λ Experiments on spread plate culture of phages
Phages originate from the infection of a single bacterium by a single phage particle. The daughter virus particles synthesized after the first infection adsorb and infect neighboring bacteria, which sequentially release another generation of daughter virus particles. The spread of such daughter virus particles is limited if the bacteria are grown on semi-solid media (e.g., containing agarose or agar). The source of this experiment is "Guide to Molecular Cloning Experiments, Third Edition" translated by Huang Peitang et al.
Operation method
Experiments on spread plate culture of λ phage
Principle
Phages originate from the infection of a single bacterium by a single phage particle. The daughter virus particles synthesized after the first infection adsorb and infect neighboring bacteria, which sequentially release another generation of daughter virus particles. The spread of such daughter virus particles is limited if the bacteria are grown on semi-solid media (e.g., containing agarose or agar).
Materials and Instruments
λ Phage Proto-Species E. coli Strain Move I. Materials For more product details, please visit Aladdin Scientific website.
MgSO4 SM and SM+ gelatin
LB or NZCYM Agar Plates Sorvall SS-34 or equivalent Circular Filter Paper Water Baths
1. Buffers and solutions
MgSO4 (10 mmol/L), SM and SM+ gelatin
2. culture medium
LB or NZCYM (50 ml in 250 ml conical flasks)
LB or NZCYM agar plates 
LB or NZCYM top agar or agarose (0.7%)
3. Centrifuge and rotor
Sorvall SS-34 or equivalent.
4. Specialized equipment
Round filter paper (for petri dishes)
Heating equipment or water bath pre-set at 47°C
5. Carriers and strains
λ Phage progeny
Escherichia coli strain 
II. METHODS
Preparation of plate spreading bacteria
1. Inoculate single colonies of suitable E. coli strains in NZCYM or LB medium (50 ml in 250 ml conical flasks) and incubate overnight at 37℃ with moderate shaking.
Some protocols recommend incubation at 30°C rather than 37°C. Growing at low temperatures increases the chance that overnight cultured cells will not reach saturation, which reduces the amount of cellular debris in the medium. As mentioned earlier, phage added to saturated cultures will adsorb to LamB proteins in the cellular debris, thus reducing the titer of the phage culture.
2. Centrifuge at room temperature, 4000 g (or 5800 r/min in Sorvall SS-34 rotor) for 10 min and collect the cells.
3. Remove supernatant and resuspend precipitate with 20 ml of 10 mmol/L MgSO4. Measure the OD600 value after 100-fold dilution and further dilute the cell suspension with 10 mmol/L MgSO4 to a final concentration of OD600 equal to 2.0.
In order to improve the survival rate of plate spreading bacteria during storage, the cell suspension was incubated in a 100 ml sterile flask at 37℃ for 1 h with moderate shaking.
4. The bacterial suspension of plate culture was stored at 4℃.
A robust E. coli culture can still be used after 2~3 weeks. However, severely weakened E. coli (e.g., recA- strain) will rapidly lose viability at 4℃ under starvation conditions. Fresh cultures are required when using such strains for λ phage culture.
5. Microwave for a short time to melt the upper layer of agar or agarose. Dispense the melted agar or agarose (3 ml for 100 mm dishes, 7 ml for 200 mm dishes) and place in a heated container or in a 47°C water bath in order to keep the solution in a molten state.
Infection of plate-laying bacteria
6. Make a 10-fold serial dilution of the original phage (in SM+ gelatin) and mix the dilutions thoroughly by slight vortexing or by tapping the side of the tube.
7. Take 0.1 ml of bacteria from the plate from step 4 into a series of sterile test tubes (13 or 17 X 100 mm).
8. Add 0.1 ml of each dilution of phage to each tube containing plate-laying bacteria and mix the bacteria and phage by shaking or slight vortexing.
9. Incubate the mixture at 37℃ for 20 min to allow phage particles to adsorb onto the bacteria. Remove the tubes from the water bath and allow them to cool to room temperature.
10. Add the melted agar or agarose from one of the dispenser tubes to the first test tube, mix by tapping or vortexing for 5 s. Immediately pour the entire contents of the tube into the center of a labeled agar plate. Try to avoid air bubbles. Gently rotate the plate to evenly distribute the bacteria and the upper agarose layer. Repeat this operation until all the contents of the tubes have been transferred to the separately labeled plates.
11. Cover the plate with a lid and leave it at room temperature for 5 min to allow the upper layer of agar or agarose to solidify, and finally invert the plate for incubation at 37 °C.
For some E. coli and phage vectors, plates incubated at room temperature rather than 37°C will form better phage spots. For example, when using Stretagene's SRBρ and SRB(P2)ρ as host bacteria, 39°C is the recommended incubation temperature. In addition, when λgt10 is incubated at 39°C, it will produce better phage plaques in E. coli hfl- strains .
12. Continue the incubation overnight and count or screen (pick) the individual phage spots.
Phage plaques appear after about 7 h of incubation and are counted or picked after 12 to 16 h. At this point, phage plaques in robust E. coli hfl- strains will not be counted. At this point, the diameter of the phage plaques formed in robust E. coli is about 2-3 mm. if the plate is too dry, the phage plaques will grow slowly and will not reach their maximum diameter. Freshly prepared plates have a different problem. During incubation at 37°C, water vapor can form droplets on the top layer of agar, which can contaminate the growing phage plaques with each other due to the movement of the droplets. To avoid this problem, leave the plates at room temperature for 2 d before use, or leave them half open in a laminar flow cabinet or incubator (less effective) at 37°C for 2 h. If the plates are to be used urgently and there is no time to dry the plates, wait for the top layer of agar to solidify before removing the beads of water from the lids of the flat dishes, and add a round piece of sterile filter paper to each lid. During inversion incubation at 37°C, the filter paper will absorb water vapor and minimize cross-contamination of phage spots.
With experience, phage spots can be adjusted to the appropriate size. For example, when screening libraries by hybridization, it is often desirable to limit the size of the phage plaque in order to screen the maximum number of phages per plate. From this point of view, plates that are older should be used and slightly more bacteria than usual should be added as phage moss. Under other conditions, e.g. when using debilitated red- gam- λ phages, it is necessary to reduce the amount of bacteria inoculated and/or use a low concentration (0.6 %) of top agar or agarose to obtain appropriately sized phage mats. When the bacterial moss is full grown and the cells have reached a stable phase, the λ-phage plaque will no longer increase in size. 
