Virginia Tech/phage lysate

ATCC λ Propagation

The wild type (WT) lambda phage was purchased from the American Type Culture Collection (ATCC) in the form of a freeze dried lysate. The phage was labeled as ATCC 23724-B2. A lambda negative strain of E. coli was purchased as well (C600 or ATCC 23724).

In order to propagate this phage, an overnight culture of C600 was prepared. The overnight culture was used to produce plates with top agar bacterial lawns and liquid cultures. It was soon discovered that it was more difficult to work with liquid cultures than bacterial lawns when the titer of the phage is unknown. Phage plaques were used to produce high titer lysates in the realm of 10^9-10^11 plaque forming units per milliliter (pfu/mL).

When working with phage in liquid cultures, it was discovered that the amount of phage added and the density of the cell culture were very important. As the multiplicity of infection (MOI) increases, so does the likelihood of lysogeny. A lower MOI typically drives the phage to the lytic pathway. Phage propagation is also more likely to occur, through lysis or lysogeny, if the cell culture is less dense. These findings were not only observed through the lab work, but they are also published in a number of different sources. This would be very helpful when testing the infection of a single population model.

λ-EYFP Propagation

The fluorescent phage provided by Phillippe Thomen was a different experience. It was provided to the team as a lysogen in NM538. The Alvarez paper, "Propagation of fluorescent viruses in growing plaques," describes the phage as having a thermosensative mutation in the gene that regulate cI. The culture was grown to an OD600 of 0.1 at 30oC and then subjected to heat shock at 42oC for 15 minutes. This effectively destabilized the cI protein, and drove the lysogens to lysis. Within 45 minutes at 37oC and good aeration, the phages had lysed the bacteria and cleared the culture (a sign that lysis did in fact occur). The phage was recovered by adding 0.1 mL chloroform to the cultures, spinning down the cell debris, and collecting the supernatant.

Serial dilutions of the lysates were used to produce plaques of phage on top agar bacterial lawns. These plaques were used to generate high titer lysates of λ-EYFP in the order of 10^9-10^11 pfu/mL. The lysates would be important for experiments determining bacteria-phage interactions as well as infections of populations of bacteria.