The University of Southern Mississippi, Gulf Coast Research Laboratory, 703 East Beach Drive, Ocean Springs, MS 39564, United States.
The University of Southern Mississippi, Gulf Coast Research Laboratory, 703 East Beach Drive, Ocean Springs, MS 39564, United States.
J Microbiol Methods. 2013 Aug;94(2):98-102. doi: 10.1016/j.mimet.2013.04.012. Epub 2013 May 6.
A new selective and differential medium, Vibrio vulnificus X-Gal (VVX), was developed for direct enumeration of V. vulnificus (Vv) from oyster samples. This agar utilizes cellobiose and lactose as carbon sources, and the antibiotics colistin and polymyxin B as selective agents. Hydrolysis of 5-bromo-4-chloro-3-indolyl- beta-d-galactopyranoside (x-gal), used in the agar as a lactose analog, produces an insoluble blue dye that makes lactose positive colonies easily distinguishable from any non-lactose fermenting bacteria. Various bacterial species were spot plated onto thiosulfate-citrate-bile salts-sucrose agar (TCBS), and CHROMagar Vibrio, two vibrio-specific selective agars, non-selective agar, and VVX to compare selectivity of VVX to other widely used media. A V. vulnificus pure culture was serially diluted on VVX and non-selective agar to determine the VVX percent recovery. Water and oyster samples were spread plated on VVX agar and allowed to incubate for 16-18 h at 33 °C. Blue and white colonies from VVX agar were picked and screened by end point PCR for the Vv hemolysin vvhA. VVX agar showed a significant improvement over TCBS and CHROMagar at preventing non-target growth. There was an 87.5% recovery compared to non-selective plating and a 98% positivity rate of blue colonies picked from oyster tissue plating. The findings suggest that this new agar is a fast, distinctive, and accurate method for enumeration of V. vulnificus from the environment.
一种新的选择性和差异化培养基，创伤弧菌 X-Gal（VVX），被开发用于直接从牡蛎样本中计数创伤弧菌（Vv）。该琼脂利用纤维二糖和乳糖作为碳源，以及抗生素黏菌素和多粘菌素 B 作为选择性试剂。在琼脂中用作乳糖类似物的 5-溴-4-氯-3-吲哚基-β-d-半乳糖吡喃糖苷（x-gal）的水解产生不溶性蓝色染料，使乳糖阳性菌落与任何非乳糖发酵细菌易于区分。将各种细菌种类点样到硫代硫酸盐-柠檬酸盐-胆汁盐-蔗糖琼脂（TCBS）、两种创伤弧菌特异性选择性琼脂、非选择性琼脂和 VVX 上，以比较 VVX 与其他广泛使用的培养基的选择性。将创伤弧菌纯培养物在 VVX 和非选择性琼脂上连续稀释，以确定 VVX 的回收率。将水和牡蛎样本涂抹在 VVX 琼脂上，并在 33°C 下孵育 16-18 小时。从 VVX 琼脂中挑选蓝色和白色菌落，并通过终点 PCR 筛选 Vv 溶血素 vvhA。VVX 琼脂在防止非目标生长方面明显优于 TCBS 和 CHROMagar。与非选择性平板相比，回收率为 87.5%，从牡蛎组织平板中挑选的蓝色菌落的阳性率为 98%。这些发现表明，这种新的琼脂是一种快速、独特且准确的方法，可用于从环境中计数创伤弧菌。
