Bacteriophage Isolation and Life Cycle

Study Notes

Bacteriophage Isolation

Bacteriophages are obligate intracellular parasites of bacteria. They use bacterial resources for replication.
Bacteriophages have a life cycle.
Lytic (virulent) phages: Multiply within bacteria, killing the cells by lysis at the end of the cycle. The presence of clear zones, known as plaques, on bacterial lawns indicates lytic phage infection.
Lysogenic (temperate) phages: Integrate their genome into the bacterial chromosome, replicating with the host for many generations. The phage genome exists in a repressed state, called a prophage. The cell containing the prophage is not affected by it and is termed lysogeny.

Isolation of Phages

General Considerations:
- The bacterial host must be free of prophage.
- A good source should have a high abundance of the specific host bacteria.
- The host bacterium is best used in exponential growth phase.
- Optimal growth conditions (medium, oxygen, temperature and incubation time) are critical for phage isolation.

Isolation of Lytic Phages

Three sequential steps for isolation:
- Enrichment (Phage amplification): Increasing phage concentration.
- Centrifugation and filtration: Separating phages from the bacteria.
- Phage cultivation: Growing phages in a medium.
Several phage cultivation methods exist, including spot test, double layer, and turbidity techniques.

Isolation of Coliphages from Sewage

Coliphages infect Escherichia coli.
Sewage is a good source for coliphage isolation due to high E. coli counts.
Isolation steps:
- Enrichment of coliphage:
  - Filtering raw sewage.
  - Adding nutrient broth to filtered sewage.
  - Incubating with shaking for 24 hours.
  - Inoculating the mixture with an overnight E. coli culture.
- Centrifugation and filtration:
  - Centrifuging the sewage-bacteria-bacteriophage mixture (5,000 rpm/10 minutes).
  - Separating the supernatant (containing phage suspension) from the pellet (containing bacteria).
  - Filtering the supernatant to sterilize (using a 0.45 µm pore size filter).
- Phage cultivation:
  - Spot test: Detecting phage presence using a water bath to melt nutrient agar, followed by cooling to 50°C. Pouring into a petri dish, allowing solidification, spreading an E. coli culture on the agar surface, then spotting the phage filtrate. Incubating at 37°C for 24 hours. Observing clear zones (plaques) indicates infective phage.
  - Double Layer Technique: Melting nutrient agar, cooling to 50°C, pouring the cooled medium into a plate, allowing the plate to set, mixing the phage suspension with bacterial broth culture and incubating. Pouring melted semi-solid agar over the pre-solidified agar layer. Circular movement and incubation of the plate at 37°C for 24 hours. Observing clear zones indicates infective phage,
  - Turbidity technique: Mixing the phage suspension with bacterial broth, incubating at 37°C for 24 hours. Absence of turbidity indicates bacterial cell lysis (infective phage is present)

Isolation from Solid Samples (Food and Soil)

For solid samples, prepare a sample suspension in a suitable culture medium for the host bacterium.
Centrifuge the suspension to remove solids, and process the supernatant for phage isolation.