Phage Isolation Methods PDF

Isolation of Bacteriophages  What is the bacteriophage? Obligate intracellular parasites of bacteria, using the resources of bacterial cell for phage replication. According to the phage life cycle Lytic...

Isolation of Bacteriophages  What is the bacteriophage? Obligate intracellular parasites of bacteria, using the resources of bacterial cell for phage replication. According to the phage life cycle Lytic Lysogenic or or Virulent phages Temperate phages Lytic or Virulent phages: ✓Phages that can multiply within bacteria and kill the cell by lysis at the end of the life cycle. ✓Phages infect bacteria with formation of a clearing or plaque on a lawn of susceptible bacteria. This is meaning the bacteria are killed by lysis as newly produced phages are released from the damaged cells. Lysogenic or Temperate phages: ✓Phages that can grow and integrate its genome into the bacterial chromosome replicating with the host for many generations. ✓The phage genes are not transcribed; the phage genome exists in a repressed state. ✓The phage DNA in this repressed state is called a prophage because it is not a phage but it has the potential to produce phage. ✓The cell with a prophage is not affected by the presence of the prophage. ✓The cell which has a prophage is termed a lysogeny.  Isolation of phages General considerations: 1. The bacterial cell must be free of prophage (the bacterial cell is not lysogeny). 2. The suitable source for phage isolation is a source where its specific host is abundant. Wherever the bacteria are found, it is likely that phages are also present 3. It is the best to use the host bacterium in the exponential growth phase. 4. The growth conditions (medium, oxygen level, temperature, incubation time, etc.) applied during the phage isolation must be optimal for the host bacterium.  Isolation of lytic phages The practical steps to isolate the virulent phages include three sequential steps: Phage cultivation methods Enrichment of phage (Phage amplification) Centrifugation and filtration Phage cultivation  Isolation of coliphage from sewage ✓The coliphage is a bacteriophage that infects Escherichia coli. ✓The sewage is a very suitable natural source to isolate the coliphages because E. coli are present normally in the intestines of the mammals (the intestines are the normal habitat for E. coli).  Isolation of coliphage from sewage 1. Enrichment of coliphage Addition of 5 ml of 10 x Filtration of raw nutrient broth to 40 ml of sewage filtered sewage Incubation at 37 °C/24 h. Inoculating the mixture using shaking incubator with 5 ml of an overnight E. coli culture  Isolation of coliphage from sewage 2. Centrifugation & filtration Centrifugation of sewage – bacteria – bacteriophage culture at 5000 rpm / 10 minutes Pellet Supernatant (remaining bacterial (Phage suspension) cells) Sterilization using a membrane filter with 0.45 µm pore size  Isolation of coliphage from sewage 3. Phage cultivation to detect the presence of coliphage in the prepared phage suspension A. Spot test 1. Melting the nutrient agar medium at 100°c using a water bath. 2. Cooling the melted medium to 50°c. 3. Pouring the medium into the plate. 4. Allowing the plate to set. 5. Spreading 1 ml of the E. coli broth culture on the surface of nutrient agar. 6. Spotting five µL of the filtrate on the surface of the nutrient agar layer inoculated with E. coli. 7. Incubating the plate at 37 °C / 24 h.  Isolation of coliphage from sewage A. Spot test After the incubation, appearance of a clear zone (plaque) on the bacterial lawn is used as an indicator for the presence of lytic phages infecting E. coli  Isolation of coliphage from sewage B. Double layer technique 1. Melting the nutrient agar medium at 100°c using a water bath. 2. Cooling the melted medium to 50°c. 3. Pouring the medium into the plate. 4. Allowing the plate to set. 5. Mixing 0.3 mL of phage suspension with 0.5 mL of E. coli broth culture. 6. Incubating the mixture at 37 °C / 10 min.  Isolation of coliphage from sewage 7. Mixing the phage – bacteria suspension with 3.0 mL of melted semi-solid nutrient agar at 46°C. 8. Pouring the phage - bacteria - melted semi-solid nutrient agar mixture on pre-solidified base nutrient agar layer. 9. Circular movement of the plate. 10. Allowing the plate to solidify the upper layer. 11.Incubating the plate at 37 °C / 24 h.  Isolation of coliphage from sewage B. Double layer technique After the incubation, appearance of a clear zone (plaque) on the bacterial lawn is used as an indicator for the presence of lytic phages infecting E. coli  Isolation of coliphage from sewage C. Turbidity technique 1. Mixing 0.3 mL of phage suspension with 0.5 mL of E. coli broth culture. 2. Incubating the mixture at 37 °C / 24 h. 3. After the incubation, there is no turbidity in the tube as a result of the bacterial cell lysis.  For phage isolation from solid sample (food and soil) Preparation of the Centrifugation of the sample suspension in The supernatant suspension to the suitable medium could be processed remove the bulk for the host for phage isolation solids bacterium  In the lab Detection of coliphage in the phage suspension prepared from sewage applying the spot test.