实验五 肠道致病菌的微生物学检查结核分枝杆菌的培养及形态58

1、实验五肠道致病菌的微生物学检查结核分枝杆菌的培养及形态观察 医学微生物学实验实验内容：1.肠道致病菌的微生物学检查2.结核分枝杆菌的培养及形态观察抗酸染色1.肠道致病菌的微生物学检查 1实验目的Aaaaaaaaaaaaaaaa示教：常见肠道细菌2常用培养基介绍主要包括：麦康克培养基、伊红美蓝培养基、中国蓝琼脂平板、SS琼脂平板、双糖铁培养基示教：伊红美蓝培养基 双糖铁培养基 SS平板别离肠道病原菌的强选择培养基成分和作用：根底培养基：提供氮源和碳源胆盐、枸橼酸钠、煌绿：可抑制肠道非致病菌的生长，而对肠道致病菌抑制作用弱。乳糖，中性红pH6.8-8.0，红橙黄 ：致病菌不分解乳糖，但分解蛋白胨产

2、生碱性物质菌落淡黄色；大肠杆菌分解乳糖产酸，菌落呈红色病原菌:小,无色非致病菌:大,红色病原菌:透明,无色，小菌落 非致病菌:紫黑色EMBEosin Methyl Blue)平板别离肠道病原菌的弱选择培养基克氏双糖培养基接种方法如何观察上层含乳糖局部乳糖发酵：致病菌红色，非致病菌黄色H2S然后观察下层发酵葡萄糖产酸产气动力乳糖硫酸亚铁固体葡萄糖半固体指示剂:酚红3肠道致病菌的别离鉴定标本收集别离培养初步鉴定血清学鉴定生化反响标本选择鉴别培养基 SS平板生化反响血清学鉴定双糖管等Ab检测未知Ag (玻片凝集试验)3724h？和球菌的不同粪便标本肠道致病菌别离鉴别流程 乳糖 葡萄糖 动力 硫化氢大

3、肠杆菌 黄色 + -痢疾杆菌 红色 + - -伤寒杆菌 红色 + + +/-鉴别肠道致病菌的生化反响糖发酵试验 甲基红试验VP实验枸橼酸盐利用试验硫化物硫化氢生成试验吲哚生成试验尿素水解试验示教：生化反响举例 糖发酵试验(fermentaton test)示教：单糖发酵管 甲基红试验methyl red test- + - +VPVoges-Proskauer试验 +- Urease Test 尿素酶试验：H2S Production 硫化氢试验 - +IMViC试验(Indole Test; Methyl Red (MR) Test; Voges-Proskauer (VP) Test; C

4、itrate Utilization Test) 大肠埃希菌 产气杆菌 2.结核分枝杆菌的培养及形态观察发病趋势和临床意义：aaaaaaaaa结核分枝杆菌形态：细长，略弯曲的杆菌，多聚集成团。无鞭毛,芽胞。染色性：不易着色，齐尼抗酸染色阳性。菌体呈红色，背景中其他物质蓝色。培养特性：专性需氧，最适37 ，营养要求高，生长缓慢，18h分裂一代。罗氏(Lowenstein-Jensen)培养基含蛋黄、甘油、马铃薯、无机盐、孔雀绿，26周可见菌落生长，菜花、颗粒或结节状菌落，乳白色或黄色，不透明。在液体培养基，生长于外表，有皱褶。结核分枝杆菌的培养方法1痰标本培养前处理1酸处理法：痰标本参加4硫酸稀

5、释24倍，置室温下30min，期间振荡23次，使痰液化后即可接种。2碱处理法：痰标本参加2氢氧化钠溶液稀释24倍，置37温箱内30min，期间振荡23次，痰液化后即可接种。2接种培养法 取上述处理过的痰液0.1ml均匀接种于改进罗氏培养基斜面，37孵育34周后观察菌落特点。菌落为乳白色或米黄色，外表粗糙呈颗粒状或菜花状抗酸染色 结核杆菌对苯胺染料一般不易着色，但经加温或延长染色时间后能抵抗3盐酸乙醇的脱色作用。经此法染色后，结核杆菌及其他分枝杆菌呈红色，非抗酸菌呈蓝色。示教：结核分枝杆菌抗酸染色 实验步骤：1用竹签挑取开放性肺结核病人晨痰标本中干酪样小粒或脓性局部，或用取菌环挑取经酸、碱处理后

6、的痰标本，置载玻片中央，均匀涂布成1.52.0cm卵圆形痰膜，枯燥，固定。2滴加石炭酸复红液于涂片上，玻片置于酒精灯火焰缓缓加热，至有蒸汽冒出，约维持5min切勿沸腾或使染液干涸于玻片上。如有染液干涸趋势，应补加染液。然后自然冷却，用流水漂去多于染液。3滴加3盐酸乙醇脱色，至涂片较厚处无颜色脱出为止，约30s，水洗。4滴加碱性亚甲蓝液复染1min，水洗。5滤纸吸干后镜检。实验材料痰液1管/小组，抗酸染色染液1套/小组，玻片1个/人实验报告内容 抗酸染色法实验原理、实验步骤、实验结果绘图和结果分析。0B3F6I9LdOgSjVmYq!t&w-z1C4G7JbMePhTkWnZr$u(x+A2E5

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18、#s&v)y0C3F6IaLdPgSjVnYq!t*w-A1D4G8JbMeQhTlWoZr%u(x+B2E6H9KcOfRiUmXp#s&v)z0C3F7IaLdPgSkVnYq$t*w-A1D5G8JbNeQhTlWo#r%u(y+B2E6H9LcOfRjUmXp!s&w)z0C4F7IaMdPhSkVnZq$t*x-A1D5G8KbNeQiTlWo#r%v(y+B3E6H9LcOgRjUmYp!s&w)z1C4F7JaMdPhSkWnZq$u*x-A2D5H8KbNfQiTlXo#s%v(y0B3E6I9LdOgRjVmYp!t&w)z1C4G7JaMePhSkWnZr$u*x+A2D5

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20、%u(y+B2E6H9KcOfRjUmXp!s&v)z0C4F7IaMdPgSkVnZq$t*x-A1D5G8JbNeQiTlWo#r%u(y+B3E6H9LcOfRjUmYp!s&w)z0C4F7JaMdPhSkVnZq$u*x-A2D5GcOfRjUmXp!s&v)z0C4F7IaMdPgSkVnYq$t*x-A1D5G8JbNeQiTlWo#r%u(y+B3E6H9LcOfRjUmYp!s&w)z0C4F7JaMdPhSkVnZq$u*x-A2D5G8KbNfQiTlXo#r%v(y+B3E6I9LcOgRjUmYp!t&w)z1C4F7JaMePhSkWnZq$u*x+A2D5H8KbNfQiUlXo#s%v(y0B3F6I9LdOgRjVmYq!t&w-z1C4G7JbMePhTkWnZr$u*x+A2E5H8KcNfQiUlXp#