I've been trying to isolate bacteriophages specific to soil bacteria (isolated from low, moderate and highly saline soils from research extension centers) for the last couple of months using the enrichment method followed by plaque assay. However, I'm always ending up with no plaques in my diluted plates (with complete lysis in the undiluted one). I'm keeping positive controls (w/o phages) and negative controls (media only). I'm using the following protocol:

• O/N Incubation of soil samples with host bacteria (Bacillus toyonensis) in LBMC (2.5 mM MgSO4 and CaCl2 in final media) at 30 degree C, followed by centrifugation @ 4700 rpm for 20 mins, adding chloroform to supernatant, 5 mins incubation and 5 mins centrifugation @ 4700 rpm for 5 mins and flitering using a 0.22 micron filter.
• Multiple rounds of enrichment (with chloroform sterilization and filtering) of the lysate with host until OD drops approx 10 times relative to +con. I also prepared another control by treating the +con with the same lysate collection treatment as with the one containing phages, since I was suspicious something in the media is killing my host before they can be infected by the phages. Interestingly, I found this control showing almost similar drop in OD relative to +con, although no phages were added here, thus confirming my suspicion.
• Plaque assay with 8-10 ten-fold dilutions of the final lysate added to host in early stationary phase (OD=0.2-0.3), in 1.5% LBMC plates, 0.7% soft LBMC agar kept at 42 degrees (I also tried 0.3% and 0.2%), incubated at 30 degrees as well as 37 degrees O/N. No plaques in dilution plates, undiluted stock showing complete lysis. Tried keeping the plates for longer times as well (upto 5 days) with similar results.

In my understanding, some sort of endolysins/toxins might be getting enriched in the culture media and killing the hosts, thereby not letting the phages infect the bacteria and multiplying. I also tried using a second host (Serratia plymuthica) and ended up with the same results. I'm relatively new to this and I would really appreciate any help/thoughts/suggestions on how to troubleshoot this.