I am planning to investigate the relationship between phage infection in Pseudomonas and disinfectant resistance
Dear Maria,
Please read the following text:
Phage Isolation
Phages were isolated from sewage following a simple enrichment procedure. Samples from sewage were centrifuged for 5 min at 4100 x g (Biofuge fresco). Ten ml of the supernatant were mixed with 5 ml of an P. aeruginosa overnight culture and incubated in 50 ml LB broth at room temperature. After an incubation of 48 h the cells were sedimented by centrifugation at 4100 x g (Biofuge fresco) for 10 min and the supernatant was transferred to a clean tube. To kill the remaining bacteria,
several drops of chloroform were added to the supernatant and the mixture was
mixed for 30 s. To separate the phages, appropriate dilutions of the phage lysate
were spotted onto bacterial lawns of top-agar plates. Top-agar plates were produced by adding approximately 5 x 108 cells/ml of P. aeruginosa from an overnight LB broth to 3.5 ml of LB top agar (0.75 %). The inoculated top-agar was overlaid on an LB agar plate and allowed to solidify. After incubation at 37 ℃ for 10 to 16 h zones of lysis were recognized. Single plaques, derived from a single phage, were separated by stinging with a pipette tip into the plaque followed by resuspending the phages in SM buffer. The resulting phage lysate was stored at 4 ℃.
This text was taken from pages 27-28 of the publication contained in the following link:
http://digisrv-1.biblio.etc.tu-bs.de:8080/docportal/servlets/MCRFileNodeServlet/DocPortal_derivate_00008650/Doktorarbeit.pdf
Hoping this will be helpful,
Rafik
Dear Rafik:
I thank you very much. I will try with your protocol,
María Angélica
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