Bx3 25nd25

Four-day old biofilm of A. hydrophila dislodged from the substrates by vortexing for 5 min and mounted fresh on to the slide, was observed under the phase contrast microscope (Olympus, BX3-25ND25, Japan) and microphotographed.
Biofilm cells were characterized in FE-SEM according to the manufacturer's instruction. BF cells were separated from chitin after vortexing for 3 min, the supernatant, with the cluster of biofilm was centrifuged at 200 rpm for 5 min to remove chitin particles, biofilm in the supernatant was pelletized at 1000 rpm for 10 min and the pellet was resuspended in sterile PBS. A small quantity of prepared biofilm cells were dispersed on a SEM specimen mount. Enough argon was maintained in the chamber so that the vacuum reads 0.08 mbar. Sample was coated with gold (Eiko 1B-3 at 0.15 torr) as appropriate for a specified period of time and current to obtain an acceptable coating. Finally sample was transferred back to the specimen box after coating for analyzing at Carl Zeiss Sigma VP Field Emission Scanning Electron Microscope in the Central Laboratory of DST-PURSE PROGRAMME, Mangalore University.

For histological study, healthy fishes were euthanized and mid gut were separated, washed in normal saline and preserved in 10% neutral buffered formalin for 72 h for fixation. All the histological procedures were followed as detailed by Bullock [42 ]. The tissues on the glass slides were analysed through the light microscope (Olympus, BX3-25ND25, Japan) to observe the gut associated lymphoid tissue (GALT).

Immunofluorescence staining was performed following procedures that were previously detailed.^[18b,c] Pancreas were extracted and paraffin‐embedded before being sectioned (5 µm width) onto microscopic slides. For immunofluorescence staining, antigen retrieval was performed followed by exposure to primary antibodies at 4 ℃ overnight. The pancreas sections were then washed with PBS and staining with secondary antibody for 1–2 h at room temperature and examined under a fluorescence microscope (Olympus BX3‐25ND25, Japan).