Medium data

Search for medium no. = [1232].
Unless otherwise stated, sterilize media by autoclaving at 121°C for 15 min.

1232   VIBRIO SEAWATER MEDIUM

Concentrated Vibrio suspension (see below)  10.0     ml
Seawater (autoclaved)  100.0     ml
Aseptically combine. Cultivate with shaking (150 - 200 rpm) for 16 - 24 hr. Separate from the prey bacteria physically (e.g., centrifugation), if necessary.
Concentrated Vibrio suspension:
Cultivate Vibrio alginolyticus JCM 32963 (or any Vibrio strain that can be served as prey bacteria) in 100 ml Marine Broth 2216 (Medium No. 41) with shaking at 150 - 200 rpm for 24 hr. Aseptically centrifuge the culture at 8,000 x g for 5 min. Remove the supernatant and suspend precipitated cells in 10 ml sterile seawater (pH 7.6 - 8.0). The cell suspension can be preserved at 4C for a couple of days.
Comment: For cultivation on solid media, the double-layer agar technique can be used. Typically, pour autoclaved seawater containing 1.5% agar (cool to 50 - 60C) to 9 cm petri dishes (15.0 ml per dish) and allow to solidify (bottom agar medium). Autoclave seawater containing 0.7% agar, cool to 60 - 65C and distribute 10 ml each of the molten agar solution into tubes kept at 50C (top agar medium). Then, add 1 ml of inoculum (bacterial cell suspension) and 1 ml of the concentrated Vibrio suspension to the top agar medium. Quickly mix the top agar medium and pour onto the bottom agar medium.