| 产品名称： | 维生素B12测定培养基；B12测定肉汤；维生素B12测定液体培养基 |
|---|---|
| 英文名称： | Vitamin B12 Assay Medium；B12 Assay Medium |
| 培养基类型： | 营养培养基 |
| 级别： | for microbiology |
| 品牌： | ELITE-MEDIA（艾礼培养基） |
| 产品目录号： | M342-01、M342-02 |
| 产品规格： | 100g、250g |
| 产品外观： | 乳白色或淡黄色粉末，易出现结块。 |
| 灭菌后颜色与澄清度： | 淡黄色或浅琥珀色透明液体，有少许沉淀。 |
| 保存条件： | 密封，2-25°C保存。制备好的培养基2-8°C避光保存。 |
| 注意事项： | 避免摄入、吸入、皮肤接触。 |
| 相关产品： |
B12培养琼脂 B12接种肉汤 B12测定琼脂 |
产品描述：
维生素B12测定培养基(Vitamin B12 Assay Medium)在维生素B12的微生物法测定实验中作为测定培养基。本配方采用USP和AOAC引用配方，用于测定食品、动物饲料及其它材料中B12含量。测定用标准菌株为德氏乳杆菌乳酸亚种ATCC7830工作原理
在维生素微生物测定法中，用到三类培养基：护理培养基、接种培养基和测定培养基。B族维生素和氨基酸的微生物测定法中检测用细菌有莱希曼氏乳杆菌（Lactobacillus leichmanni ATCC7830）、植物乳杆菌（Lactobacillus plantarum ATCC8014）、干酪乳杆菌（Lactobacillus casei ATCC7469）、希氏肠球菌（Enterococcus hirae ATCC8043）和其它必需维生素B的菌株。用途：
维生素B12测定。配方与配制方法
配制方法：
1. 称取8.3g本产品，加入100ml超纯水和2ml 10%吐温80。
2. 加热煮沸2-3min，使其完全溶解。冷却至室温后检测ph，如果需要，可以调整至6.0。
3. 分装5ml到试管中，将沉淀均匀分装到每个试管中。
4. 加人待测样品，用超纯水定容至10ml。
5. 115°C灭菌10min，或者121°C灭菌5min。
实验方法
Sample preparation:In case all the vitamin B12 is present in a free form the examination material (e.g. powders or levigated tablets) can be simply extracted with water. Should the sample contain bonded vitamin B12, decomposition with buffer solution or enzymatic hydrolysis is necessary.
Decomposition with buffer solution:
Buffer: Dissolve 1.29 g disodium hydrogen phosphate, 1.1 g citric acid and 1.0 g sodium metabisulfite in 100 ml of distilled water.
Homogenize 1 g of sample in 50 ml buffer and autoclave for 10 minutes at 121°C. Let cool down and adjust the pH to 6.0, fill up to 100 ml with sterile distilled water. Filter or centrifuge to get a solution without particles.
Decomposition with enzymatic hydrolysis
Homogenize 1 g sample in 80 ml of acetate buffer. Papain (76222), amylase (10080) and a few drops of chloroform or toluene are added to the homogeneous suspension. The two enzymes can be replaced by a corresponding diastase. Incubate for about 24 hours at 37°C, then heat for 30 minutes at 100°C. Let cool down and adjust the pH to 6.6 with a sodium hydroxide solution and fill up to 100 ml with acetate buffer (72749; pH 4.65). The suspension is filtered or centrifuged to separate particles. It is recommended to perform a preliminary test, if the content of vitamin B12 is completely unknown. For this preliminary test, a concentrated extract is prepared and examined in different dilutions, a dilution factor of 10 is recommended.
Preparatory culture of test organism
Lactobacillus delbrueckii subsp. lactis (ATCC 7830) is used as a test organism. Inoculate the Lactobacillus in Micro-Inoculum-Broth (composition: Proteose peptone 5 g/l, yeast extract 20 g/l, D(+)-glucose 10 g/l, potassium dihydrogen phosphate 2 g/l, Tween 80 0.1 g/l) and incubate for 20 hours at 37°C. Then centifuge the culture and wash three times with physiological saline and adjust to a microbial count of 108 bacteria/ml.
Calibration
Dissolve 100 mg of vitamin B12 (Sigma V2876) in 1 litre of distilled water (100 ug/ml). This stock solution is diluted to 100 pg/ml to give the reference solution.
Prepare the following calibration concentrations:
End volume: 5 ml
Preparation of sample and controls
Culture and sterility controls only contain 5 ml of water. The samples are also prepared as dilution series and filled up to 5 ml with distilled water.
Test
Dissolve 83 g of dehydrated Vitamin B12 Assay Medium together with 2 ml Tween- 80 in 1 litre distilled water. Heat gently to a boil to dissolve the medium completely. Check the pH and correct if necessary to 6.0 (at 25°C). Add 5 ml of culture medium to all tubes with control, sample or calibration solution and close them with caps. Sterilisation for 10 min at 115°C. Let cool down and inoculate all test tubes, excluding the sterile controls, with 1 drop of preparatory culture. Incubate for 24 hours at 37°C.
结果与分析
Examination, evaluationThe calibration standards and samples are measured photometrically at 546 nm against the culture control. A calibration curve is recorded with the optical density (OD) values on the linear ordinate against the vitamin B12 concentration on the logarithmic abscissa. To get reproducible results the control culture measured against water should have an OD546 nm, 1 cm below 0.150. There must be no growth by the sterile controls.
Principle and Interpretation:
The growth of Lactobacillus delbrueckii var. lactis is limited by the concentration of vitamin B12 in this defined medium. Due to the limitation the vitamin B12 concentration can be calculated with a calibration curve.
