Oxoid CM0945R TBX琼脂 TBX MEDIUM 2.5kg

Oxoid CM0945R TBX琼脂 TBX MEDIUM 2.5kg

Dehydrated Culture Media

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TRYPTONE BILE X-GLUCURONIDE MEDIUM (TBX)

Code: CM0945

A selective, chromogenic medium for the detection and enumeration of Escherichia coli in food and animal feed. This medium meets the formulation and performance criteria laid down in ISO 166491,2,3 and ISO 11133:20144.

一种选择性显色培养基,用于检测和计数食品和动物饲料中的大肠杆菌。 该培养基符合 ISO 166491,2,3 和 ISO 11133:20144 中规定的配方和性能标准。

Typical Formula* gm/litre
Tryptone 20.0
Bile Salts No. 3 1.5
Agar 15.0
X-glucuronide 0.075
pH 7.2 ± 0.2 @ 25°C
* Adjusted as required to meet performance standards

Directions
Suspend 36.6g of TBX Medium in 1 litre of distilled water. Sterilise by autoclaving at 121°C for 15 minutes. Cool to 50°C and pour the medium into sterile Petri dishes.

方向
将 36.6 克 TBX 培养基悬浮在 1 升蒸馏水中。 通过在 121°C 高压灭菌 15 分钟进行灭菌。 冷却至 50°C,将培养基倒入无菌培养皿中。

Description
TBX Medium is based on Tryptone Bile Agar (CM0595). Tryptone Bile Agar was originally formulated to improve on earlier methods used to detect Escherichia coli in foods5.6 in terms of speed, reliability, better recovery from frozen samples and the detection of poor lactose fermenters.
TBX Medium builds on these advantages through the addition of a chromogenic agent – X-glucuronide – which detects glucuronidase activity. This is the same enzyme detected by MUG reagent7, and has been shown to be highly specific for E. coli8. However, approximately 3-4% of E. coli are glucuronidase negative, notably E. coli O157 strains9.

Most E. coli strains can be differentiated from other coliforms by the presence of the enzyme glucuronidase. The chromogen in TBX Medium is 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-glucuronide), and is targeted by this enzyme. E. coli cells are able to absorb this complex intact and intracellular glucuronidase splits the bond between the chromophore and the glucuronide. The released chromophore is coloured and builds up within the cells, causing E. coli colonies to be coloured blue/green.

描述
TBX 培养基基于胰蛋白胨胆汁琼脂 (CM0595)。胰蛋白胨胆汁琼脂最初是为了改进早期用于检测食品中大肠杆菌的方法5.6 在速度、可靠性、更好地从冷冻样品中恢复和检测不良乳糖发酵罐方面而配制的。
TBX 培养基通过添加显色剂 – X-葡糖苷酸 – 来检测葡糖苷酸酶活性,从而建立在这些优势之上。这与 MUG 试剂 7 检测到的酶相同,并且已被证明对大肠杆菌具有高度特异性8。然而,大约 3-4% 的大肠杆菌是葡萄糖醛酸酶阴性的,尤其是大肠杆菌 O157 菌株 9。

大多数大肠杆菌菌株可以通过葡萄糖醛酸酶的存在与其他大肠菌群区分开来。 TBX 培养基中的色原是 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-glucuronide),并被该酶靶向。大肠杆菌细胞能够完整地吸收这种复合物,并且细胞内的葡糖苷酸酶会分裂发色团和葡糖苷酸之间的键。释放的发色团是有颜色的并在细胞内积聚,导致大肠杆菌菌落呈蓝色/绿色。

Technique
Dry the surface of the medium of the prepared plates. Dilute the food sample according to the method being followed for example 1:10 with Maximum Recovery Diluent (CM0733). Homogenise in a stomacher or a laboratory blender. Further dilute the sample as required.

The following incubation techniques may be used (consult the relevant standard for the complete method):
1. Pipette 0.1ml of the homogenate on to the plate and spread over the surface with a sterile spreader. Incubate the plates for 24 hours at 37°C10.
2. Pipette 0.5ml of the homogenate on to the plate and spread over the surface with a sterile spreader. Incubate the plates for 4 hours at 30°C, then 18 hours to 24 hours at 44°C11.
3. Place a cellulose membrane on to the surface of a Minerals Modified Glutamate Agar plate. Pipette 1ml of the homogenate on to the membrane. Incubate for 4 hours at 37°C. Transfer the membrane to a TBX plate and incubate for 18 hours to 24 hours hours at 44°C1.
4. Pipette 1ml of the homogenate into a sterile Petri dish. Add TBX Medium, cooled to 45°C. Mix well and allow to set. Incubate for 18 hours to 24 hours at 44°C. If the presence of stressed cells is suspected pre-incubate the plates for 4 hours at 37°C2.

Multiply the numbers of blue/green colonies by the dilution factor and express the result as the number of E. coli per gram of food.

Storage conditions and Shelf life
Store the dehydrated medium at 10-30°C and use before the expiry date on the label.
Store the prepared plates of medium at 2-8°C.

Appearance  
Dehydrated medium: Straw coloured, free-flowing powder
Prepared medium: Straw coloured gel

技术
干燥准备板的介质表面。根据所遵循的方法(例如 1:10)用最大回收稀释剂 (CM0733) 稀释食品样品。在胃部或实验室搅拌机中均质化。根据需要进一步稀释样品。

可以使用以下孵育技术(完整方法请参阅相关标准):
1. 将 0.1 ml 匀浆移液到板上,并用无菌涂抹器涂抹在表面上。将板在 37°C10 下孵育 24 小时。
2. 将 0.5 ml 匀浆移液到板上,并用无菌涂抹器涂抹在表面上。将板在 30°C 下孵育 4 小时,然后在 44°C 下孵育 18 小时至 24 小时11。
3. 将纤维素膜置于矿物改性谷氨酸琼脂平板的表面。移取 1ml 匀浆到膜上。在 37°C 下孵育 4 小时。将膜转移到 TBX 板上并在 44°C1 下孵育 18 小时至 24 小时。
4. 将 1ml 匀浆移液到无菌培养皿中。加入 TBX 培养基,冷却至 45°C。充分混合并使其凝固。在 44°C 下孵育 18 小时至 24 小时。如果怀疑存在应激细胞,则将板在 37°C2 下预孵育 4 小时。

将蓝色/绿色菌落的数量乘以稀释因子,并将结果表示为每克食物中大肠杆菌的数量。

储存条件和保质期
将脱水培养基储存在 10-30°C 并在标签上的有效期之前使用。
将准备好的培养基板储存在 2-8°C。

外貌
脱水介质:稻草色、自由流动的粉末
制备培养基:稻草色凝胶

Quality control

Positive controls: Expected results
Escherichia coli ATCC®25922 *
WDCM 00013
Good growth; blue/green coloured colonies
Escherichia coli NCTC 13216
WDCM 00202
Good growth; blue/green coloured colonies
Negative controls:
Citrobacter freundii ATCC® 43864
WDCM 0006
Growth; white to green beige colonies
Enterococcus faecalis ATCC®29212*
WDCM 00087
No growth
* This organism is available as a Culti-Loop®

References 参考文献
1. ISO 16649-1:2001 Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli – Part 1: Colony-count technique at 44 degrees C using membranes and 5-bromo-4-chloro-3-indolyl beta-D-glucuronide
2. ISO 16649-2:2001 Microbiology of food and animal feeding stuffs – Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli – Part 2: Colony-count technique at 44 degrees C using 5-bromo-4-chloro-3-indolyl beta-D-glucuronide
3. ISO 16649-3:2015 Microbiology of the food chain – Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli – Part 3: Detection and most probable number technique using 5-bromo-4-chloro-3-indolyl-ß-D-glucuronide
4. ISO 11133:2014 Microbiology of food, animal feed and water – Preparation, production, storage and performance testing of culture media
5.Gross R.J. and Rowe B. (1985) J. Hyg. Lond. 95. 531-550.
6. Anderson J.M. and Baird-Parker A.C. (1975) J. Appl. Bact. 39. 111-117.
7. Feng P.C.S. and Hartman P.A. (1982) Appl. Environ. Microbiol. 43. 1320-1329.
8. Hansen W. and Yourassowsky E. (1984) J. Clin. Microbiol. 20. 1177-1179.
9. Ratnam S., March S.B., Almed R., Bezanson G.S. and Kasatiya S. (1988) J. Clin. Microbiol. 26. 2006-2012.
10. Donovan T.J. et al (1998) Communicable Disease and Public Health 1 : 188-196.
11. PHLS Standard Methods for Microbiological Examination of Food, Dairy and Water Samples. F20: Direct Enumeration of Escherichia coli.

细菌学蛋白胨 (LP0037)
麦芽提取物 (LP0039)
真菌胨 (LP0040)
酸水解酪蛋白 (LP0041)
胰蛋白胨 (LP0042)
胰蛋白胨 T (LP0043)
中性大豆蛋白胨 (LP0044)
胰蛋白月示 (LP0047)
乳蛋白水解产物 (LP0048)
蛋白胨 P (LP0049)
胆盐 (LP0055)
3号胆盐 (LP0056)
细菌学乳糖 (LP0070)
细菌学葡萄糖 (LP0071)
特殊蛋白胨 (LP0072)
月示蛋白胨 (LP0085)
谷氨酸钠 (LP0124)
40%尿素溶液 (SR0020)
10%乳酸 (SR0021)
3.5%亚碲酸钾 (SR0030)
番茄汁 (SR0032)
马血清 (SR0035)
50%乳酸钾 (SR0037)
Fildes 提取物 (SR0046)
蛋卵黄乳剂 (SR0047)
血球已溶解的马血 (SR0048)
去纤维蛋白的马血 (SR0050)
去纤维蛋白的羊血 (SR0051)
亚碲酸盐蛋卵黄乳剂 (SR0054)
支原体添加剂 G (SR0059)
支原体添加剂 P (SR0060)
Tinsdale 添加剂 (SR0065)
弯曲菌选择培养基添加剂 (Skirrow) (SR0069)

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