Hajna tetrathionate broth

A total of 235 local chicken products (liver, breast, thigh, neck skin, and minced meat) were collected from 25 retail stores and 6 chicken processing plants between January 2018 and October 2021. The products were transported under refrigeration to the National Institute of Health Sciences. Each sample was tested within 24 h after arrival at the laboratory. Twenty-five grams of the sample was mixed in 225 mL of buffered peptone water (Oxoid Ltd., Hampshire, UK) and incubated at 37 °C for 18 h for pre-enrichment. After incubation, 0.1 and 1 mL of the culture was added to 10 mL of Rappaport–Vassiliadis broth (Oxoid) and 10 mL of Hajna tetrathionate broth (Eiken Chemical), respectively, then incubated at 42 °C for 20 h. After incubation, each culture was streaked onto two selective isolation agar plates: xylose-lysine-deoxycholate agar (Oxoid) and CHROMagar^TM Salmonella (CHROMagar, Paris, France) then incubated at 37 °C for 24 h. For the selective isolation from minced meat, Mannitol-Lysine-Crystal-Violet-Brilliant-Green agar (Nissui Pharmaceutical, Tokyo, Japan) was used in place of xylose-lysine-deoxycholate agar. Putative Salmonella colonies were biochemically identified as mentioned above. One strain per sample was suspended in 20% glycerol and stored at −80 °C until ready for serotyping and antimicrobial susceptibility testing.

Samples were collected using sterile techniques, placed in sterile plastic sampling bags, and chilled with ice blocks during transport. Samples were delivered to the Laboratory of Veterinary Public Health, Kagoshima University, and cultured on the day of arrival. Approximately 1 g of cecal contents was aseptically mixed with 5 mL of sterilized distilled water and homogenized by vortexing. Then, 1 mL of the suspension was pre-enriched in 5 mL of Hajna tetrathionate broth (Eiken Chemical Co., Ltd., Tokyo, Japan) and incubated in a water-bath at 42 °C. After 24 h incubation, a loopful of the culture was streaked onto a selective Rambach agar plate, which was incubated at 37 °C for 24 h [16 (link)].
Suspected pink colonies were selected from each plate and streaked on nutrient agar slants. Salmonella identification was confirmed by biochemical tests, including fermentation of glucose, lactose and sucrose, hydrogen sulfide production, citrate utilization, lysine decarboxylation, methyl red and indole tests. Serotyping of isolated Salmonella strains was performed with reliable commercial antisera (Difco, Detroit, MI, USA), and the results were interpreted according to the Kaufmann-White scheme [26 ].