Isolation and Identification of Aeromonas Bacteria

Ten grams of the samples were mixed with 90 mL sterile normal saline and flapped in BagMixer (Interscience, St. Nom, France) for 1 min. Then, the treated sample was continuously diluted and counted on Plate Count Agar (PCA, Aoboxing Bio-Tech, Beijing, China) and incubated at 28 °C for 48 h. After 48 h, all obviously distinct bacterial colonies were appraised and each colony was streaked onto Aeromonas Selective Medium Base (Ryan) followed by incubation at 28 °C for 24 h. The color, outline, size and other optical properties of the colonies were observed and recorded. The VITEK-2 Compact system (BioMerieux, Marcy l’Etoile, France) was used to identify the single colony [23 ]. Further study on the colony was validated with 16S rRNA PCR using the forward primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and the reverse primer 1492R (5′-ACGGCTACCTTGTTACGACTT-3′) amplify the 16S rRNA gene [24 (link)]. The gene sequences were analyzed by comparing the closely related sequences by BLASTN program in the GenBank database and phylogenetic analyses using MEGA 5.0 software.