| 產品名稱： | 維生素B12測定培養基；B12測定肉湯；維生素B12測定液體培養基 |
|---|---|
| 英文名稱： | Vitamin B12 Assay Medium；B12 Assay Medium |
| 培養基類型： | 營養培養基 |
| 級別： | for microbiology |
| 品牌： | ELITE-MEDIA（艾禮培養基） |
| 產品目錄號： | M342-01、M342-02 |
| 產品規格： | 100g、250g |
| 產品外觀： | 乳白色或淡黃色粉末，易出現結塊。 |
| 滅菌后顏色與澄清度： | 淡黃色或淺琥珀色透明液體，有少許沉淀。 |
| 保存條件： | 密封，2-25°C保存。制備好的培養基2-8°C避光保存。 |
| 注意事項： | 避免攝入、吸入、皮膚接觸。 |
| 相關產品： |
B12培養瓊脂 B12接種肉湯 B12測定瓊脂 |
產品描述：
維生素B12測定培養基(Vitamin B12 Assay Medium)在維生素B12的微生物法測定實驗中作為測定培養基。本配方采用USP和AOAC引用配方，用于測定食品、動物飼料及其它材料中B12含量。測定用標準菌株為德氏乳桿菌乳酸亞種ATCC7830工作原理
在維生素微生物測定法中，用到三類培養基：護理培養基、接種培養基和測定培養基。B族維生素和氨基酸的微生物測定法中檢測用細菌有萊希曼氏乳桿菌（Lactobacillus leichmanni ATCC7830）、植物乳桿菌（Lactobacillus plantarum ATCC8014）、干酪乳桿菌（Lactobacillus casei ATCC7469）、希氏腸球菌（Enterococcus hirae ATCC8043）和其它必需維生素B的菌株。用途：
維生素B12測定。配方與配制方法
配制方法：
1. 稱取8.3g本產品，加入100ml超純水和2ml 10%吐溫80。
2. 加熱煮沸2-3min，使其完全溶解。冷卻至室溫后檢測ph，如果需要，可以調整至6.0。
3. 分裝5ml到試管中，將沉淀均勻分裝到每個試管中。
4. 加人待測樣品，用超純水定容至10ml。
5. 115°C滅菌10min，或者121°C滅菌5min。
實驗方法
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.
結果與分析
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.