Phage plaque plates

Agar Plate Sizes and Number of Phage Plaques Plated"... 

Replica plating. A technique in which an impression of a culture is taken from a master plate and transferred to a fresh plate. The impression can be of bacterial clones or phage plaques. 

Mutagenicity (revertants/plate) Phage-inducing activity (plaques/plate)... 

With a ball point pen, draw a circle round each plaque to be picked on both sides of the filter. Mark them so that they can be identified, e.g., the phage from plate 2 could be called 2.1, and so on. 

Master plate of X phage plaques on E. coli lawn... 

Master plate of bacteria colonies (or phage plaques)... 

Fig. 3.4 Plaques formed by a phage on a plate seeded viith Bacillus subtilis. 

When a temperate phage is mixed with sensitive indicator bacteria and plated as described above, the reaction at each focus of infection is generally a combination of lytic and lysogenic responses. Some bacteria will be lysed and produce phage, others will survive as lysogenic cells, and the plaque becomes visible as a partial area of clearing in the bacterial lawn. It is possible to pick off cells from the central areas of these plaques and demonstrate that they carry prophage. 

Make serial dilutions of 10°, 10-3, 10-6, and 10"9 of the phage in SM buffer, and spot 5-pL aliquots of each dilution onto the solidified top agar. Tilt and rotate the plate to allow each spot to spread to a diameter of approx 10 mm, and allow the phage suspension to dry into the top agar. Incubate the plate overnight at 37°C. The approximate titer of the library is then be estimated from the number of plaques at each dilution. 

Infect Y1090 with the purified recombinant phage as described in the screening protocol, and plate at a density of about 50-100 PFU. Pick a single, well-isolated plaque with a Pasteur pipet, and remove the agar plug... 

Infect 200 pL E coli TG1 at OD = 02 with 10-pL serial dilutions of helper phage in order to obtain well-separated plaques. Add to 3 mL H-top agar (42°C), and pour onto warm TYE plates. Allow to set, and then incubate overnight. 

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