Imvic test

Approximately 0.01 ml of milk from each sample was cultured on 5% sheep Blood Agar Plates and MacConkey Agar Plates (Oxoid, UK) [19 ]. The agar plates were initially incubated at 37°C for 18-24 h. If no growth appeared after 24 h, incubation was extended for another 24 h before final judgment was taken. From growth-positive plates, isolates were sub-cultured on nutrient agar (HiMedia, India) plates at 37°C for 24 h to obtain pure cultures. Microorganisms were identified based on colony morphology, hemolysis characteristics on blood agar, and Gram-staining [19 ]. Gram-positive bacteria were then confirmed based on the results of the catalase test, oxidase test, DNase test, growth on mannitol salt agar, and Microbact 12S (Thermo Fisher Scientific, USA). Gram-negative bacteria were confirmed based on lactose fermentation on MacConkey Agar, Kligler Iron Agar, catalase test, oxidase test, and IMViC test (HiMedia Laboratories, India).

Genomic DNA was extracted and purified using PureDirex genomic DNA isolation kit (Bio-helix, Taiwan) and was used for PCR amplification. 16S rRNA gene was amplified by KOD One™ PCR master mix (Toyobo, Japan) using the forward primer 27F (5′-AGA GTT TGA TCM TGG CTC AG-3′) and the reverse primer 1492R (5′-TAC GGY TAC TTG TTA CGA CTT-3′). The PCR reaction was performed in a thermal cycler (Analytikjena, Germany). The amplified PCR products were purified according to the manufacturer’s protocol (Bio-helix, Taiwan). 16S DNA sequencing was conducted by ATCG Co., Ltd. (Pathumthani, Thailand). The nucleotide sequence was aligned with the related sequences in NCBI database. Phylogenetic tree was constructed using Molecular Evolutionary Genetics Analysis (MEGA, version 7.0.26) software with the Neighbor-joining method.
The genomic DNA of the CB15 strain was used as a template for PCR amplification of PHA synthase (phaC) with forward primer G-D (5′-GTG CCG CCS YRS ATC AAC AAG T-3′) and reverse primer G2-R (5′-GTA GTT CCA SAY CAG GTC GTT-3′) [20 (link)].
IMViC-test (Himedia, India), citrate test, and catalase test were performed. The ability to grow on various sugars such as glucose, xylose, arabinose, sucrose, and fructose were conducted by inoculating the bacterium into Phenol Red Broth Base (PRBB) medium (Himedia, India) containing 1% (w/v) of each sugar.