Tris–HCl, 0.01 M MgCl2, pH 7.4. A clarified, concentrated plate scattering during fluorescent lamp, white light illumination from lysate of infected cells was layered on at the top of the density the bottom. During centrifugation, the bacteriophage particles gradient, as illustrated in Figure 5 (left). Then, centrifugation was sedimented through the 35%/65% sucrose interface and pro- performed at 33,000 rpm, 5°C, for 60 min. After centrifugation, duced a band of light scattering when illuminated (Fig. 5). The the centrifuge tube was placed in a blackened aluminum holder gradient was then fractionated by pipeting from the top. with a hole at the bottom. A photograph was taken of the light Isolation and propagation of deletion mutants. To select for deletion mutants, a clarified, concentrated, wild type plate lysate was diluted by at least 1:200 into buffer that contained 0.1 M NaCl, 0.01 M Tris-Cl, 0.002 M EDTA. The temperature was raised to 52°C for 10 min. The result was loss of titer by a factor of 1 106. The surviving bacteriophages were propagated as single plaques. Bacteriophages from a single plaque were then subjected to the same inactivation; the loss of titer was deter- mined by dilution and traditional plating44 in a plaque-supporting 0.1% agarose overlay. If a plaque yielded bacteriophage particles significantly more temperature-resistant than the wild type bacteriophage, then two additional single-plaque purifications were performed and the elevated temperature-resistance of the more purified mutant was again tested. Two deletion mutants © 2012 Lande18s9,531), were isolated Bwhich and, wias characterized, oalso msore recsistanitby D(118,397–124,982), eafacntorof c~106 which ethan was . more resistant to inactivation by a factor of ~106 and D(181,412– the wild type bacteriophage. To compare D and I for a mutant to D and I for wild type bacteriophage, plaques were formed by dilution and traditional plating44 in a plaque-supporting 0.1% agarose overlay. Procedure, Do not diswere tfor rwilditbypebaucteriopthagee.Th.e plates (10 for each host preparation and media were the same for mutants as they bacteriophage) were incubated overnight, at room temperature (25 ± 2°C) in thermal contact with each other, so that the tem- perature did not vary by more than 0.2°C among the 30 plates. The values of plaque diameter were measured from digital photographs of the plates. The average (i.e., D) and standard deviation are reported after measurement of a total of 100 plaques for each bacteriophage. To determine I, 0.2 ml of each of 10 plaques was removed from the center of a plaque and diluted in growth medium for plating and plaque formation in a 0.1% agarose supporting gel. The average (i.e., I) and standard deviations are reported for the number of plaque-formers per ml of plaque pipeted. Extraction and restriction endonuclease analysis of bacterio- phage DNA. To extract bacteriophage 0305w8-36 DNA, we made clarified, concentrated plate lysates. We then digested host DNA and, next, purified bacteriophage DNA via phenol extrac- tion, by use of procedures previously described.8 These procedures do not include purification of the bacteriophage particles. To approximately map deletions, purified bacteriophage Figure 5. Revised purification of bacteriophage 0305w8-36. After DNA was digested with the following restriction endonucleases: fractionation of a lysate of 0305w8-36-infected cells in a sucrose density BamH1, BstN1, SpeI. DNA fragments in a digest were frac- tgurbadeiewnats, tphheoptoogstr-acpehnetdri.fuTghaetiloignhltigshctatstceartintegrifnrgomfrobmactthereiocpehnatrgifeuge tionated by electrophoresis through a horizontal, submerged particles is indicated at the left near the tube bottom by “phage.” 0.7% agarose gel (Seakem, LE agarose, Lonza). The electro- The pre-centrifugation composition of the tube contents is also phoresis buffer used was 0.09 M TRIS-acetate, pH 8.4, 0.001 M indicated at the left. The “65% shelf” is the top of the 65% sucrose layer; EDTA. The region of 0305w8-36 genome covered by each the “10–35% sucrose gradient” was poured above the shelf; the lysate restriction endonuclease-generated DNA fragment was deter- bwoatshlathyeermedenabisocuves athned 1th0e–3b5o%ttosmucroofstehgercaednietrniftu.gLieghtutbsecaatrteeriinndgicfarotemd. mined from the 0305w8-36 sequence and the fragment length. We approximately located the ends of a deletion by observing www.landesbioscience.com Bacteriophage 33