Microbank freezer kit

Serial ten-fold dilutions of the larvae homogenate, at each sampling point, were plated on 90 mm Marine Agar plates (Difco Laboratories, Detroit, MI, USA). The remaining homogenate was stored at −80 °C for the microbiota characterization, as described above. The Petri dishes were thereafter incubated at room temperature (20–22 °C) for 10 days. Colonies that showed inhibition zones of growth were considered antagonistic and were sub-cultured to pure cultures using the streak plate method. These bacterial isolates were preserved at −80 °C in cryovials with ceramic beads (Microbank^TM Freezer kit, PRO-LAB Diagnostics, Richmond Hill, ON, Canada).
Similarly, samples were taken from live food organisms (Artemia sp. and rotifers) for microbiological analysis. The sampled live food cultures were washed with 50 mL filtered autoclaved seawater using a mesh with 50 μm pore size and thereafter homogenized in one mL of autoclaved seawater in a glass homogenizer. The homogenates were plated on Marine Agar and the same procedure was followed for the isolation of antagonistic bacteria as previously described for the fish larvae samples.

Samples of five larvae were taken 0, 7, 14, and 20 DAH and were anesthetized with 3-aminobenzoic acid ethyl ester 0.2 mg/mL (0.2% MS-222, Sigma). The larvae were washed once with 50 mL sterile seawater through a mesh with 250 μm pore size, were subsequently individually homogenized, and then plated after serial 10-fold dilutions on Marine Agar plates (Difco Laboratories, Detroit, MI, USA). The plates were incubated for 7 days at 20 °C in the dark and colonies were counted after one, two, and seven days. Frequently encountered, similar-in-appearance colonies were considered dominant and were selected from all sampling days from Marine Agar plates, pure-cultured, and thereafter stored by use of a MicrobankTM Freezer kit (PRO-LAB Diagnostics, Richmond Hill, Canada) at −80 °C.