Api 20ne kit

A loopful of all the previously inoculated broths was cultured on eosin methylene blue (EMB) agar (Oxoid, Oxoid Ltd., London) for isolation of Enterobacteriaceae other than salmonellae. Pseudomonas agar base with CN supplement (Oxoid) was used for the selective isolation of P. aeruginosa. All the inoculated plates were incubated aerobically at 37 °C for 24 h. Suspected colonies were picked and examined for morphological and culture characteristics according to Quinn et al. [20 ].
The API 20E and API 20NE kits (BioMerieux, France) were used according to the manufacturer’s instructions to detect the biochemical profiles of the isolated Enterobacteriaceae and P. aeruginosa, respectively.

To identify strain FP607^T, the physiological and biochemical characteristics were determined using Biolog GEN III MicroPlates™ (Biolog, Hayward, CA, USA). The utilization pattern was monitored on an OmniLog^®® Incubator/Reader (Biolog, Hayward, CA, USA). API 20NE kits (bioMérieux, Marcy l’Etoile, France) were used to determine the properties of strain FP607^T according to the manufacturer’s instructions. Polar lipids were detected using thin-layer chromatography (TLC). The different spots were primarily distinguished using chromogenic reagents (molybdenum phosphate, molybdenum blue, ninhydrin, D-reagent, and α-naphthol) [29 (link)]. Respiratory quinones were extracted and analyzed using high-performance liquid chromatography (HPLC; Shimadzu LC-20A) [30 (link)]. Cellular fatty acids were extracted and identified using gas chromatography (GC) (Agilent 7890 B) according to the protocol of the MIDI Sherlock Microbial Identification System and the RTSBA 6.1 database [31 (link)].

The bacterial strain was characterized on the basis of cell morphology including colony color, shape, size, and growth on certain media. To study the cell motility and shape, single colonies isolated from agar plates were prepared by using a JEM-1400, JEDL scanning electron microscope (SEM) and SU8010, Hitachi transmission electron microscope (TEM).
The ability to use common nitrogen and carbon sources, and resistance to common antibiotics, were tested as described by Gao et al. (1994 (link)). Additional enzyme activities and biochemical features were determined by using API ZYM, API 20NE kits and API 50 CH at 25 °C as recommended by the manufacturer (bioMérieux). The temperature range and salinity tolerance was investigated by incubating the bacterium on R2A agar at different temperatures (4, 10, 20, 25, 28, 30, 37 and 40 °C) and in medium containing 1, 2, 3, 4, 5, 6, 7, 8% (w/v) of NaCl. The pH range for strain 36-5-1^T was measured in R2A broth adjusted with HCl or NaOH to different pH values across the range from 4.0 to 13.0 at intervals of 1.0 pH unit. Dye and chemical resistance were investigated by using methyl orange, methyl red, methylene blue, neutral red, congo red, malachite green, bromothymol blue and sodium deoxycholate at concentrations of 1 and 2% (w/v) in R2A medium.