Jem 100c

Ten microliters of the CsCl-purified phage suspension (10⁹ PFU/mL) were applied to 400-mesh carbon-coated copper grids, negatively stained with 1% uranyl acetate and analyzed using a JEM-100C (JEOL, Akishima, Japan) transmission electron microscope at 80 kV accelerating voltage. Images were taken on Kodak film SO-163 (Kodak, Cat. \# 74144, Hatfield, PA, USA). Phage particle dimensions were measured using ImageJ version 1.53e [https://imagej.nih.gov/ij/index.html, accessed on 1 June 2021] in relation to the scale bar generated from the microscope [14 ].

Salivary gland polytene chromosome squashes were prepared for electron microscopy analysis and examined as described earlier [31] , [70] (link). The 120–150 nm sections were cut using an LKB-IV (Sweden) ultratome and examined under a JEM-100C (JEOL, Japan) electron microscope at 80 kV.

Cell morphology was examined using an Axio Imager D1 light microscope (Zeiss, Oberkochen, Germany) equipped with a phase-contrast unit. Electron micrographs were obtained by using a JEOL model JEM-100C transmission electron microscope and negative staining with 2% (w/v) sodium phosphotungstate. Gram reaction was determined using the Gram-staining kit (Deltalab, Barcelona, Spain).

Following pentobarbital injection (100 mg/kg), animals to have EM examination of their brains underwent transcardiac perfusion with heparinized 0.9% NaCl, followed by 500 mL of 4% paraphormaldehyde and 2.5% glutaraldehyde in 0. 1 M phosphate buffer (PB), pH 7.4, at a perfusion pressure 120 mm Hg. The brains were removed from skull and placed in the same fixative overnight. The right hemispheric tissue blocks containing the inferior colliculi (IC) and the medial geniculate body (MGB)were cut into 400 micron-thick coronal slices. Slices were washed in cold 0.1 M PB and kept in 2.5% glutaraldehyde in 0.1 M PB until processing; when processing, the slices were washed in cold PB, post-fixed in 1% osmium tetroxide in cold PB for 2 h and again washed in 0.1 M PB. The IC and GMB were identified with an optical microscope Leica MM AF, cut out from the coronal slices, dehydrated in graded series of ethanol and acetone, and embedded in araldite. Blocks were trimmed and 70–75 nm thick sections were cut with an ultra-microtome Reichert, picked up on 200-mesh copper grids, double-stained with uranyl-acetate and lead-citrate, and examined with a JEM 100 C (JEOL, Japan), HF 3300 (Hitachi, Japan) and Tesla BS 500, Czechoslovakia) transmission electron microscopes. For each case, 120 sections were observed.

VLP-containing fractions from the column or VLP preparations in PBS were subjected to electron microscopy (EM) analysis immediately or after storage at +4 °C for 1–2 days. Samples were adsorbed to carbon-formvar-coated copper grids and negatively stained with a 1% aqueous solution of uranyl acetate. The grids were examined at 100 kV using a JEM-1230 electron microscope or a JEM-100C electron microscope at 80 kV (both from Jeol Ltd., Tokyo, Japan). Dynamic light scattering (DLS) was used to determine the size distribution profiles of the particles and performed on a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Malvern, UK). The DLS results were analyzed using the DTS software (Malvern, version 6.32). The ImageJ program was used for precise measurements of the HBc particle sizes.

For TEM observations, ookinetes were pelleted at 720xg and fixed in 2% glutataraldehyde/2% paraformaldehyde in 0.1 M sodium cacodylate buffer pH 7.4 for 45 min at 4°C, post fixed in 1% osmium tetroxide, dehydrated in graded ethanol, stained with uranyl acetate 1% and finally embedded in graded Durcupan:propylene oxide series (1:3, 1:1, 3:1, 4:0). Ultrathin-sections (50–100nm) were taken on a Leica LKB2088 ultramicrotome and examined under JEM 100C/JEOL/Japan Transmission Electron Microscope. Microphotographs were obtained with an ES500W Erlangshen camera and analysed by the DigitalMicrograph software (Gatan, Germany).