2.5. One-Step Phage Growth Curve, Adsorption Assay, and Killing Efficacy

One-Step Phage Growth Curve

A. Preparation of the bacterial host

1. Grow the bacterial host culture at 37 °C with shaking until the exponential growth phase is reached (OD₆₀₀=0.5–0.6).
2. Adjust or estimate the bacterial concentration to approximately 10⁸ CFU/mL.

B. Phage infection

1. Mix 0.9 mL of the bacterial suspension (approximately 10⁸ CFU/mL) with 0.1 mL of phage suspension (approximately 10⁷ PFU/mL) to obtain an MOI of 0.01.
2. Incubate the phage–host mixture for 10 min at 37 °C to allow phage adsorption.
3. Centrifuge the mixture at 13,000 rpm for 4 min.
4. Carefully remove the supernatant and resuspend the bacterial pellet in 1 mL LB medium.
5. Repeat the centrifugation step at 13,000 rpm for 4 min to remove remaining non-adsorbed phage particles.
6. Resuspend the bacterial pellet in 1 mL LB medium.

C. Initiation of the one-step growth experiment

1. Dilute the infected bacterial suspension 1:10,000 into 50 mL pre-warmed LB medium.
2. Incubate the culture at 37 °C with shaking.

D. Time-resolved sampling

1. Collect 500 µL aliquots at 3-min intervals from 0 to 60 min (0, 3, 6, ... 60 min).
2. Add chloroform and right after sampling to each aliquot at a final chloroform:sample ratio of 1:50 v/v (10 µl to 500 µL). Shake each sample vigorously after chloroform addition.
3. Incubate the samples at 4 °C overnight.
4. Centrifuge the samples at 12,000 rpm for 1 min.
5. Transfer the supernatant to a fresh sterile tube without disturbing the pellet.

E. Phage titration by double-agar overlay plaque assay

1. Prepare appropriate serial dilutions of each sample in sterile diluent (e.g. PBS).
2. Prepare approximately 4 mL in separate tubes of molten 0.4% agar and maintain at 55 °C.
3. Add 100 µL log phase bacterial suspension and 100 µL phage dilution to the molten agar tube.
4. Mix gently and immediately pour the suspension onto an LB agar plate.
5. Allow the overlay to solidify.
6. Incubate the plates overnight at 37 °C.
7. Count plaques on plates containing a suitable number of well-separated plaques.

F. Growth curve construction

1. Calculate the phage titer (PFU/mL) for each sampling time point based on the plaque counts and corresponding dilution factor. For example, if 100 µL 10^-4 phage dilution resulted in 15 separate plaques on the plate, it means the sample titer is 1,5×10⁶ PFU/mL.
2. Plot phage titer (PFU/mL) against time after infection to generate the one-step growth curve.
3. Perform the experiment using at least three independent biological replicates and report the mean phage titer for each time point.

Phage Adsorption Assay

1. Grow the bacterial host culture to the exponential growth phase.
2. Adjust the bacterial suspension to approximately (10⁸) CFU/mL.
3. Transfer 1 mL of the bacterial suspension to a sterile tube.
4. Add 10 µL of diluted phage suspension at approximately (10⁷) PFU/mL to obtain an MOI of 0.01.
5. Mix gently and incubate for 10 min at 37 °C to allow phage adsorption.
6. Centrifuge the mixture at 13,000 rpm for 5 min to pellet the bacterial cells and adsorbed phages.
7. Carefully collect the supernatant without disturbing the bacterial pellet.
8. Determine the residual phage titer in the supernatant by plaque assay.
9. Determine the initial phage titer using the phage suspension before incubation with the bacterial host.
10. Calculate the phage adsorption efficiency using:(initial phage titer—residual titer in the supernatant)/initial phage titer.
11. Perform the experiment using appropriate biological and technical replicates.

Phage Killing Efficacy Assay

1. Grow the bacterial host culture to the exponential growth phase.
2. Prepare the bacterial suspension at approximately (10³) CFU/mL in LB medium.
3. Prepare phage suspensions at the required concentrations to obtain the desired MOIs.
4. Add 180 µL of bacterial suspension to the appropriate wells of a sterile 96-well tissue culture plate.
5. Prepare three replicate wells for each experimental condition.
6. Include the following controls:
   • LB medium without bacteria as a negative control.
   • Bacterial culture without phage as a positive growth control.

7. Add 20 µL of phage suspension to the experimental wells to obtain the following final phage concentrations and MOIs:

   Phage concentration	MOI
   (107) PFU/mL	10,000
   (106) PFU/mL	1,000
   (105) PFU/mL	100
   (104) PFU/mL	10
   (103) PFU/mL	1
   (102) PFU/mL	0.1
   (101) PFU/mL	0.01

8. Mix the plate contents gently.
9. Incubate the plate at 37 °C with shaking at 180 rpm for 24 h.
10. Monitor bacterial growth using a multimode microplate reader.
11. Measure OD₆₀₀ every 5 min throughout the incubation period.
12. Plot OD₆₀₀ as a function of time for each phage concentration and control condition.
13. Compare the growth kinetics of phage-treated cultures with the bacteria-only control to evaluate the killing efficacy of the phage.