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Fiche Technique DỮ LIỆU KỸ THUẬT XLT4 AGAR PHÁT HIỆN SALMONELLA 1 MỤC ĐÍCH XLT4 (Xylose-Lysine-Tergitol 4) Agar là một môi trường phân lập chọn lọc để phát hiện Salmonella, ngoại trừ Salmonella Typhi và Paratyphi XLT4 Agar có thể được sử dụng như môi trường thứ hai được lựa chọn trong các phương pháp phát hiện Salmonella thông thường trong vi sinh thực phẩm. Nó cũng được sử dụng làm môi trường phát hiện trong lĩnh vực thú y để phát hiện Salmonella. Thành phần điển hình tương đương với thành phần trong các tiêu chuẩn NF U47-102. 2 LỊCH SỬ Năm 1991, Miller và Tate đã chứng minh rằng việc sử dụng các XLT4 Agar tăng tần suất phát hiện Salmonella non-Typhi trong mẫu gia cầm có chứa một lượng lớn vi sinh vật thứ cấp, và môi trường này cho phép phân biết tốt giữa Salmonella và Citrobacter. Các nhà nghiên cứu tạo ra môi trường bằng cách kết hợp Tergitol 4 trong môi trường Xylose-Lysine cải tiến để ức chế phạm vi rộng của hệ thực vật cạnh tranh (Proteus, Pseudomonas, Providencia) mà trước đó đã cản trở việc phát hiện Salmonella. Những nghiên cứu tiếp theo do Dusch và Altwegg thực hiện đã khẳng định rằng XLT4 Agar có thể được sử dụng để phát hiện Salmonella trong các mẫu lâm sàng, ngoại trừ Salmonella Typhi và Salmonella Paratyphi. 3 NGUYÊN TẮC Xylose được lên men bởi các vi khuẩn gây bệnh đường ruột (ngoại trừ Shigella), do đó được phân biệt với các vi khuẩn khác. Sau khi đã cạn kiệt các xylose, Salmonella carboxylate lysine tạo thành cadaverine, làm tăng pH. Trong môi trường kiềm, Salmonella hình thành các khuẩn lạc màu đỏ khi có mặt chất chỉ thị pH, màu đỏ phenol. Các khuẩn lạc màu đen do sự xuất hiện của các sunfua sắt ở trung tâm khuẩn lạc được hình thành thông qua việc khử ferric ammonium citrate bởi tác nhân sinh hydrogen sulfide gây bệnh. Môi trường có chứa hai loại đường bổ sung là lactose và sucrose. Quá trình lên men của một hoặc cả hai loại đường dẫn đến axit hóa môi trường và dẫn đến sự hình thành các khuẩn lạc màu vàng khi có mặt chất chỉ thị phenol red. Chủng không gây bệnh mà không có decarboxylase lysine tạo axit hóa từ quá trình lên men đường. Sự giảm pH dẫn đến ngăn cản sự tạo màu đen của các khuẩn lạc. Tergitol 4 là tên thương mại của dung dịch tẩy rửa anionic 26-28%, 7-ethyl 2-methyl 4-undecyl sulfate là dạng muối natri. Nó ức chế vi khuẩn Gram dương và nhiều chủng vi khuẩn Gram âm, đặc biệt là Proteus. 23/30 Đường C1, phường 12, quận Tân Bình Thành phố Hồ Chí Minh Điện thoại: (+84.8) 6292.3745 - 6292.3731 - 6292.3773 Page 1 of 5 Email: [email protected] - Website: www.dongnamlab.com 4 THÀNH PHẦN Có thể được điều chỉnh để có được hiệu suất tối ưu Trong 1 lit môi trường: - Peptone....................................................................................................................... 1,6 g - Yeast extract ............................................................................................................... 3,0 g - L-Lysine....................................................................................................................... 5,0 g - Lactose........................................................................................................................ 7,5 g - Sucrose ....................................................................................................................... 7,5 g - Xylose........................................................................................................................ 3,75 g - Sodium chloride ......................................................................................................... 5,0 g - Sodium thiosulfate....................................................................................................... 6,8 g - Ferric ammonium citrate ............................................................................................. 0,8 g - Red phenol............................................................................................................. 80,0 mg - Tergitol 4 .................................................................................................................. 4,6 mL - Bacteriological agar................................................................................................... 18,0 g pH của môi trường hoàn chỉnh ở 250C: 7,4 ±0,2 5 CHUẨN BỊ • Cân 59,0 g môi trường bột khô (BK156) trong 1 lit nước cất vô trùng hoặc nước khử ion • Thêm 4,6 ml Tergitol 4 Selective Supplement (BS039). • Đun từ từ cho đến khi sôi, khuấy cho đến khi môi trường tan hoàn toàn. • Không để quá nhiệt, không hấp tiệt trùng môi trường • Duy trì môi trường ở trạng thái lỏng ở 44-47oC. • Đổ vào đĩa petri vô trùng và để đông lại trên bề mặt phẳng, mát. 6 HƯỚNG DẪN SỬ DỤNG • Làm khô đĩa trong tủ ấm bằng cách mở hé nắp. • Trên bề mặt đĩa đã chuẩn bị như trên hoặc các đĩa chuẩn bị sẵn (BM036) đưa về nhiệt độ phòng, lấy vòng khuẩn từ môi trường tăng sinh để phát hiện Salmonella, cấy ria trên bề mặt môi trường • Ủ ở 37 ± 2°C trong 24 ± 3 giờ. • Lưu ý: Trong lĩnh vực thú y, cần ủ trong 48 giờ để phát hiện Salmonella Abortusovis 7 KẾT QUẢ Khuẩn lạc Salmonella điển hình (dương tính H2S) có màu đỏ với một tâm màu đen. Chúng có thể xuất hiện một quầng sáng màu vàng sau 24 giờ ủ. Trong trường hợp ủ kéo dài, các khuẩn trở thành màu đỏ đến màu hồng với một tâm màu đen hoặc đen hoàn toàn. Salmonella âm tính với H2S có màu đỏ đến màu hồng, không có tâm màu đen. Citrobacter, Klebsiella và Enterobacter cloacae tạo khuẩn lạc vàng. Sự phát triển của Enterobacter aerogenes và Escherichia coli thì bị ức chế một phần, khuẩn lạc hiện trên môi trường có màu vàng. Proteus, Pseudomonas và Providencia bị ức chế một phần đến hoàn toàn. Shigella phát triển chậm và có khuẩn lạc hồng. 23/30 Đường C1, phường 12, quận Tân Bình Thành phố Hồ Chí Minh Điện thoại: (+84.8) 6292.3745 - 6292.3731 - 6292.3773 Page 2 of 5 Email: [email protected] - Website: www.dongnamlab.com Xem phụ lục 1: Hình ảnh minh họa 8 KIỂM SOÁT CHẤT LƯỢNG Môi trường dạng bột: bột màu hồng nhạt, chảy tự do và đồng nhất. Tergitol 4 Supplement: Dung dịch hơi vàng, trong Môi trường hoàn chỉnh đã được chuẩn bị sẵn: thạch màu đỏ cam Phản ứng nuôi cấy đặc trưng sau 24 giờ ủ ở 37 °C Tăng trưởng Vi sinh vật Đặc trưng (Tỉ suất: PR) Salmonella Typhimurium WDCM 00031 Tốt, score 2 khuẩn lạc đỏ với tâm đen Salmonella Enteritidis WDCM 00030 Tốt, score 2 khuẩn lạc đỏ với tâm đen Escherichia coli WDCM 00013 Yếu, score 0-1 khuẩn lạc vàng Enterococcus faecalis WDCM 00087 ức chế, score 0 - Staphylococcus aureus WDCM 00034 ức chế, score 0 - 9 BẢO QUẢN Môi trường dạng bột (không chứa Tergitol 4): 2-30 °C Tergitol 4 Selective Supplement: 2 - 25°C Môi trường đổ sẵn trong đĩa petri : 2-8 °C - Hạn sử dụng được ghi trên nhãn. Môi trường hoàn chỉnh chuẩn bị sẵn trong đĩa với supplement (*): 15 ngày 2-8 °C Môi trường chuẩn bị sẵn trong ống (*): không khuyến nghị (*) Benchmark value được xác định theo điều kiện tiêu chuẩn, theo hướng dẫn của nhà sản xuất 10 ĐÓNG GÓI Môi trường bột khô: Chai 500 g ..........................................................................................................................................BK156HA Tergitol 4 Selective Supplement ống 50 mL...........................................................................................................................................BS03908 Môi trường hoàn chỉnh trong đĩa petri (Ø 90 mm): 20 đĩa …………..................................................................................................................................BM03608 11 THAM KHẢO Miller, R.G., Tate, C.R., Mallinson, E.T., and Scherrer, J.A.. 1991. Xylose-Lysine-Tergitol 4: An improved selective agar medium for the isolation of Salmonella. Poultry Science, 70 : 2429-2432. Miller, R.G., Tate, C.R., Mallinson, E.T., and Scherrer, J.A.. Erratum. Xylose-Lysine-Tergitol 4: An improved selective agar medium for the isolation of Salmonella. Poultry Science, 71 : 398. Tate, C.R., Miller, R.G., and Mallinson E.T.. 1992. Evaluation of two isolation and two no-isolation methods for detecting naturally occurring Salmonellae from broiler flock environmental drag-swab samples. Journal of Food Protection, 55 : 964-967. 23/30 Đường C1, phường 12, quận Tân Bình Thành phố Hồ Chí Minh Điện thoại: (+84.8) 6292.3745 - 6292.3731 - 6292.3773 Page 3 of 5 Email: [email protected] - Website: www.dongnamlab.com Dusch, H., and Altwegg, M.. 1995. Evaluation of five new plating media for isolation of Salmonella species. Journal of Clinical Microbiology, 33 : 802-804. Wallace, H.A.. 1996. Evolution of Methods for the Detection of Salmonella in Foods. Journal of A.O.A.C. International, 79 : 4-12. NF U47-102. Janvier 2008. Méthodes d’analyse en santé animale. Isolement et identification de tout sérovar ou de sérovar(s) spécifié(s) de salmonelles chez les mammifères. NF EN ISO 6579-1. Avril 2017.Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche, le dénombrement et le sérotypage des Salmonella - Partie 1 : recherche des Salmonella spp. 12 THÔNG TIN BỔ SUNG Thông tin được cung cấp trên nhãn được ưu tiên hơn các công thức hoặc hướng dẫn được mô tả trong tài liệu này và có thể dễ bị sửa đổi bất cứ lúc nào mà không cần cảnh báo. Mã tài liệu: XLT4_EN_V7 Ngày tạo: 03-2003 Cập nhật: 02-2018 Nguồn gốc của sửa đổi: Tài liệu tham khảo. 23/30 Đường C1, phường 12, quận Tân Bình Thành phố Hồ Chí Minh Điện thoại: (+84.8) 6292.3745 - 6292.3731 - 6292.3773 Page 4 of 5 Email: [email protected] - Website: www.dongnamlab.com PHỤ LỤC 1: HÌNH ẢNH MINH HỌA XLT Agar Phát hiện Salmonella spp. Kết quả: Tăng trưởng đạt được sau 24 giờ ủ ở 37 °C. 23/30 Đường C1, phường 12, quận Tân Bình Thành phố Hồ Chí Minh Điện thoại: (+84.8) 6292.3745 - 6292.3731 - 6292.3773 Page 5 of 5 Email: [email protected] - Website: www.dongnamlab.com .
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