1、實驗五腸道致病菌的微生物學檢查結核分枝桿菌的培養及形態觀察 醫學微生物學實驗實驗內容：1.腸道致病菌的微生物學檢查2.結核分枝桿菌的培養及形態觀察抗酸染色1.腸道致病菌的微生物學檢查 1實驗目的Aaaaaaaaaaaaaaaa示教：常見腸道細菌2常用培養基介紹主要包括：麥康克培養基、伊紅美藍培養基、中國藍瓊脂平板、SS瓊脂平板、雙糖鐵培養基示教：伊紅美藍培養基 雙糖鐵培養基 SS平板別離腸道病原菌的強選擇培養基成分和作用：根底培養基：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

2、生堿性物質菌落淡黃色；大腸桿菌分解乳糖產酸，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基克氏雙糖培養基接種方法如何觀察上層含乳糖局部乳糖發酵：致病菌紅色，非致病菌黃色H2S然后觀察下層發酵葡萄糖產酸產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本收集別離培養初步鑒定血清學鑒定生化反響標本選擇鑒別培養基 SS平板生化反響血清學鑒定雙糖管等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尿素水解試驗示教：生化反響舉例 糖發酵試驗(fermentaton test)示教：單糖發酵管 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 產氣桿菌 2.結核分枝桿菌的培養及形態觀察發病趨勢和臨床意義：aaaaaaaaa結核分枝桿菌形態：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，背景中其他物質藍色。培養特性：專性需氧，最適37 ，營養要求高，生長緩慢，18h分裂一代。羅氏(Lowenstein-Jensen)培養基含蛋黃、甘油、馬鈴薯、無機鹽、孔雀綠，26周可見菌落生長，菜花、顆粒或結節狀菌落，乳白色或黃色，不透明。在液體培養基，生長于外表，有皺褶。結核分枝桿菌的培養方法1痰標本培養前處理1酸處理法：痰標本參加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：痰標本參加2氫氧化鈉溶液稀釋24倍，置37溫箱內30min，期間振蕩23次，痰液化后即可接種。2接種培養法 取上述處理過的痰液0.1ml均勻接種于改進羅氏培養基斜面，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 結核桿菌對苯胺染料一般不易著色，但經加溫或延長染色時間后能抵抗3鹽酸乙醇的脫色作用。經此法染色后，結核桿菌及其他分枝桿菌呈紅色，非抗酸菌呈藍色。示教：結核分枝桿菌抗酸染色 實驗步驟：1用竹簽挑取開放性肺結核病人晨痰標本中干酪樣小粒或膿性局部，或用取菌環挑取經酸、堿處理后

6、的痰標本，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/人實驗報告內容 抗酸染色法實驗原理、實驗步驟、實驗結果繪圖和結果分析。0B3F6I9LdOgSjVmYq!t&w-z1C4G7JbMePhTkWnZr$u(x+A2E5

7、H8KcNfRiUlXp#s%v)y0C3F6IaLdOgSjVnYq!t*w-z1D4G8JbMeQhTkWoZr%u(x+B2E5H9KcOfRiUmXp#s&v)y0C3F7IaLdPgSjVnYq$t*w-A1D4G8JbNeQhTlWoZr%u(y+B2E6H9KcOfRjUmXp!s&v)z0C4F7IaMdPgSkVnZq$t*x-A1D5G8JbNeQiTlWo#r%u(y+B3E6H9LcOfRjUmYp!s&w)z0C4F7JaMdPhSkVnZq$u*x-A2D5G8KbNfQiTlXo#r%v(y0B3E6I9LcOgRjVmYp!t&w)z1C4F7JaMePhSkW

8、nZq$u*x+A2D5H8KbNfQiUlXo#s%v(y0B3F6I9LdOgRjVmYq!t&w-z1C4G7JbMePhTkWnZr$u(x+A2E5H8KcNfQiUlXp#s%v)y0B3F6IaLdOgSjVmYq!t*w-z1D4G7JbMeQhTkWoZr$u(x+B2E5H9KcNfRiUmXp#s&v)y0C3F7IaLdPgSjVnYq!t*w-A1D4G8JbMeQhTlWoZr%u(x+B2E6H9KcOfRiUmXp!s&v)z0C3F7IaMdPgSkVnYq$t*x-A1D5G8JbNeQiTlWo#r%u(y+B3E6H9LcOfRjUmXp!s&w)z0C

9、4F7IaMdPhSkVnZq$t*x-A2D5G8KbNeQiTlXo#r%v(y+B3E6I9LcOgRjUmYp!t&w)z1C4F7JaMePhSkWnZq$u*x-A2D5H8KbNfQiTlXo#s%v(y0B3E6I9LdOgRjVmYp!t&w-z1C4G7JaMePhTkWnZr$u*x+A2E5H8KcNfQiUlXp#s%v)y0B3F6I9LdOgSjVmYq!t&w-z1D4G7JbMePhTkWoZr$u(x+A2E5H9KcNfRiUlXp#s&v)y0C3F6IaLdPgSjVnYq!t*w-A1D4G8JbMeQhTlWoZr%u(x+B2E5H9KcOfRi

10、UmXp#s&v)z0C3F7IaLdPgSkVnYq$t*w-A1D5G8JbNeQhTlWo#r%u(y+B2E6H9LcOfRjUmXp!s&w)z0C4F7IaMdPgSkVnZq$t*x-A1D5G8KbNeQiTlWov)z0C3F7IaLdPgSkVnYq$t*w-A1D5G8JbNeQhTlWo#r%u(y+B2E6H9LcOfRjUmXp!s&v)z0C4F7IaMdPgSkVnZq$t*x-A1D5G8KbNeQiTlWo#r%v(y+B3E6H9LcOgRjUmYp!s&w)z1C4F7JaMdPhSkWnZq$u*x-A2D5G8KbNfQiTlXo#r%v(y0B3E

11、6I9LcOgRjVmYp!t&w)z1C4G7JaMePhSkWnZr$u*x+A2D5H8KcNfQiUlXo#s%v)y0B3F6I9LdOgRjVmYq!t&w-z1C4G7JbMePhTkWnZr$u(x+A2E5H8KcNfRiUlXp#s%v)y0C3F6IaLdOgSjVnYq!t*w-z1D4G8JbMeQhTkWoZr%u(x+B2E5H9KcNfRiUmXp#s&v)y0C3F7IaLdPgSjVnYq$t*w-A1D4G8JbNeQhTlWoZr%u(y+B2E6H9KcOfRjUmXp!s&v)z0C4F7IaMdPgSkVnYq$t*x-A1D5G8JbNeQiTl

12、Wo#r%u(y+B3E6H9LcOfRjUmYp!s&w)z0C4F7JaMdPhSkVnZq$u*x-A2D5G8KbNfQiTlXo#r%v(y0B3E6I9LcOgRjUmYp!t&w)z1C4F7JaMePhSkWnZq$u*x+A2D5H8KbNfQiUlXo#s%v(y0B3F6I9LdOgRjVmYq!t&w-z1C4G7JbMePhTkWnZr$u*x+A2E5H8KcNfQiUlXp#s%v)y0B3F6IaLdOgSjVmYq!t*w-z1D4G7JbMeQhTkWoZr$u(x+B2E5H9KcNfRiUmXp#s&v)y0C3F6IaLdPgSjVnYq!t*w-A1

13、D4G8JbMeQhTlWoZr%u(x+B2E6H9KcOfRiUmXp!s&v)z0C3F7IaMdPgSkVnYq$t*x-A1D5G8JbNeQiTlWo#r%u(y+B2E6H9LcOfRjUmXt*w-A1D4G8JbMeQhTlWoZr%u(x+B2E6H9KcOfRiUmXp!s&v)z0C3F7IaMdPgSkVnYq$t*w-A1D5G8JbNeQhTlWo#r%u(y+B2E6H9LcOfRjUmXp!s&w)z0C4F7IaMdPhSkVnZq$t*x-A2D5G8KbNeQiTlXo#r%v(y+B3E6I9LcOgRjUmYp!s&w)z1C4F7JaMdPhSkW

14、nZq$u*x-A2D5H8KbNfQiTlXo#s%v(y0B3E6I9LdOgRjVmYp!t&w-z1C4G7JaMePhTkWnZr$u*x+A2D5H8KcNfQiUlXo#s%v)y0B3F6I9LdOgSjVmYq!t&w-z1D4G7JbMePhTkWoZr$u(x+A2E5H9KcNfRiUlXp#s&v)y0C3F6IaLdOgSjVnYq!t*w-z1D4G8JbMeQhTkWoZr%u(x+B2E5H9KcOfRiUmXp#s&v)z0C3F7IaLdPgSkVnYq$t*w-A1D5G8JbNeQhTlWo#r%u(y+B2E6H9KcOfRjUmXp!s&v)z0C

15、4F7IaMdPgSkVnZq$t*x-A1D5G8KbNeQiTlWo#r%v(y+B3ELdPgSkVnYq$t*w-A1D4G8JbNeQhTlWoZr%u(y+B2E6H9KcOfRjUmXp!s&v)z0C4F7IaMdPgSkVnZq$t*x-A1D5G8KbNeQiTlWo#r%v(y+B3E6H9LcOgRjUmYp!s&w)z0C4F7JaMdPhSkVnZq$u*x-A2D5G8KbNfQiTlXo#r%v(y0B3E6I9LcOgRjVmYp!t&w)z1C4G7JaMePhSkWnZr$u*x+A2D5H8KbNfQiUlXo#s%v(y0B3F6I9LdOgRjVmY

16、q!t&w-z1C4G7JbMePhTkWnZr$u(x+A2E5H8KcNfRiUlXp#s%v)y0C3F6IaLdOgSjVmYq!t*w-z1D4G7JbMeQhTkWoZr$u(x+B2E5H9KcNfRiUmXp#s&v)y0C3F7IaLdPgSjVnYq$t*w-A1D4G8JbNeQhTlWoZr%u(y+B2E6H9KcOfRiUmXp!s&v)z0C3F7IaMdPgSkVnYq$t*x-A1D5G8JbNeQiTlWo#r%u(y+B3E6H9LcOfRjUmYp!s&w)z0C4F7JaMdPhSkVnZq$t*x-A2D5G8KbNeQiTlXo#r%v(y+B3E

17、6I9LcOgRjUmYp!t&w)z1C4F7JaMePhSkWnZq$u*x+A2D5H8KbNfQiUlXo#s%v(y0B3F6I9LdOgRjVmYp!t&w-z1C4G7JaMePhTkWnZr$u*x+A2E5H8KcNfQiUlXp#s%v)y0B3F6IaLhSkWnZq$u*x+A2D5H8KbNfQiTlXo#s%v(y0B3E6I9LdOgRjVmYp!t&w-z1C4G7JaMePhTkWnZr$u*x+A2E5H8KcNfQiUlXp#s%v)y0B3F6IaLdOgSjVmYq!t*w-z1D4G7JbMePhTkWoZr$u(x+A2E5H9KcNfRiUlXp

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

19、cOgRjUmYp!s&w)z1C4F7JaMdPhSkWnZq$u*x-A2D5H8KbNfQiTlXo#r%v(y0B3E6I9LcOgRjVmYp!t&w)z1C4G7JaMePhSkWnZr$u*x+A2D5H8KcNfQiUlXo#s%v)y0B3F6I9LdOgSjVmYq!t&w-z1D4G7JbMePhTkWnZr$u(x+A2E5H8KcNfRiUlXp#s%v)y0C3F6IaLdOgSjVnYq!t*w-z1D4G8JbMeQhTkWoZr%u(x+B2E5H9KcOfRiUmXp#s&v)y0C3F7IaLdPgSjVnYq$t*w-A1D4G8JbNeQhTlWoZr

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#