| 產品名稱： | 緩沖炭酵母提取物瓊脂；軍團菌BCYE瓊脂；BCYE培養基 |
|---|---|
| 英文名稱： | Buffered charcoal yeast extract agar base；Legionella BCYE Agar Base；BCYE Agar Base |
| 培養基類型： | 選擇性培養基 |
| 級別： | for microbiology |
| 品牌： | ELITE-MEDIA（艾禮培養基） |
| 產品目錄號： | M133-01、M133-02 |
| 產品規格： | 250g、500g(培養基添加劑需另購) |
| 產品外觀： | 灰色至黑色均一粉末。 |
| 滅菌后顏色與澄清度： | 灰黑色至藍黑色，不透明凝膠。 |
| 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： | 軍團菌添加劑（Legionella Supplement） |
產品描述：
緩沖炭酵母提取物瓊脂基礎(Buffered charcoal yeast extract agar,base)簡稱軍團菌BCYE瓊脂基礎（Legionella BCYE Agar, Base）加入添加劑后用于選擇性培養軍團菌。BCYE瓊脂也稱軍團菌生長平板。本培養基配方與ISO 11731-2 一致。工作原理
軍團菌為革蘭氏陰性桿菌，專性需氧，胞內寄生菌。軍團菌最初是從以軍團菌病人肺組織感染的豚鼠中分離出來的。這種方法雖然有較高的可靠性，但非常昂貴，而且費時費力，不久就被平板培養法所取代。目前標準培養基為活性碳酵母浸膏瓊脂平板，也稱軍團菌生長平板(BCYE)。接種在BCYE平板上的樣品在溫度為35-37ºC，培養10天，在濃度為2.5% CO2的環境下培養更有利于軍團菌的生長。軍團菌的菌落通常呈白色、灰色、有熒光。軍團菌在BCYE平板上生長而在平板和半光氨酸缺失BCYE-Cys平板上不生長。軍團菌BCYE培養基用于選擇性培養軍團菌，滿足軍團菌生長所需的特殊要求。活性炭降解過氧化氫和其它代謝毒素。ACES緩沖劑是為了維持穩定的最適pH。α-酮戊二酸單鉀鹽刺激微生物生長。L-半胱氨酸是必需的氨基酸，也是軍團菌重要的能量物質。焦磷酸鐵是鐵的來源。
軍團菌屬的主要特征見下表：
| 特征 | L.pneumophila | L.bozemanii | L.dumoffii | L.micdadei | L.gormanii | L.longbeachae | L.jordanis |
| BCYE瓊脂 | + | + | + | + | + | + | + |
| 血瓊脂 | - | - | - | - | - | - | - |
| MWY瓊脂上菌落顏色 | 白色綠色 | 藍灰色 | 綠色 | 綠色 | 綠色 | 白色綠色 | 白色綠色 |
| 革蘭氏反應 | -ve | -ve | -ve | -ve | -ve | -ve | -ve |
| 抗酸組織 | - | - | - | - | - | - | - |
| 鞭毛 | + | + | + | + | + | + | + |
| 氧化酶 | + | - | - | + | - | + | + |
| 過氧化氫酶 | + | + | + | + | + | + | + |
| 青霉素酶 | + | + | + | - | + | + | + |
配方與配制方法
| 成分 | g/L |
| 酵母提取物(細菌學級) | 10.0 |
| 活性炭 | 2.0 |
| α-酮戊二酸單鉀鹽 | 1.0 |
| N-(2-乙酰氨基)-2-氨基乙烷磺酸 （ACES緩沖劑） | 10.0 |
| 瓊脂 | 17.0 |
| Final pH | 6.9 ± 0.2 |
軍團菌添加劑(Legionella Supplement)配方如下：
| L-半胱氨酸鹽酸鹽 | 400 mg |
| 焦磷酸鐵 | 250 mg |
本產品配方與美國Sigma-Aldrich出品的BCYE瓊脂（Sigma貨號86558）配方完全一致。
配制方法：
1. 稱取40 g本產品，用900ml去離子水重懸。
2. 煮沸使培養基完全溶解。
3. 121°C滅菌15min。
4. 冷卻至50°C時，加入過濾除菌的軍團菌添加劑（見上表），混勻。
5. 倒平板時不斷攪動，確保活性炭顆粒均勻分布。
實驗方法：
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 Legionella BCYE Agar 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.
結果與分析
35-37°C ，90%濕度，培養3-4天。ATCC標準菌株在緩沖炭酵母提取物瓊脂基礎培養基上的生長情況如下:| ATCC標準菌株 | 生長情況 | 菌落顏色 |
| 大腸桿菌 ATCC25922 | -/+ | - |
| 杜氏軍團菌 ATCC33343 | +++ | 淺藍色至灰色 |
| 嗜肺性軍團病桿菌 ATCC33153 | +++ | 白灰色至藍灰色 |
| 表皮葡萄球菌 ATCC12228 | -/+ | - |