Tcbs agar

Gut samples were streaked onto thiosulphate-citrate-bile-salts-sucrose (TCBS) agar (Oxoid, Hampshire, UK) and were incubated at 30 °C for 24 h. Following incubation, Vibrio isolates, from their colonies that appeared rounded and medium-size yellow or green, were identified using morphological characterization and biochemical tests (API 20E, BioMerieux UK Ltd., Basingstoke, UK). The rate of bacterial isolation was calculated based on the percentage (%) of the number of vaccinated and non-vaccinated fish that were infected with Vibrio sp.

All samples were treated by adding 25 ml of the seawater sample into 225 ml enrichment medium of alkaline peptone water broth (APW) supplemented with 3% NaCl and incubated at 37 °C for 24 h. On the second day, a loop full of each enriched sample was streaked on Thiosulfate citrate bile salts sucrose (TCBS) agar (Oxoid, Basingstoke, UK) and CHROM agar (CHROM, France) and incubated at 37 °C for 24 h.

Intestine samples were collected from the group of shrimps fed with MOS and used as sources for isolation of mannanase-producing bacteria. Each shrimp intestine was chopped into fine pieces and added to 200 μL of 1X phosphate buffer pH 7.4. Each sample was homogenized using sterilized micro pestles. Homogenate sample (60 μL) was inoculated into 3 mL of M9 minimal media (3 mM Na₂HPO₄, 2 mM KH₂PO₄ 1 mM NaCl, 1 mM NH₄Cl, 2 mM MgSO₄, 0.1 mM CaCl₂) with 1% MOS (copra meal hydrolysate) as a sole carbon source. The bacteria were grown under aerobic conditions by shaking at 250 rpm for 16–18 h at 30 °C.
For primary screening, M9 culture broth was serially diluted and spread on M9 agar containing 1% MOS and incubated at 30 °C for 24 h. The colonies were further screened on TCBS agar (Oxoid, UK). Our bacterial candidates were those that were able to grow on M9 containing 1% MOS as the sole carbon source but not on TCBS to avoid potential pathogenic Vibrio. The selected colonies were grown in LB containing 1% locust bean gum (LBG, Sigma-Aldrich, St. louis, MO, Sigma) at 30 °C for 24 h. Colonies with a clear zone indicating mannanase activity were confirmed by the 0.5% Congo red staining method [38 (link),39 (link)] and bacterial candidates were stored in 20% glycerol at −80 °C.

Bacterial strains used in this study are shown in Table 3. V. parahaemolyticus strains were initially cultured aerobically onto selective media Thiosulphate Citrate Bile Sucrose (TCBS) agar (Oxoid) at 37°C for 24 h. For enumeration of colony counts, routine sub culturing and growth on Marine Agar (Conda Labs, Spain) was used and incubated at 30°C for 24 h.

Bacterial strains used in this study are shown in Table 1. V. parahaemolyticus strains were initially cultured aerobically onto selective media Thiosulphate Citrate Bile Sucrose (TCBS) agar (Oxoid) at 37°C for 24 h to check for contamination. For enumeration of colony counts we used Marine Agar (Conda lab, India) at 30°C for 24 h and for routine subculturing and growth Luria-Bertani (LB) agar at 37°C for 18 h was used. For growth curves, wildtype T024 and T024:ΔmutT were grown in minimal media with aeration at 30°C and optical readings and colony counts were carried out on Marine Agar at 0, 2, 4, 6 and 8 h. For virulence assays the V. parahaemolyticus strains were grown in Marine Broth (Conda Lab, India) at 37°C.