In a one-step growth curve experiment using a lytic bacteriophage against bacterial strain, we consistently observe a sudden and significant increase in phage titer after 60 minutes of incubation at different MOIs. This increase results in an uncountable number of plaques on agar plates and notably, the phage titer does not reach a plateau even after 100 minutes. We are trying to determine the potential reasons for this unusual growth pattern during One-Step Growth Curve Without Plateau Phase. The experimental protocol followed is outlined below:
1. Bacterial cultures are grown in the respective broth medium at 37 °C until mid-log phase.
2. 1 mL of bacterial culture is harvested by centrifugation at 10,000 × g for 5 minutes.
3. The supernatant is discarded, and the bacterial pellet is resuspended in 0.1 mL of phage lysate at a multiplicity of infection (MOI) of 0.1.
4. This mixture is incubated at 37 °C for 10 minutes to allow initial phage adsorption.
5. The mixture is centrifuged again at 10,000 × g for 5 minutes to collect the phage-bacteria complex.
6. The supernatant is discarded, and the pellet is resuspended in 10 mL of fresh broth medium.
7. The suspension is incubated at 37 °C with shaking to continue the infection cycle.
8. At 5-minute intervals, 100 µL samples are collected for up to 100 minutes, plated on a fresh bacterial lawn, and incubated overnight to quantify plaques.
How many 10-fold (or 100-fold) dilutions did you make? At some dilution you really should have a countable number of phages.
I wonder if perhaps you have phage contamination somewhere so that your plating cells are just getting contaminated? It might help if you showed some pictures.
Michael J. Benedik, Thank you for your response. We have performed a 10-fold dilution, and at concentrations up to 10^-6, we found countable phage plaques. In the case of a one-step growth curve of phage, plaque counts increased progressively at different MOIs up to 50–60 minutes, followed by an increase in uncountable plaques or complete lysis from 60 minutes onward (70–100 minutes), making it difficult for us to determine the burst size and latent period precisely. For your reference, I am sharing my data.
One-step growth curve experiments:
MOI 1
TIME (mins) PLAQUE COUNT
00 ---no plaque
10 ---no plaque
20 ---no plaque
30 ----------25
40 --- -------172
50--- --------241
60----------- Uncountable
70 to 100---- Uncountable (full clear/ lysed)
MOI 0.5
TIME (mins) PLAQUE COUNT
00 ---no plaque
10 --- no plaque
20 ------------2
30------------ 21
40 ------------107
50 ------------433
60 ------------ Uncountable
70 to 100 ------Uncountable (full clear/ lysed)
MOI 0.25
TIME (mins) PLAQUE COUNT
00 ---no plaque
10 ---no plaque
20 --------1
30 --------25
40-------- 65
50 ---------210
60 ---------Uncountable
70 to 100 --Uncountable (full clear/ lysed)
It is still not clear what you are doing. What dilutions are you using for each time point?
Michael J. Benedik, we have followed this protocol:
1. Bacterial cultures were grown in the respective broth medium at 37 °C until mid-log phase.
2. 1 mL of bacterial culture was harvested by centrifugation at 10,000 × g for 5 minutes.
3. The supernatant was discarded, and the bacterial pellet was resuspended in 0.1 mL of phage lysate at a 10^-6 dilution.
4. This mixture was incubated at 37 °C for 10 minutes to allow initial phage adsorption.
5. Then the mixture was centrifuged again at 10,000 × g for 5-10 minutes to collect the phage-bacteria complex.
6. The supernatant was discarded, and the pellet was resuspended in 10 mL of fresh broth medium.
7. The suspension was incubated at 37 °C with shaking to continue the infection cycle.
8. At every 10-minute interval, 100 µL samples were collected for up to 100 minutes, plated on a fresh bacterial lawn, and incubated overnight to quantify plaques.
we have followed this protocol, Please suggest if any changes need to be made.
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