In situ fluorescence microscopy of bacteriophage aggregates

Abstract

SummaryVirus aggregation is analyzed because of its potential use for both classifying viruses and understanding virus ecology and evolution. Virus aggregation is, however, problematic because aggregation causes loss of virions during processing for microscopy of any type. Thus, here we detect virus aggregation by fluorescence microscopy of wet-mounted dissections of dilute gel-supported plaques (in situ fluorescence microscopy) of a test virus, the long-tail aggregating Bacillus thuringiensis bacteriophage, 0305φ8-36. Background fluorescence is reduced to nonproblematic levels by using the dye, DAPI (4 ,6-diamidino-2-phenylindole), to stain viral nucleic acid. In situ fluorescence microscopy reveals two in situ phases, one weakly fluorescent. Most bacteriophages partition to the weakly fluorescent phase. Aggregates of bacteriophage 0305φ8-36 are detected during fluorescence microscopy via the following. (1) Coordinated motion is found for visibly separate particles in solution; the motion is either thermally generated, fluid drift-induced or mechanical pressure-induced. (2) Aggregate dissociation is observed. Some of the larger aggregates are elastic and entangled with material of the weakly fluorescent phase. The larger aggregates sometimes sink at 1-g within specimens. In situ fluorescence microscopy rapidly distinguishes 0305φ8-36 from nonaggregating bacteriophages.

Aggregation/dispersion transitions of T4 phage triggered by environmental ion availability

“…The state of phage aggregation advances knowledge of the phage ecology, life cycle, in the context of virus classification as reported by others [10, 16, 18, 24]. We propose that low-cation triggered phage aggregation reflects a new mechanism in an evolutionary adaptation to environment [16]. …”

Phages Preying on Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis: Past, Present and Future

“…Plaques are usually clear, except for the border that seems to contain larger phage aggregates than the ones in the clear zone. Clear plaques can also contain roughly circular opaque spots that may resemble phage-resistant host colonies, but instead are aggregates of phage particles [191,195]. The formation of these aggregates seems not to require the presence of either host cells or nucleic acid [195].…”

Enhancing and initiating phage-based therapies

“…(2) Plaque size and border sharpness, coupled with response to changing the concentration of the plaque-supporting gel, provide a preliminary estimate of phage size. 29 These results are tested and refined, within hours, by in situ (in-plaque) fluorescence microscopy 30 and, within 2 days, by PFGE of expelled DNA obtained without isolating phage particles.…”

“…This is shown by the formation of clear, large plaques in overlays of dilute (0.05-2.0%) agarose. 29,30 Empirically, »0.02% agarose (below the minimum gelling concentration) is the lower limit for the agarose concentration that is needed for phage-induced clearing of bacterial turbidity in the upper layer of Petri plates (unpublished data). Hydrated polymer-sensitivity of this type potentially provides the following advantage: increase in propagation when a phage particle receives a signal of the presence of susceptible host cells sufficiently concentrated to expand the phage population.…”

“…A preliminary indication can also be obtained by visually observing plaques. 30 (4) Informatic analysis of DNA sequences (again, obtained without phage purification) and cryo-EM analysis, together with 3D reconstruction at high resolution, will generate new criteria for selecting phages for phage cocktail construction. (5) Testing effects of both polymers and other non-phage cocktail components optimizes the type and amount of these components, given the phages of the cocktail and the information in (1)-(4).…”