pre-dialyzed versions of the same specimens. The procedures are resolution limit of the fluorescence microscope) in some pro- described in the Materials and Methods Section. A presumed jections. These particles were undergoing tumbling and trans- dimer was revealed via two separate, resolution-limited bright lational motion in the specimen. A series of video images, framed regions that, during motion, were tethered together and separated to include the same particle (0.5 sec between frames), is in by about two tail lengths (about 1,000 nm or about 4 the Figure 3D. The two bright regions are seen separately from each other in frames indicated with an arrow; rotation brings these regions into coincidence in the other frames. The dimer was translating throughout this video series. In the second frame from the left, the leftmost bright region is from a second particle that drifted into apparent contact with the dimer; the framed particle is distinct in the other images. By this criterion, ~10% of the particles observed were dimers. If, in analogy with the behavior of other myoviruses,35 0305w8-36 attaches to a host cell via its tail, then dimerization will inhibit the infection of host cells. Bacteriophage adsorption to a host cell. The tail of bacterio- phage 0305w8-36 is long enough so that we determined the orientation of host-attached bacteriophage particles by in situ (in-plaque) fluorescence microscopy of DAPI-stained (Materials © 2012 Landetiosnwill and Methods be Breveailed Section) obya samples DsAPI-cstaineid, of 0305w8-36 eresolutnion-limcited plaques. If espot the . bacteriophage adsorbs tail tip-to-cell surface, then this orienta- (packaged DNA) at a distance from the cell surface that is maintained during translational and tumbling motion. Given the 486 nm rigid tail and the potentially flexible, wavy tail fibers of 0305w8-36, this distance depends on the extent to which the Do not disinanytcasre.ibute. tail fibers are extended, but would be between 486 and ~700 nm Figure 4 shows a single host cell with at least eight bacterio- phages attached, the latter as revealed by resolution-limited spots derived from DNA-bound dye fluorescence. One spot is at the upper right and five distinct spots are at the lower left of the bacterial cell. Two of these latter spots are produced by fluo- rescence from two bacteriophages. The additional bacteriophages (1) were obscured by superposition in the frame in Figure 4, Figure 3. Electron microscopy of bacteriophage 0305w8-36 after improved fractionation. After fractionation of reduced aggregation bacteriophage particles in a sucrose step gradient, fluorescence microscopy was performed with particles from the phage band-region of the gradient (undialyzed). The sucrose was then removed by dialysis and the bacteriophage particles were prepared for electron microscopy (EM) by negative staining, by use of procedures described in the Materials and Methods Section. (A) Low magnification EM image of a field of several bacteriophages. (B and C) Higher magnification EM images of bacterio- phage dimers. (D) Captured video frames from fluorescence microscopy of DAPI-stained particles; the frames are separated by 0.5 sec. Legend for (B and C): S, segment of tail sheath; T, exposed segment of tail tube; M, probable fragment of the host; F, tail fiber.” Figure 4. In situ fluorescence microscopy of host-bacteriophage attachment. A portion of the clear region of a 0.1% agarose-supported plaque was pipeted, mixed with dye and examined by fluorescence microscopy, as described in the Materials and Methods Section. The bar is 1,000 nm (1 mm) long. 30 Bacteriophage Volume 2 Issue 1