Isolating bacteriophages, particularly those that infect **Pseudomonas aeruginosa**, can be accomplished using relatively low-cost methodologies. Here are some effective techniques:

1. Enrichment Culture Method

- Materials Needed:

- Pseudomonas aeruginosa culture

- Nutrient broth or tryptic soy broth

- Agar plates

- Procedure:

1. Inoculate nutrient broth with Pseudomonas aeruginosa and incubate overnight.

2. Mix the overnight culture with a sample suspected of containing bacteriophages (e.g., water, soil).

3. Incubate the mixture at room temperature or 37°C for a few hours.

4. Centrifuge to remove debris, then filter the supernatant using a 0.22 µm filter.

5. Prepare agar plates and overlay them with the filtered supernatant mixed with a soft agar medium containing Pseudomonas aeruginosa.

6. Incubate the plates and look for plaque formation.

2. Direct Isolation from Environmental Samples

- Materials Needed :

- Environmental samples (e.g., soil, water)

- Pseudomonas aeruginosa culture

- Agar plates

- Procedure :

1. Collect environmental samples and prepare dilutions.

2. Add the diluted samples directly to soft agar mixed with Pseudomonas aeruginosa.

3. Pour the mixture onto agar plates and allow it to solidify.

4. Incubate the plates and monitor for plaques.

3. Plaque Assay

- After obtaining plaques from the enrichment cultures, you can further isolate and characterize the bacteriophages:

1. Pick individual plaques using a sterile tool.

2. Inoculate them into a fresh soft agar overlay with Pseudomonas aeruginosa.

3. Repeat the process to purify the phage.

4. Storage and Maintenance

- Once isolated, bacteriophages can be stored in a buffer (e.g., SM buffer) at 4°C or frozen at -80°C for long-term storage.

- Cost-Effective : Most materials needed are commonly found in microbiology labs, making these methods relatively low-cost.

These techniques should help you successfully isolate bacteriophages from your cultures.

@Mehrab Dashtdar thank You!! Greetings from México