Oxoid SR0147E Oxoid 幽门螺杆菌选择性添加剂(Dent)
Oxoid SR0147E H.PYLORI SELECTIVE SUPP.(DENT)
幽门螺杆菌选择性分离培养基Helicobacter pylori Selective Medium
| 货号 | 产品描述 | 规格 | 可配制培养基 | |
| CM0331B | 哥伦比亚血琼脂基础 Columbia lood Agar Base |
500g | 12.8L | |
| SR0147E | 幽门螺杆菌选择性添加剂（Dent） Helicobacter pylori Selective Supplement (Dent) |
10小瓶 | 每小瓶500ml |
哥伦比亚血琼脂基础，CM0331B：
| 成分 | 含量 g/L |
| 特殊蛋白胨 | 23.0 |
| 淀粉 | 1.0 |
| 氯化钠 | 5.0 |
| 琼脂 | 10.0 |
| pH 7.3±0.2 |
幽门螺杆菌选择性添加剂（Dent），SR0147E：
| 每小瓶成分 | 每小瓶含量 | 每L培养基含量 |
| 万古霉素 | 5.0mg | 10.0mg |
| 甲氧苄氨嘧啶 | 2.5mg | 5.0mg |
| 头孢磺啶 | 2.5mg | 5.0mg |
| 两性霉素B | 2.5mg | 5.0mg |
使用方法：
将39g的CM0331B加入1L无菌水中，加热溶解，然后121℃灭菌15分钟。
按照说明书将1瓶SR0147E溶解，加入约50℃时的哥伦比亚血琼脂基础中，然后向培养基中加入35ml裂解马血，混匀，倒平板。
产品描述：
幽门螺杆菌一般在胃中生长， 主要伴随胃炎和消化性溃疡。幽门螺杆菌选择性添加剂（Dent）是由Dent的将幽门螺杆菌从胃粘膜分离的分离培养基改进而来。这个添加剂是对 Skirrow培养基的改进版，将多粘菌素B替换为头孢磺啶和两性霉素B，这样能抑制念珠菌。当我们用这种培养基进行常规的100个胃粘膜活检时，Dent的培养基表现出比Skirrow培养基和巧克力血琼脂2更加高的幽门螺杆菌提取率，非目标菌株的生长也更加少了。
技术要点：
1、按照指导制备培养基，平板可以在4℃下保存3周，但一定要使培养基保持湿润，可以将平板放在一个塑料包装内保持水分。
2、将样本涂布在培养基上，注意，在进行胃粘膜活检复苏幽门螺杆菌时，提取出的幽门螺杆菌要尽快进行培养，如涉及到运输，要讲胃粘膜放在一个小的瓶子中，瓶子内要含0.1ml无菌盐水，这样可以防止胃粘膜脱水。
3、在微需氧条件下35℃培养3-5天，可以实用Oxoid的微需氧产气袋CN0025和CN0035（不需要加水）。
4、对培养基上单独的，半透明的和不连在一起的菌落镜检，注意幽门螺杆菌并不类似那些弯曲杆菌。
5、对分离出来的菌株按照以下形状进行对比：革兰氏阴性，弯曲或螺旋状，35℃生长，25℃不生长，42摄氏度variable growth，尿素酶阳性，过氧化氢酶阳性，氧化酶阳性，马尿酸阴性。
保存条件：
干粉在10-30℃保存，在标签上的效期之前使用。制成的培养基在2-8℃保存。
质控：
阳性对照：Helicobacter pylori ATCC® 43526 生长，无色菌落
阴性对照：Candida albicans ATCC® 10231 抑制或不生长
HELICOBACTER PYLORI SELECTIVE MEDIUM
A selective supplement for the isolation of H. pylori from clinical specimens.
COLUMBIA BLOOD AGAR BASE
Code: CM0331
| Formula |
gm/litre |
| Special peptone |
23.0 |
| Starch |
1.0 |
| Sodium chloride |
5.0 |
| Agar |
10.0 |
| pH 7.3 ± 0.2 |
Directions
Add 39g to 1 litre of distilled water. Boil to dissolve and sterilise by autoclaving at 121°C for 15 minutes.
HELICOBACTER PYLORI SELECTIVE SUPPLEMENT (DENT)
Code: SR0147
| Vial contents (each vial is sufficient for 500 ml of medium) |
per vial
|
per litre
|
| Vancomycin |
5.0 mg |
10.0 mg |
| Trimethoprim |
2.5 mg |
5.0 mg |
| Cefsulodin |
2.5 mg |
5.0 mg |
| Amphotericin B |
2.5 mg |
5.0 mg |
Directions
Reconstitute one vial as directed, aseptically add the contents to 500 ml of Columbia Blood Agar Base cooled to approximately 50°C. Add 35 ml of Laked Horse Blood SR0048 and mix well before pouring into sterile Petri dishes.
Description
Helicobacter pylori is associated with a number of gastric conditions, chiefly gastritis and peptic ulcers1,2,3.
Helicobacter pylori Selective Supplement (Dent) was developed from Dent’s selective medium described for the isolation of H. pylori from gastric biopsies2. This is a modification of Skirrow’s medium4 in which polymixin B is replaced by cefsulodin and amphotericin B is added to inhibit Candida species.
When used routinely in the laboratory for 100 gastric biopsies, Dent’s medium achieved a higher isolation rate for H. pylori and lower contamination by other organisms when compared with Skirrow’s medium and chocolate blood agar2. The provision of a good selective medium for H. pylori will help establish the role of this organism in the aetiology of gastric disease.
Technique
1. Prepare the medium as directed. The plates can be stored at 4°C for three weeks but it is essential that they are kept moist. This can be achieved simply
by keeping the plates in a plastic bag.
2. Smear the specimen on to the medium.
Note – the recovery of H. pylori from gastric biopsies is improved by direct cultivation as soon as possible after collection. If transportation is necessary, then place the biopsy against the neck of a small, sterile glass bottle containing 0.1 ml of sterile saline2. The biopsy should adhere to the glass but be protected from dehydration by water vapour.
3. Incubate at 35°C for three to five days under microaerophilic conditions. Use Campylobacter Gas Generating Kit BR0056 or BR0060 with an active
catalyst BR0042. Alternatively use CampyGen CN025 or CN035. CampyGen does not require the addition of water or a catalyst.
4. Examine for the presence of discrete, translucent and non-coalescent colonies. Note that colonies of H. pylori do not resemble those of Campylobacter
species.
5. Confirm the identity of the isolates with the following tests:
Gram negative, curved or spiral bacillus. Growth at 35°C, no growth at 25°C, variable growth at 42°C. Urease positive, Catalase positive, Oxidase positive, Hippurate negative.
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 medium at 2-8°C.
Appearance
Dehydrated Medium: Straw coloured, free-flowing powder.
Prepared medium: Opaque red-coloured gel.
Quality control
| Positive controls: | Expected results |
| Helicobacter pylori ATCC® 43526 | Good growth; colourless colonies. |
| Negative control: | |
| Candida albicans ATCC® 10231 | Inhibited or no growth |
* This organism is available as a Culti-Loop®
References
1. Marshall B. K., Warren J. R., Blincow E. D., Phillips M., Goodwin C. S., Murray R., Blackbourne S. J. and Waters T. E. (1988) The Lancet, December 24/31, No 8626/8627.
2. Dent J. C. and McNulty C. A. M. (1988) Eur. J. Clin. Microbiol. Infec. Dis. 7. 555±568.
3. Buck G. E. (1988) Laboratory Management, 26, No.9.
4. Skirrow M. B. (1977) BMJ, 1. 9-11.