I want to isolation phages to infecting S. ausreus, from nasal sawb samples, but i don´t found a protocols. Thank´s
Dear Dayan,
Please read the following text:
Identification of S. aureus and MRSA
One of the nasal swabs was plated on CNA agar (Columbia CNA w/5% Sheep Blood w/Colistin, Nalidixic Acid) and incubated at 37°C for 24 hours. Beta-hemolytic colonies from the CNA plates were subcultured on blood agar plates (BAP) and incubated at 37°C. After 24 hours, any beta-hemolytic colonies on the BAP were screened for S. aureus using gram stain, catalase and coagulase tests (using the Staphaurex Plus latex agglutination test [Remel]). All tentative S. aureus thus identified were confirmed by S. aureus-specific 16S rRNA gene PCR.29 Confirmation of methicillin resistance was by mecA PCR, and the presence of Panton-Valentine leukocidin (PVL) toxin genes by lukSF-PV PCR.30,31 All S. aureus isolates were then genotyped by spa typing32 and multi-locus sequence typing.33
Lytic Bacteriophage Assay
The assay method was modified from Sambrook and Russell.34 Briefly, a nasal swab was suspended into a 5 ml tube containing 3 ml of trypticase soy broth (TSB), vortexed, and incubated at room temperature for 20 minutes. The supernatant was filtered through a 0.22 μm filter into microcentrifuge tubes to remove the bacterial cells and then stored at 4°C. Simultaneously, TSA (tryptic soy agar) base plates were prepared by pouring 15 mls of molten TSA (Trypticase Soy broth with 1.5% bacto-agar and calcium chloride to a final concentration of 400 μg/ml) into sterile petri plates. Two colonies of host cell (methicillin-sensitive S. aureus, ATCC 29213) grown on BAP were inoculated into 2 ml of TSB and incubated at 37°C for 3.5 hours. A 100 μl aliquot of this log phase culture (OD 0.5 at A600) was added to 2.5 ml molten top agar (TSB+0.6% agar+CaCl2 [400 μg/ml]), vortexed briefly, and poured onto the TSA base plates. The plates were allowed to cool. The base of the plate was marked to divide it into 16 squares. A 30 μl aliquot of filtrate was spotted onto each square. The plates were left to stand in a laminar air hood until the spotted filtrates were absorbed into the agar. They were then incubated at 37°C and checked for plaques at 12, 24, 36, and 48 hours. Suspected plaques on plates were photographed.
ATCC 29213 is a reference strain isolated from a human wound that is used in antimicrobial sensitivity testing and quality control in most clinical microbiology laboratories.35 To test the suitability of this reference, we used it to screen for lytic bacteriophages in clinical (abscesses, pustules, and wounds) and sewage materials in preliminary experiments. Three phages (C13–14, C21, and U-11) were thus isolated and used as positive controls in the lytic bacteriophage assay (data not shown). Consequently, it was chosen as a suitable host cell to screen for lytic bacteriophages.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3134439/
Hoping this will be helpful,
Rafik
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