In situ fluorescence microscopy of bacteriophage aggregates

Virus 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.