細菌感染不論在醫院、自然環境中一直都是威脅人類身體健康的議題，然而在臨床上細菌感染的即時診斷是困難的，即使科學家不斷各種抗生素來治療細菌感染，但敗血症仍然是全球主要死因之一。現有細菌檢測主要可分成兩大類:(1)傳統培養基檢測法:細菌經過特殊培養基培養1~2天後觀察菌落顏色變化。(2)免疫分析法:利用抗體或抗原進行相關檢測，可獲得高專一性之檢測結果。目前細菌檢測上尚未具有低成本、簡單操作、不需額外設備協助之快速檢測方法，以即時提供樣品中細菌濃度之資訊。在本研究我們選擇大腸桿菌及金黃色葡萄球菌作為偵測細菌，大腸桿菌為自然環境中常見的指標性細菌，而金黃色葡萄球菌經常是造成細菌感染的原因。本研究利用MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 及PMS (Phenazine methosulfate)與細菌內酵素產生氧化還原反應，並添加氫氧化鈉增強反應呈色效果。實驗結果顯示，細菌與反應試劑在595 nm波長下隨著細菌濃度越高有較高的吸收度。我們也利用伊紅Y-甲基藍複合染劑(Eosin Y-Methylene blue)及Triton X-100來分辨不同濃度的細菌，在菌液中依序加入添加Triton X-100之伊紅Y溶液、亞甲基藍染劑，根據細菌成分化學性質不同，細菌細胞質透過離子鍵與酸性染料伊紅Y結合呈粉紅色，細菌核酸透過離子鍵與鹼性染料亞甲基藍結合成藍紫色。隨著樣本中細菌量越多被染成藍紫色的核酸越多，因此，菌量越多顏色越深，反之菌量越少則顏色越淡。

Bacterial infections have been a threat to human health in hospitals and the natural environment. However, immediate diagnosis of bacterial infections is difficult. Even if various antibiotics have been administrated to treat bacterial infections, sepsis is still one of the major causes of death worldwide. Nowadays, the bacteria detection methods can be mainly divided into two categories: (1) traditional medium detection method: bacteria are cultured in a special medium for 1 to 2 days to observe the colony color change. (2) Immunoassay: The detection of antibodies or antigens can be used to obtain highly specific test results. At present, there is no rapid detection method for low-cost, simple operation and no additional equipment requirement for bacterial detection. In an addition, current methods are unable to provide instant information about the concentration of bacteria in the sample. In our study, we chose Escherichia coli and Staphylococcus aureus as the materials. We used MTT - PMS reagent to produce redox with enzymes in bacterium, and add Sodium hydroxide to enhance the color of the reaction. The absorbance value in 595 nm increases with the increasing concentration of bacteria. We use Eosin Y - Methylene blue reagent to identify different concentrations of bacteria.

Abstract Ⅰ
摘要 Ⅱ
誌謝辭 Ⅲ
目錄 Ⅳ
圖目錄 Ⅷ
第一章、緒論 1
1.1研究動機與目的 1
1.1.1細菌檢測於臨床上重要性 1
1.2細菌檢測技術 4
1.3細菌檢測技術分類 5
1.3傳統培養法 5
1.3.2分生分析法(PCR放大法) 7
1.3.3酵素免疫分析法 9
1.3.4以 MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)及吩嗪硫酸甲酯 (Phenazine methosulfate, PMS)檢測細菌 11
1.3.5 以2-(2-甲氧基-4-硝苯基)-3-(4-硝苯基)-5-(2,4-二磺基苯)-2H四唑單鈉 (2-(2-Methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium sodium salt, WST-8)及1-甲氧基-5-甲基酚嗪硫酸甲酯鹽 (1-Methoxy-5-methylphenazinium methyl sulfate, 1-Methoxy PMS) 檢測細菌 13
1.3.6以伊紅y(Eosin Y) 及亞甲基藍 (Methylene blue) 檢測細菌 14
1.4細胞破碎技術 15
1.5革蘭氏陽性菌(Gram positive) 16
1.6革蘭氏陰性菌(Gram negative) 17
1.7大腸桿菌 (Escherichia coli) 18
1.8金黃色葡萄球菌(Staphylococcus aureus) 19
1.9伊紅Y ( Eosin Y) 20
1.10亞甲基藍(Methylene blue) 21
1.11曲拉通X-100（Triton X-100) 21
第二章、材料與方法 22
2.1 實驗藥品與材料 22
2.1.1 實驗藥品 22
2.1.2實驗材料 23
2.2實驗儀器 23
2.2.1 TECAN Sunrise酵素免疫分析儀測讀儀(ELISA Reader) 23
2.2.2 Thermo超微量核酸定量光譜儀(Nanodrop) 24
2.2.3 顏色感測器(Color sensor) 25
2.3菌液制備 25
2.3.1固態培養基制備 25
2.3.2細菌培養 26
2.4 藥品制備 27
2.4.1 MTT - PMS試劑制備 27
2.4.2 WST-8 - 1-Methoxy PMS 試劑制備 27
2.4.3 細菌複合染劑制備 27
2.5 實驗方法 27
2.5.1以酵素免疫分析儀測讀儀檢測MTT-PMS試劑在各種系統中與不同濃度菌液595 nm吸光值變化 27
2.5.2以超微量核酸定量光譜儀檢測MTT-PMS試劑與不同濃度菌液450nm吸光值變化 29
2.5.3以顏色感測器檢測 MTT - PMS試劑與不同濃度菌液顏色變化 30
2.5.4以酵素免疫分析儀測讀儀檢測WST-8 -1-Methoxy PMS試劑與不同濃度菌液450nm吸光值變化 30
2.5.5以伊紅Y (Eosin Y)及亞甲基藍(Methylene blue)檢測不同濃度菌液 31
第三章、實驗結果與討論 33
3.1以酵素免疫分析儀測量MTT-PMS試劑與不同濃度細菌於不同系統595nm吸光值 33
3.1.1緩衝溶液系統 33
3.1.2尿液系統 35
3.1.3血清系統 36
3.1.4以超微量核酸定量光譜儀測量MTT - PMS試劑與不同濃度細菌於595 nm吸光值 37
3.1.5以顏色感測器連續測量MTT-PMS試劑與不同濃度菌液顏色變化 39
3.2以細菌核酸染色劑法鑑別不同濃度菌液並以酵素免疫分析儀測讀儀測量595nm吸光值 39
3.3以細菌核酸染色劑法鑑別不同濃度菌液並以酵素免疫分析儀測讀儀測量595nm吸光值 40
第四章、結論及未來展望 42
4.1結論 42
4.2未來展望 43
參考文獻 44

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