| 产品名称： | 军团菌GVPN选择性琼脂；GVPN培养基 |
|---|---|
| 英文名称： | Legionella GVPN Selective Agar；GVPN Medium |
| 培养基类型： | 选择性培养基 |
| 级别： | for microbiology |
| 品牌： | ELITE-MEDIA（艾礼培养基） |
| 产品目录号： | M135-01、M135-02 |
| 产品规格： | 250g(添加剂需另购)、20个平板/包(即用型9cm平板) |
| 产品外观： | 灰色至黑色均一粉末。 |
| 灭菌后颜色与澄清度： | 灰黑色至蓝黑色，不透明凝胶。 |
| 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： | -- |
产品描述：
军团菌GVPN选择性琼脂(Legionella GVPN Selective Agar)是军团菌选择性培养基。本培养基配方与ISO 11731-2 一致。工作原理
军团菌为革兰氏阴性杆菌，专性需氧，胞内寄生菌。军团菌最初是从以军团菌病人肺组织感染的豚鼠中分离出来的。这种方法虽然有较高的可靠性，但非常昂贵，而且费时费力，不久就被平板培养法所取代。目前标准培养基为活性碳酵母浸膏琼脂平板，也称军团菌生长平板(BCYE)。接种在BCYE平板上的样品在温度为35-37ºC，培养10天，在浓度为2.5% CO2的环境下培养更有利于军团菌的生长。军团菌的菌落通常呈白色、灰色、有荧光。军团菌在BCYE平板上生长而在平板和半光氨酸缺失BCYE-Cys平板上不生长。军团菌BCYE培养基用于选择性培养军团菌，满足军团菌生长所需的特殊要求。活性炭降解过氧化氢和其它代谢毒素。ACES缓冲剂是为了维持稳定的最适pH。α-酮戊二酸单钾盐刺激微生物生长。L-半胱氨酸是必需的氨基酸，也是军团菌重要的能量物质。焦磷酸铁是铁的来源。
配方与配制方法
| 成分 | g/L |
| 酵母提取物(细菌学级) | 10.0 |
| 活性炭 | 2.0 |
| α-酮戊二酸单钾盐 | 1.0 |
| N-(2-乙酰氨基)-2-氨基乙烷磺酸 （ACES缓冲剂） | 10.0 |
| 琼脂 | 12.0 |
| Final pH | 6.9 ± 0.2 |
军团菌添加剂(Legionella Supplement)： for 1L
| L-半胱氨酸盐酸盐 | 400 mg |
| 焦磷酸铁 | 250 mg |
军团菌GVPN选择性添加剂(Legionella GVPN Selective Supplement):for 1L
| 硫酸多粘菌素B | 80,000 IU |
| 甘氨酸 | 3.0 g |
| 万古霉素 | 1.0 mg |
| 纳他霉素 | 40 mg |
配制方法：
1. 称取36g本产品，用990ml去离子水重悬。
2. 煮沸使培养基完全溶解。
3. 121°C灭菌15min。
4. 冷却至50°C时，加入过滤除菌的军团菌添加剂（见上表）和军团菌MWY选择性添加剂，混匀。
5. 倒平板时不断搅动，确保活性炭颗粒均匀分布。
实验方法：
Clinical SamplesFor the isolation of Legionella spp. from patients with clinical evidence of Legionnaires' disease, greatest success has been achieved by the examination of lung tissue and bronchial aspirate.
1. Homogenise the patient's specimen in sterile distilled water.
2. Examine microscopically for Legionella by the Fluorescent Antibody Method (FA) and for other bacteria by Gram's stain.
3. Inoculate specimens that are FA-positive but with no other organisms present on to plates of BCYE Medium. Both FA-positive and FA-negative specimens in which other organisms have been detected by the Gram stain, are inoculated on to the selective medium BMPA.
4. Incubate the plates at 35°C in a 90% relative humidity atmosphere.
5. Growth usually appears in 2-3 days but continue to examine daily for 14 days before discarding the plates.
Environmental Samples
1. Take 10 ml of the concentrated sample and centrifuge at 2,500 rpm for 20 minutes (using sealed buckets).
2. Remove the supernatant to leave approximately 1ml of fluid. Resuspend the deposit. This constitutes the inoculum.
3. Spread 0.1 ml on to plates of BCYE Medium with and without selective agents using a sterile spreader.
4. Add 9 ml of HCl-KCl buffer* (pH 2.2); shake gently and leave for 5 minutes.
*HCl-KCl buffer: 3.9 ml of 0.2 M HCl; 25 ml of 0.2 M KCl; Adjust the pH to 2.2 using 1M KOH.
Alternatively, heat 10 ml of the sample concentrate in a 50°C water bath for 30 minutes.
Important：
ACID AND HEAT PRETREATMENT OF SAMPLES MUST NOT BE COMBINED.
5. Spread 0.1 ml on to plates of BCYE Medium using a sterile spreader.
6. Incubate the plates at 35° C and examine daily for up to seven days.
Colonies suspected of being Legionella are subcultured to Tryptone Soya Agar containing 5% sheep blood and BCYE Agar. Isolates that grow on BCYE Agar but fail to grow on TSA Blood Agar and have characteristic morphology, may be presumed to be Legionella. Confirmation must be made by biochemical and serological tests.
As the media described are not completely selective for Legionella species, it is recommended that the following criteria are used for the examination of plates:
1. The colonies must have characteristic colour, size and appearance when examined under a dissecting microscope.
2. The isolates should not grow on blood agar or GVPN Medium without L-cysteine.
3. The organisms should show characteristic Gram morphology.
Legionella spp. cannot be identified solely on growth characteristics on various media or by biochemical tests. Further studies with DNA homology, cellular fatty acids and serotyping must be undertaken.
结果与分析
35-37°C ，90%湿度，培养3-4天。ATCC标准菌株在军团菌GVPN选择性琼脂培养基上的生长情况如下:| ATCC标准菌株 | 生长情况 | 菌落颜色 |
| 大肠杆菌 ATCC25922 | -/+ | - |
| 杜氏军团菌 ATCC33343 | +++ | 浅蓝色至灰色 |
| 嗜肺性军团病杆菌 ATCC33153 | +++ | 白灰色至蓝灰色 |
| 表皮葡萄球菌 ATCC12228 | -/+ | - |
