Detection of Polyhedra from Insect Virus Diseases on Filter Membranes

Polyhedra filtered directly from the air or from aqueous suspensions by means of Millipore filter membranes, are stained on the membranes as follows: To a microscope slide mount consisting of a small piece of membrane filter on which the polyhedra are retained, are added 1 drop of a 1:4 dilution of saturated aqueous picric acid and 1 drop of staining solution: 0.1 gm of naphthol blue-black, C.I. 246 (Hartman-Leddon Co.) dissolved in a mixture of 98% methanol, 5; distilled water, 4 and glacial acetic acid, 1—parts by volume (Grosset et al. Proc. Soc. Exp. Biol. Med., 97, 72-7, 1958). The slide is placed on a hot plate at 600° C (dull red heat) until evaporation takes place and the filter membrane turns blue. Before the membrane begins to burn, the slide is removed and allowed to cool. For stain-sensitive polyhedra the above procedure is adequate. However, for stain-resistant polyhedra it is sometimes necessary to heat the mount with the picric acid alone, followed by the stain and a second heating. For highly resistant polyhedra it can be necessary to heat the untreated mount, follow with a second heating with double strength picric acid; and follow this with a third heating with stain. Revealing the polyhedra, stained dark lilac-blue or green blue, for bright-field illumination, is effected by clearing the membrane with media such as Euparal, aniline, linseed oil or clove oil. This method is suitable for the detection and observation of polyhedra dispersed in nature. Groups of different size can be separated by graduated pore-size filtration during concentration and purification. Enumeration and morphological studies are thereby facilitated.