臺灣博碩士論文加值系統

為了解生物附著形成機制，挑選由台灣基隆八尺門所分離出的海洋黏液細菌V. alginolyticus HT01作為本論文之實驗菌株，分別討論此菌於飢餓環境下之生菌數、形態及疏水性的變化，並利用隧道型循環測試系統測試四種流速對細菌附著於固體表面的影響，利用掃描式電子顯微鏡(SEM)及穿透式電子顯微鏡(TEM)觀察菌體形態及附著於固體表面的細微構造。菌株的形態由未飢餓時的桿形逐漸變成球形，生菌數在飢餓28天內由108 降至106 CFU/ml，OD650 值亦隨飢餓時間的增加有下降的現象。飢餓46天的細胞具有單鞭毛，形態為球形及桿形，細胞外圍有一層黏性物質包覆，生菌數由108 降至104 CFU/ml。利用溫度轉移使細胞復甦，3天內其生菌數增加至108 CFU/ml。利用BATH及 HIC測試飢餓細菌的表面疏水性變化，兩種方法顯示菌體的疏水性並不隨飢餓時間增加，反而有下降的現象。
選用四種不同流速測定細菌在玻璃片(coverglass)及聚脂樹脂片(polyester disks)上的附著情形，以掃描式電子顯微鏡觀察菌體表面及附著的細微變化。正常細胞附著於玻璃片上的細菌數，在36 h以流速0.38及0.65 km/h 較佳，在聚脂樹脂片上以流速0.38 km/h 高於其他三種流速。以SEM 觀察細菌附著於玻璃片及聚脂樹脂片時，菌體表面有胞外多醣類物質的形成，而菌體型態則隨著附著時間的增長由桿形變成球形，甚至有凹陷的現象。1週飢餓的細胞附著於玻璃片上的細菌數並不隨著流速的改變而有所差異，其在聚脂樹脂片上的最佳附著流速與正常細胞相同，而以SEM 觀察1週飢餓細菌附著於玻璃片時，細胞形態則隨附著時間的增加逐漸變成圈形，在聚脂樹脂片上則無此現象，但菌體表面的胞外多醣類物質較玻璃片者多。流速對2週飢餓細胞的附著情形與1週飢餓細菌相似，而 SEM的觀察，在玻璃片上及聚脂樹脂片上的附著情形與1週飢餓細菌相似，但其胞外多醣類物質則皆明顯增多。

Changes in viable bacterial cell number, cell morphology and hydrophobicity during starvation were determined with a mucoid Vibrio alginolyticus HT01 isolated from Patsu Mesn pier, Keelung, Taiwan. Cell adhesion on solid surface were determined with four flow rates in a tunnel circulation flow system. The fine structure of cell adhesion on solid surface was determined by both scanning electron microscopy and transmitting electron microscopy. Rods cells in normal culture condition was changed to sphere cells under starvation. A normal viable cell count of 108 CFU/ml was reduced to 106 CFU/ml when cells entered in starvation and the cell density of OD650 was decreased accordingly. Cells at 46 days starvation appeared as spheres or rods and still possessed monotrichous flagellum. A mucoid substance surrounded the cell appeared and the cell number was reduced from 108 to 104 CFU/ml. After temperature up-shift for 3 days the starved cells were resuscinated to reach 108 CFU/ml again. Changes in hydrophobicity of starved cells were determined and found decreased when measured with both bacterial adhesion hrdrocarbon (BATH) and hydrophobic interaction chromatography (HIC) methods.
Cell adhesion on both coverglass and polyester disks with four different flow rates were determined. Adhesion of normal cells on coverglass was found highest at 36 h adhesion with both 0.38 and 0.65 km/h flow rates among the four different flow rates determined. For the adhesion on polyester disks, flow rate at 0.38 km/h was also found highest than the other three flow rates. A layer of extracellular polysaccharides was appeared when cell adhesion on both coverglass and polyester disks was examined with SEM. Cell morphology was graduately changed from rods to spheres and even intent shapes as adhesion process prolonged. Cells aderent to coverglass were not varied with changes in flow rate in one week starved cells. These one week starved cells had a similar adhesion rate on coverglass similar to that of normal cells as more cells became sphere when adhesion time was extended. It was not found in the case of adhesion on polyester disks. Nevertheless, more extracellular polysaccharide substance was found on polyester disks than on coveglass. Cell adhesion for two week starved cells was similar to that of one week starved cells. Under SEM observation cells adhesion of two week starvation cells to both coverglass and polyester disks were similar to that of one week starvation cells. But, more extracellular polysaccharides were evidently increased.

目 錄
頁次
圖表目錄.................................................................................................................................................................Ⅳ
中文摘要..................................................................................................................................................................1
英文摘要..................................................................................................................................................................3
前言..........................................................................................................................................................................5
第一章 文獻整理.....................................................................................................................................................7
一、 附著機制.........................................................................................................................................................8
1. 胞外多醣物質......................................................................................................................................................8
2. 鞭毛及纖毛..........................................................................................................................................................9
3. 外膜蛋白質.........................................................................................................................................................10
三、 飢餓變化對附著之影響................................................................................................................................10
四、 影響微生物附著的環境因子........................................................................................................................12
1. 營養濃度.............................................................................................................................................................12
2. 流速.....................................................................................................................................................................13
3. 溫度.....................................................................................................................................................................14
4. 材質.....................................................................................................................................................................14
5. 電解質種類及濃度.............................................................................................................................................15
6. 化學藥劑.............................................................................................................................................................16
五、 生物膜之物性、化性與生物學性質............................................................................................................18
1. 物理性質.............................................................................................................................................................19
2. 化學性質.............................................................................................................................................................19
3. 生物學性質.........................................................................................................................................................20
六、 偵測方法........................................................................................................................................................20
1. 附著試驗的計數方法.........................................................................................................................................20
(1) 稀釋與平板計數法(Dilution and plating methods.............................................................................................20
(2) 顯微鏡檢查法(Microscopy examination)...........................................................................................................21
(3) 分光光度比色法(Spectrophotometry)................................................................................................................22
(4) ATP分析法(ATP assay).......................................................................................................................................22
(5) 放射性標示技術法(Radiolabelling technique methods).....................................................................................22
(6) 生化分析法(Biochemical assay).........................................................................................................................23
2. 細胞外圍物質形成的偵測..................................................................................................................................23
(1) 多醣類................................................................................................................................................................23
(2) 疏水性與電荷....................................................................................................................................................26
中文摘要..................................................................................................................................................................26
英文摘要..................................................................................................................................................................28
壹、 前言.................................................................................................................................................................30
貳、 材料與方法.....................................................................................................................................................31
一、 材料.................................................................................................................................................................31
1.實驗菌株...............................................................................................................................................................31
2.培養基...................................................................................................................................................................31
3.緩衝溶液...............................................................................................................................................................31
4.化學藥品...............................................................................................................................................................32
5.鞭毛與莢膜染色...................................................................................................................................................33
6.電顯固定液..........................................................................................................................................................33
7.試驗材料..............................................................................................................................................................33
8.主要儀器設備......................................................................................................................................................34
二、 方法.................................................................................................................................................................35
1.菌種的保存與活化...............................................................................................................................................35
2.菌體的收集...........................................................................................................................................................35
3.菌株自然變異.......................................................................................................................................................35
4.疏水性試驗...........................................................................................................................................................36
(1)疏水性交互層析法.............................................................................................................................................36
(2)細菌吸附於碳氫化合物.....................................................................................................................................37
5.掃瞄式電子顯微鏡之樣品製備與觀察...............................................................................................................37
6.鞭毛負染色法之製備與觀察...............................................................................................................................38
7.鞭毛與莢膜兩用染色法.......................................................................................................................................38
參、 結果與討論.....................................................................................................................................................39
一、 菌株之形態及生化特性.................................................................................................................................39
二、 菌齡與疏水性之關係.....................................................................................................................................40
三、 低溫無營養條件對生菌數及形態之變化.....................................................................................................41
四、 低溫飢餓對細胞之疏水性變化.....................................................................................................................43
1. BATH (Bacteria adhesion to hydrocarbon)............................................................................................................43
2. HIC (Hydrophobic interaction chromatograpy).....................................................................................................44
第三章 不同流速對飢餓細胞附著於固體表面之影響.........................................................................................47
中文摘要..................................................................................................................................................................47
英文摘要..................................................................................................................................................................49
壹、 前言.................................................................................................................................................................51
貳、 材料與方法.....................................................................................................................................................52
一、 材料............................................................................................................................................................... .52
1. 實驗菌株..............................................................................................................................................................52
2. 緩衝溶液..............................................................................................................................................................52
3. 化學藥品..............................................................................................................................................................52
4. 電顯固定液..........................................................................................................................................................52
5. 試驗材質..............................................................................................................................................................52
6. 主要設備及儀器..................................................................................................................................................53
二、 方法.................................................................................................................................................................53
1.菌株的保存與活化...............................................................................................................................................53
2.菌體的收集...........................................................................................................................................................53
3.附著試驗...............................................................................................................................................................53
4.掃描式電子顯微鏡之樣品製備與觀察...............................................................................................................55
參、 結果與討論.....................................................................................................................................................56
一、隧道式循環測試槽及系統..............................................................................................................................56
二、不同流速對飢餓細胞附著於固體表面之影響..............................................................................................56
1. 正常細胞..............................................................................................................................................................57
2. 飢餓1週的細菌....................................................................................................................................................59
3. 飢餓2週的細菌.....................................................................................................................................................61
第四章 參考文獻......................................................................................................................................................64
圖表目錄
第二章
Table 2-1. Some biochemical characteristics of Vibrio alginolyticus HT01 ..................................................................80
Table 2-2. Effect of 3 media on colony appearance of V. alginolyticus HT01..............................................................81
Table 2-3. Viable count, OD and size change of V. alginolyticus HT01 in MMS buffer during starvation at 4℃ and resuscitation of 46d starved cells at 25℃......................................................................................................................82
Table 2-4. Relative hydrophobicity of V. alginolyticus HT01 determined by BATH in starvation state........................83
Table 2-5. Relative hydrophobicity of V. alginolyticus HT01 determined by HIC in starvation state............................84
Fig. 2-1. V. alginolyticus HT01 possessed a single polar flagellum with flagella stain and negative stain.......................85
Fig. 2-2. The opaque and transparent colonies of V. alginolyticus HT01 possessed different type of capsules ..............86
Fig. 2-3. V. alginolyticus HT01 was grown in TSB.......................................................................................................87
Fig. 2-4. Plate count and OD changes of V. alginolyticus HT01 in MMS buffer during starvation at 4℃......................88
Fig. 2-5. Scanning electron micrographs of V. alginolyticus HT01….…89
Fig. 2-6. V. alginolyticus HT01 in MMS buffer during 7week starvation changed in morphology by negative stain.....91
第三章
Fig. 3-1. Biofilm reactor circulation flow system...........................................................................................................92
Fig. 3-2. Long tunnel flow chambe...............................................................................................................................93
Fig. 3-3. Adhesion of V. alginolyticus HT01 on solid surfaces at various flow rates......................................................94
Fig. 3-4. Adhesion of V. alginolyticus HT01 on coverglass at different exposed times..................................................95
Fig. 3-5. Adhesion of V. alginolyticus HT01 on polyester disks at different exposed times...........................................96
Fig. 3-6. Adhesion of one week starved cells on solid surfaces at various flow rates......................................................98
Fig. 3-7. Adhesion of one week starved cells on coverglass at different exposed times............................................... ..99
Fig. 3-8. Adhesion of one week starved cells on polyester disks at different exposed times..........................................101
Fig. 3-9. Adhesion of two week starved cells on solid surfaces at various flow rates....................................................103
Fig. 3-10. Adhesion of two week starved cells on coverglass at different exposed times..............................................104
Fig. 3-11. Adhesion of two week starved cells on polyester disks at different exposed times.......................................106

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