分枝杆菌实验室的标准化操作.ppt

直接涂片法步骤 染色（1） 玻片放在染色架上，玻片间距保持10毫米以上的距离，染色架下空间足够，方便加热 火焰固定（5秒钟将玻片过火4次） 滴加石炭酸复红染液，盖满玻片。 加热至出现蒸汽，勿使沸腾。脱离火焰，保持5分钟。 高海拔地区（西藏、青海）：定期过滤染色液；如室温低则用前热水浴加热染液；火焰加热，由于海拔高、沸点低，需加热多次 染色(2) 脱色： 轻缓冲洗 沥水。 滴加脱色液，保持1分钟。 如不彻底，重复。 1.接通电源，打开开关上升聚光镜至最高，调节光圈70－80％大小2.将涂片放在载物台上，痰膜朝上 显微镜检查 读片时，首先从左至右观察； 纵向向下转换一个视野； 然后从右向左观察 分枝杆菌培养 培养基质控 前处理 接种和孵育 结果和报告 四、结果的观察与报告 接种后第3、7天各观察一次菌落生长情况 此后每周观察一次，直至满8周 每次观察记录菌落生长及污染情况 结核分枝杆菌的生长要求： 专性需氧菌 最适PH为6.5～7.2 温箱设定温度为36℃,温度范围35－37 小结 – 操作流程 涡旋振荡 1分钟 痰标本中加入 1-2倍体积 4%NaOH 分别接种0.1ml 前处理液至2 只培养基斜面 第3天、7天、此后每周 观察一次直至第九周, 记录菌落生长和污染 置36C温 箱培养 室温静置 15分钟 4 3 2 5 6 1 * * * * Smearing Sputum Using the rough end of a broken bamboo / coconut midrib stick, select and pick up the yellowish purulent particles of sputum. A wire loop (with an inner diameter of 3 mm) also can be used to transfer sputum to make a smear. Spread the sputum on the glass slide evenly by smearing, repeatedly in coil-like patterns, to an oval approximately 1-2 cm in width and 2-3 cm in length. After making the smear, place the stick into a waste bucket. Use a separate stick for each specimen. * * * * Heat Fixing the Smear Allow the smear to dry completely at room temperature. Do not dry it under the heat of the sun or over a flame. After the slide is completely dry, hold the slide using forceps with the smeared slide facing upwards. Pass the slide over the flame 2-3 times, about 2-3 seconds each. Do not heat the slide for too long or keep it stationary over the flame, or else the slide will be scorched. * * * * A Good Smear Before staining, check a smear for acceptable thickness by looking at printed letters through the smear. Hold the smeared glass slide 4-5 cm over a piece of printed paper. If the letters cannot be read, the smear is too thick. 是凝固的苯酚，可用较热的水（或热水浴脱色液）使之融解，洗去 Drying the Stained Slides Tilt and place the slide on the slide rack to dry in the air. Do not place the stained slide under the sun to dry.  * * * * Applying Immersion Oil? Put one drop of immersion oil