Jem 1200 ex 80 kv tem

TEx and TMv fractions were fixed in 2% paraformaldehyde (Serva) and allowed to adsorb onto formvar carbon-coated grids for 20 min. The grids were then washed in PBS (IIET), fixed in 1% glutaraldehyde (Sigma-Aldrich) for 5 min and washed with water (7 × 2 min). Then each grid was transferred to a drop of uranyl-oxalate (4% uranyl acetate and 0.15 M oxalic acid in 1:1 v:v ratio; Sigma-Aldrich) at pH 7 for 5 min. At this stage, samples were counterstained using two protocols: 1. with uranyl acetate or 2. with methylcellulose. 1: The grid was transferred to a drop of 2% uranyl acetate (Chemapol) for 5 min and washed with a drop of water 3 times. Then the grids were allowed to air dry for 10 min. 2: The grids were then embedded in 2% methylcellulose (Sigma-Aldrich) with uranyl acetate (9:1 v:v ratio) for 10 min on ice. The excess of methylcellulose was removed from grids by filter paper and grids were allowed to air dry for 20 min. Preparations were visualized using a JEOL JEM-1200 EX 80 kV TEM.

YeO:3 was grown for 24 h at 28ºC, after this time the bacteria were multiplied by transferring to fresh LB medium and incubated until the culture reached OD600 of 0.4–0.5. The culture was centrifuged (3000×g, 10 min), the pellet was washed with PBS and centrifuged again. The preparation was applied to a 200-mesh nickel grid with carbon film for 15 min. Next the excess liquid was sucked away, the H/MTFP-Gp17 protein at concentration of 1 mg/ml was applied to the grid for 30 min. Then the monoclonal anti-Maltose Binding Protein (MBP) antibody (produced in mouse, SIGMA) at a concentration of 1 mg/ml was applied to the grid for 30 min and in the next stage the anti-mouse IgG1 conjugated with HRP (produced in rabbit, SIGMA) at a concentration of 1 mg/ml was applied to the grid for 30 min. The grid was rinsed with PBS buffer supplemented with BSA and the Protein A-Gold with 20 nm colloidal gold was added for 30 min. After this time, the grid was washed with PBS and Milli Q water and contrasted with 2% uranyl acetate. Samples were visualized using a JEOL JEM-1200 EX 80kV TEM.

The purified bacteriophage was applied to the surface of formvar carbon-coated copper grids and negatively stained with 2% uranyl acetate for 1 min. The excess of uranyl acetate was then removed from the grids using filter paper and the grids were allowed to air dry for 20 min (Ackermann, 2009 (link)). Preparations were visualized using a JEOL JEM-1200 EX 80 kV TEM. The dimensions of the bacteriophages were determined using RADIUS EM Imaging Software.