Phage screening - colorimetric test

Prepare LB-agar plates.

Prepare LB-broth.

To recover bacteria from stock open the tube and use a sterile loop, toothpick or pipette tip to scrap off some bacteria and streak on appropriate media plates using the overnight culture and grow at37°C.

If using lyophilized strain skip step 3 and 5 and

Next, start overnight cultures by inoculating fresh liquid media (5mL ) with a single colony and

grown for 14-16 hours at37°C in a shaker incubator.

Inoculate 5mL of fresh LB (1% inoculum) and grow at 37°C until the cells reach a density of OD₆₀₀=0.2. The amount of host culture to add is not critical, but it should be enough that would normally produce a saturated culture over the period of the enrichment (0.2 OD is enough or 0.5 McFarland Standard i.e. 1.5x 10^8 CFU/ml).

Using a pipette, aseptically add 500µL of the recommended growth medium to the freeze-dried material and mix well.

Transfer the entire suspension to a test tube containing 5mL of the recommended media.

In a new falcon tube add 1mL of filtered Sample sample to 900µL of LB-broth and inoculate with 100µL of a fresh host culture (0.2 OD) and mix gently.

In another falcon tube inoculate a positive control adding 10-50uL (this amount will depend of the concentration of your phage stock) of phage stock to 1900µL of LB-broth with 100µL of a fresh host culture and mix gently.

Incubate this enrichment culture at 37°C for at least 2 hours (or under whatever conditions the host favors). At the same time inoculate a new tube with 5mLof fresh LB (1% inoculum) and grow at37°C for 2 hours.

Filter the culture through a 0.22μm syringe filter. The filtrate is now ready for testing.

In a new falcon tube add 100µL of sample (filtrate from previous step) to 1800µLof LB-broth and inoculate with 100µL of a fresh host culture (0.2 OD) and add 50µL of (resazourin) and mix gently.

Incubate the culture at 37°C for 3 hours. Check the culture every hour to monitor color change.

If the sample is positive for phage culture will remain blue. If the sample is negative culture will change to pink color.