The " One Step Growth Curve" From:
BACTERIOPHAGES
Biology and Applications
EDITED BY
Elizabeth Kutter
Alexander Sulakvelidze
Written by Karin Carlson
Department of Cell and Molecular Biology,
University of Uppsala, Uppsala, Sweden
Plaque size is primarily determined by the nature and size of the phage, thickness of bottom and top agar layers, concentration of agar in the top layer (and resultant diffusion rate), type of medium, plating cell density, distribution of adsorption times, and burst size.
A production of about 10–15 phages per infected cell is generally sufficient for plaque formation.
Each phage particle that gives rise to a plaque is called a plaque-forming unit (PFU).
The number of PFUs in a given volume of sample gives the viable phage concentration or titer.
The plaque-forming ability of phages may differ dramatically under various environmental conditions.
Furthermore,assaying the same phage preparation against various host strains often will yield different titers with each host. In some cases this reflects the host on which the phage was initially grown and is the result of restriction-modification systems; in others, it reflects the concentration and properties of the particular host receptor under those conditions or the status of the tail fibers.
Thus, when specifying the phage titer, it is important to determine optimum plating conditions and identify confounding factors and to indicate the host strain used for the plaque assay and other parameters (e.g., the buffer in which the phages were suspended, the incubation media, etc.) that may have an impact on the assay’s results.
The soft-agar overlay contains relatively few nutrients, and the bottom layer’s nutrients are the ones that primarily support growth of the indicator bacteria. Lower soft-agar concentrations give larger plaques.Several plates (duplicates or triplicates) are required to obtain reasonably accurate titers.
Killer titer
For a phage to form a visible plaque in a lawn of permissive bacteria, at least 10–15 progeny phages must be produced in each infected cell. Sometimes the host bacteria are “killed”, i.e. no longer able to grow, divide, and form colonies, upon infection even though not enough new phages are produced to form a plaque.
In such cases, the titer of killing particles (KP) can be determined by assaying the phages’ ability to kill the host.
An estimate of killing titer is also a useful test for the “quality”of an unknown phage suspension, since damage during storage is likely to reduce the viable titer (a measure of the ability to carry out productive infection) much more rapidly than the killing titer (largely a measure only of the ability to initiate infection.
MOI actual =the average number of phage particles that have adsorbed to and killed the host bacteria.
To determine killer titers, it is important to know the exact bacterial concentration at the time of phage infection so that a precise MOI can be used.
The best killing titer estimates are obtained using an MOI between 1 and 4, which necessitates assaying phage dilutions that differ by only a factor of 2 or 3.
If the phage titer is unknown, a pilot experiment with serial tenfold dilutions will indicate the range of dilutions needed for a second more precise experiment.
The steps involved in determining a phage’s bacterial killing titer
1. Ahead of time, determine how long a time phage and bacteria need to be incubated together to achieve maximal phage adsorption to, and killing of, bacteria (the “adsorption time”).
2. Prepare 3 serial twofold dilutions of the phage to get expected MOIs of about 1–4.
3. Grow an exponential culture of the bacterial host strain. Put 3 empty flasks into the same thermostated water bath.
4. Mix 0.05 ml of the exponential culture (“0-sample”) with 5 ml of room-temperature diluent; quickly dilute to a final concentration of 1000–3000 expected CFU/ml and plate 0.1 ml in triplicate.
5. Immediately distribute 1 ml of the bacterial culture into empty flasks in the water bath.
6. At 1-min intervals, add 0.1 ml phage dilution to one 1-ml aliquot of the bacterial culture and continue incubation under the bacterial growth conditions.
7. At time A, take an aliquot (50 ml) from each infected 1-ml culture into 5 ml diluent, make a series of tenfold dilutions and plate 0.1 ml of each dilution in duplicate immediately.
Evaluation of results
Count the bacterial colonies on all plates, and calculate the fraction of surviving host bacteria (P0) for each phage dilution used to infect the bacteria. Phages and bacteria are distributed in liquid according to the Poisson distribution .
As applied to phages that infect bacteria, k is the number of phage particles adsorbing to and killing a bacterium, m (the “actual”multiplicity of infection, MOI actual) is the average of k, and P is the fraction of bacteria infected by k phage particles.
Therefore, the fraction of surviving host bacteria is:
P0 =P(0,m)=e^−m
from which one obtains:
m=−lnP0
The killer titer of the phage is then calculated from m and the dilution of the phage used to infect the bacteria. For example, if 5% of the infected cells survived the infection, P0=0.05;m = −ln0.05=3.
If one obtains this fraction of survivors after infecting 1 ml of a bacterial culture containing 2 *10^8 CFU/ml, the 0.1 ml phage dilution used for infection contains 10*3*2 *10^8 killing particles (KP) per ml, that is, 6*10^9 KP/ml.
Phage Burst Size(Demo)
