實驗五 腸道致病菌的微生物學(xué)檢查結(jié)核分枝桿菌的培養(yǎng)及形態(tài)58

1、實驗五腸道致病菌的微生物學(xué)檢查結(jié)核分枝桿菌的培養(yǎng)及形態(tài)觀察 醫(yī)學(xué)微生物學(xué)實驗實驗內(nèi)容：1.腸道致病菌的微生物學(xué)檢查2.結(jié)核分枝桿菌的培養(yǎng)及形態(tài)觀察抗酸染色1.腸道致病菌的微生物學(xué)檢查 1實驗?zāi)康腁aaaaaaaaaaaaaaa示教：常見腸道細菌2常用培養(yǎng)基介紹主要包括：麥康克培養(yǎng)基、伊紅美藍培養(yǎng)基、中國藍瓊脂平板、SS瓊脂平板、雙糖鐵培養(yǎng)基示教：伊紅美藍培養(yǎng)基 雙糖鐵培養(yǎng)基 SS平板別離腸道病原菌的強選擇培養(yǎng)基成分和作用：根底培養(yǎng)基：提供氮源和碳源膽鹽、枸櫞酸鈉、煌綠：可抑制腸道非致病菌的生長，而對腸道致病菌抑制作用弱。乳糖，中性紅pH6.8-8.0，紅橙黃 ：致病菌不分解乳糖，但分解蛋白胨產(chǎn)

2、生堿性物質(zhì)菌落淡黃色；大腸桿菌分解乳糖產(chǎn)酸，菌落呈紅色病原菌:小,無色非致病菌:大,紅色病原菌:透明,無色，小菌落 非致病菌:紫黑色EMBEosin Methyl Blue)平板別離腸道病原菌的弱選擇培養(yǎng)基克氏雙糖培養(yǎng)基接種方法如何觀察上層含乳糖局部乳糖發(fā)酵：致病菌紅色，非致病菌黃色H2S然后觀察下層發(fā)酵葡萄糖產(chǎn)酸產(chǎn)氣動力乳糖硫酸亞鐵固體葡萄糖半固體指示劑:酚紅3腸道致病菌的別離鑒定標(biāo)本收集別離培養(yǎng)初步鑒定血清學(xué)鑒定生化反響標(biāo)本選擇鑒別培養(yǎng)基 SS平板生化反響血清學(xué)鑒定雙糖管等Ab檢測未知Ag (玻片凝集試驗)3724h？和球菌的不同糞便標(biāo)本腸道致病菌別離鑒別流程 乳糖 葡萄糖 動力 硫化氫大

3、腸桿菌 黃色 + -痢疾桿菌 紅色 + - -傷寒桿菌 紅色 + + +/-鑒別腸道致病菌的生化反響糖發(fā)酵試驗 甲基紅試驗VP實驗枸櫞酸鹽利用試驗硫化物硫化氫生成試驗吲哚生成試驗?zāi)蛩厮庠囼炇窘蹋荷错懪e例 糖發(fā)酵試驗(fermentaton test)示教：單糖發(fā)酵管 甲基紅試驗methyl red test- + - +VPVoges-Proskauer試驗 +- Urease Test 尿素酶試驗：H2S Production 硫化氫試驗 - +IMViC試驗(Indole Test; Methyl Red (MR) Test; Voges-Proskauer (VP) Test; C

4、itrate Utilization Test) 大腸埃希菌 產(chǎn)氣桿菌 2.結(jié)核分枝桿菌的培養(yǎng)及形態(tài)觀察發(fā)病趨勢和臨床意義：aaaaaaaaa結(jié)核分枝桿菌形態(tài)：細長，略彎曲的桿菌，多聚集成團。無鞭毛,芽胞。染色性：不易著色，齊尼抗酸染色陽性。菌體呈紅色，背景中其他物質(zhì)藍色。培養(yǎng)特性：專性需氧，最適37 ，營養(yǎng)要求高，生長緩慢，18h分裂一代。羅氏(Lowenstein-Jensen)培養(yǎng)基含蛋黃、甘油、馬鈴薯、無機鹽、孔雀綠，26周可見菌落生長，菜花、顆粒或結(jié)節(jié)狀菌落，乳白色或黃色，不透明。在液體培養(yǎng)基，生長于外表，有皺褶。結(jié)核分枝桿菌的培養(yǎng)方法1痰標(biāo)本培養(yǎng)前處理1酸處理法：痰標(biāo)本參加4硫酸稀

5、釋24倍，置室溫下30min，期間振蕩23次，使痰液化后即可接種。2堿處理法：痰標(biāo)本參加2氫氧化鈉溶液稀釋24倍，置37溫箱內(nèi)30min，期間振蕩23次，痰液化后即可接種。2接種培養(yǎng)法 取上述處理過的痰液0.1ml均勻接種于改進羅氏培養(yǎng)基斜面，37孵育34周后觀察菌落特點。菌落為乳白色或米黃色，外表粗糙呈顆粒狀或菜花狀抗酸染色 結(jié)核桿菌對苯胺染料一般不易著色，但經(jīng)加溫或延長染色時間后能抵抗3鹽酸乙醇的脫色作用。經(jīng)此法染色后，結(jié)核桿菌及其他分枝桿菌呈紅色，非抗酸菌呈藍色。示教：結(jié)核分枝桿菌抗酸染色 實驗步驟：1用竹簽挑取開放性肺結(jié)核病人晨痰標(biāo)本中干酪樣小粒或膿性局部，或用取菌環(huán)挑取經(jīng)酸、堿處理后

6、的痰標(biāo)本，置載玻片中央，均勻涂布成1.52.0cm卵圓形痰膜，枯燥，固定。2滴加石炭酸復(fù)紅液于涂片上，玻片置于酒精燈火焰緩緩加熱，至有蒸汽冒出，約維持5min切勿沸騰或使染液干涸于玻片上。如有染液干涸趨勢，應(yīng)補加染液。然后自然冷卻，用流水漂去多于染液。3滴加3鹽酸乙醇脫色，至涂片較厚處無顏色脫出為止，約30s，水洗。4滴加堿性亞甲藍液復(fù)染1min，水洗。5濾紙吸干后鏡檢。實驗材料痰液1管/小組，抗酸染色染液1套/小組，玻片1個/人實驗報告內(nèi)容 抗酸染色法實驗原理、實驗步驟、實驗結(jié)果繪圖和結(jié)果分析。0B3F6I9LdOgSjVmYq!t&w-z1C4G7JbMePhTkWnZr$u(x+A2E5

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