatants positive in spot test were tested again by a plaque forming assay. The titer of the phages in supernatant was determined by the double-layer method [Adams, 1959] using LB agar as culture medium. The plates were incubated at 37 °C and examined for the presence of plaques after 24 h. Two more successive single-plaque isolations were performed to obtain a pure phage stock. All lysates were filter-sterilized. Form the spot test, lytic and infective phage to MDR- S.aureus was collected and high titer concentrations of the phage were purified and kept in SM buffer (50mM Tris-CL, 99 mM NACL, 8mM Mgso4, 0.01gelatin (PH7.5) at 4oC with 1 % chloroform.

Good Luck

Pallavali Roja Rani

UGC-SRF

91-7382022213

[email protected]

Thanks a lot.

 No problem Abhishek ...

Good  luck...