前人研究發現，約有30 多種菌在遭受環境壓力的改變時會進入一種活而不長
的狀態，而壓力來源主要為低溫與飢餓的環境。在過去，通常是探討進入活而不長
狀態後菌體的外形改變或對壓力的耐受性，也有針對壓力處理前期去觀察菌體的形
態變化及基因表現情形，但對於整個活而不長過程的描述是缺少的。因此，本實驗
觀察冷加飢餓誘導進入活而不長過程中的不同時期，選取誘導第0 天、產生許多形
態變異的第3 天、誘導第14 天及進入活而不長後的四個時間點，並將其置於室溫
恢復1-2 天，以光學顯微鏡、掃描式及穿透式電子顯微鏡觀察菌體的形態變化，並
以菌體在環境中可能遭遇的不同壓力進行耐受性的測試，探討兩者之間有無關聯。
實驗結果發現，冷加飢餓誘導3 天後菌體出現許多兔耳狀及棒槌狀等的形態變異，
此時菌體膨大，之後慢慢變小變圓，直到進入VBNC。在穿透式電子顯微鏡的觀察
中，可清楚看到在誘導的過程中菌體內部物質由原先的濃稠逐漸轉為疏鬆，但回溫
後，除了少數菌體之外其餘皆可恢復，且細胞壁有明顯增厚的現象。而在熱、膽鹽
及低鹽處理中，回溫後的菌體對壓力的耐受性皆明顯提高，而在氧化壓力下，除了
第0 天回溫的菌體耐受性提高之外，其餘回溫後的耐受性皆明顯下降。冷加飢餓壓
力下菌體產生的形態變異可能與細胞骨架的基因表現有關，而對壓力耐受性的改變
和細胞壁厚度有無關聯則需進一步的實驗去探討。

Combination of nutrient deprivation and low temperature stresses has been shown to be
the main causes for inducing viable but noncultirable (VBNC) state in some pathogenic
bacteria. Morphological changes and stress susceptibility of the cells in the VBNC state or
during the initial period of stress application have been investigated. In this study, we
focused on the process of the induction of VBNC state contributing to the morphological
changes and stress susceptibility. At different intervals of VBNC induction, the cells were
shifted to temperature up-shift treatment for 1-2 days, and morphological changes were
observed by light, scanning and transmission electron microscopies (SEM and TEM).
Under concomitant cold and carbon starvation for 3 days, cell shape changed from rod-like
to coccoid with budding, dumbbell and rabbit-ear-like shapes. Electron microscopy
revealed that a thick peptidoglycan cell wall in the temperature-upshifted cells. Except for
H2O2, the resistance to bile salt, heat and low salinity was also increased in the
temperature-upshifted cells. Alteration of the morphology of cells under cold and
starvation may be associated with the expression of cytoskeleton genes, and the effect of
thickening cell wall on the tolerance of temperature-upshifted cells needs further
exploration.

表目錄 ………………………………………………………………………… VI
圖目錄 ………………………………………………………………………… VI
附圖目錄 ………………………………………………………………………… VIII
中文摘要 ………………………………………………………………………… 1
英文摘要 ………………………………………………………………………… 2
第壹章 緒論 …………………………………………………………………… 3
第ㄧ節 腸炎弧菌簡介 ……………………………………………………… 4
ㄧ、ㄧ般特性及分類特性 ……………………………………………… 4
二、抗原性 ……………………………………………………………… 5
三、分佈情形 …………………………………………………………… 6
四、致病情形 …………………………………………………………… 7
五、預防與治療 ………………………………………………………… 8
第二節 環境壓力對菌體的影響…………………………………………… 8
ㄧ、張壓壓力 …………………………………………………………… 8
二、膽鹽 ………………………………………………………………… 9
三、溫度 ………………………………………………………………… 11
四、飢餓 ……………………………………………………………… 12
五、過氧化物 …………………………………………………………… 14
第三節 細菌的活而不長狀態 ……………………………………………… 14
ㄧ、活而不長菌體的形成 ……………………………………………… 15
二、活而不長狀態下菌體的形態變化 ……………………………… 16
三、活而不長狀態的恢復 ……………………………………………… 17
四、活而不長狀態對致病性的影響 …………………………………… 19
第四節 腸炎弧菌的活而不長狀態 ………………………………………… 20
一、腸炎弧菌活而不長的形態變化……………………………………… 21
二、腸炎弧菌活而不長的耐受性 22
三、腸炎弧菌活而不長的恢復 ………………………………………… 22
四、腸炎弧菌活而不長的致病性 ……………………………………… 23
第貳章 研究目的與架構 ………………………………………………………… 25
第参章 材料與方法 …………………………………………………………… 26
第一節 菌種與保存 ………………………………………………………… 26
一、菌株 ………………………………………………………………… 26
二、菌種保存 …………………………………………………………… 26
1、Working stock ………………………………………………… 26
2、短期保存 ……………………………………………………… 26
3、長期保存 ……………………………………………………… 27
第二節 藥品與器材設備 …………………………………………………… 27
第三節 生長曲線之測定 …………………………………………………… 28
ㄧ、培養基與誘導液成份 ……………………………………………… 28
1、LB-2% NaCl …………………………………………………… 28
2、Thiosulfate-citrate-bile-sucrose agar …………………………… 29
3、Modified v-5 (MV-5) medium ………………………………… 29
4、Modified v-5 without mannitol medium ………………………… 30
5、PBS-0.2% NaCl ………………………………………………… 30
第四節 顯微鏡觀察 ………………………………………………………… 30
ㄧ、位相差光學顯微鏡 ………………………………………………… 30
二、螢光染劑染色 ……………………………………………………… 31
三、螢光顯微鏡記數活菌數…………………………………………… 31
四、穿透式電子顯微鏡樣品製備……………………………………… 32
五、穿透式電子顯微鏡觀察…………………………………………… 33
六、掃描式電子顯微鏡樣品製備及觀察 ……………………………… 33
七、菌體長度與細胞壁厚度的測量與統計…………………………… 33
1、菌體長度與細胞壁厚度的測量 ……………………………… 33
2、取樣與統計 …………………………………………………… 34
第五節 冷加飢餓誘導後對環境壓力耐受性的分析 ……………………… 35
ㄧ、冷加飢餓誘導的處理 ……………………………………………… 35
二、對高溫的耐受性 …………………………………………………… 36
三、對H2O2 的耐受性 …………………………………………………… 36
四、對膽鹽的耐受性 …………………………………………………… 36
五、對低鹽的耐受性 …………………………………………………… 37
第肆章 結果 ……………………………………………………………………… 38
第一節 細菌的生長情形 …………………………………………………… 38
ㄧ、腸炎弧菌Vp1137 的生長曲線 …………………………………… 38
二、冷加飢餓誘導不同天數之菌數變化 ……………………………… 38
第二節 冷加飢餓誘導下菌體的形態變化………………………………… 38
一、光學顯微鏡觀察結果 ……………………………………………… 38
二、電子顯微鏡觀察結果 ……………………………………………… 40
1、掃描式電子顯微鏡 …………………………………………… 40
2、穿透式電子顯微鏡 …………………………………………… 41
三、冷加飢餓誘導不同天數後菌體長度之變化 ……………………… 42
四、冷加飢餓誘導不同天數後菌體細胞壁厚度之變化 ……………… 43
第三節 冷加飢餓誘導不同時期菌體對壓力的耐受情形………………… 43
一、對熱處理的耐受性 ………………………………………………… 43
二、對H2O2 處理的耐受性……………………………………………… 43
三、對膽鹽處理的耐受性 ……………………………………………… 44
四、對低鹽處理的耐受性 ……………………………………………… 44
第伍章 討論 ……………………………………………………………………… 45
第一節 冷加飢餓誘導對菌體的影響……………………………………… 45
一、對細菌生長的影響 ………………………………………………… 45
二、對細菌形態變化的影響…………………………………………… 46
第二節 冷加飢餓誘導對壓力耐受性的影響 ……………………………… 49
一、對高溫耐受性的影響 ……………………………………………… 49
二、對H2O2 耐受性的影響……………………………………………… 49
三、對膽鹽耐受性的影響 ……………………………………………… 50
四、對低鹽耐受性的影響 ……………………………………………… 51
第六章 參考文獻 ………………………………………………………………… 52
表目錄
表一、冷加飢餓誘導下菌體細胞壁厚度的變化及對於壓力耐受性的增減 … 60
圖目錄
圖一、腸炎弧菌 Vp1137 之生長曲線 ………………………………………… 61
圖二、腸炎弧菌Vp1137 冷加飢餓誘導不同天數之菌數變化 ……………… 62
圖三、腸炎弧菌Vp1137 經冷加飢餓誘導進入VBNC 過程光學顯微鏡觀察結
果 …………………………………………………………………………… 63
圖四、腸炎弧菌Vp1137 經冷加飢餓誘導進入VBNC 過程掃描式電子顯微鏡
觀察結果 …………………………………………………………………… 64
圖五、腸炎弧菌Vp1137 經冷加飢餓誘導進入VBNC 過程掃描式電子顯微鏡
觀察結果 …………………………………………………………………… 65
圖六、腸炎弧菌Vp1137 經冷加飢餓誘導進入VBNC 過程掃描式電子顯微鏡
觀察結果 …………………………………………………………………… 66
圖七、腸炎弧菌Vp1137 經冷加飢餓誘導進入VBNC 過程掃描式電子顯微觀
察結果 ……………………………………………………………………… 67
圖八、腸炎弧菌Vp1137 經冷加飢餓誘導進入VBNC 過程穿透式電子顯微鏡
觀察結果 ……………………………………………………………… 68
圖九、腸炎弧菌Vp1137 經冷加飢餓誘導進入VBNC 過程穿透式電子顯微
鏡觀察結果 ………………………………………………………………
69
圖十、腸炎弧菌Vp1137 經冷加飢餓誘導進入VBNC 過程穿透式電子顯微鏡
觀察結果 …………………………………………………………………… 70
圖十一、腸炎弧菌Vp1137 經冷加飢餓誘導進入VBNC 過程穿透式電子顯微
鏡觀察結果 ……………………………………………………………… 71
圖十二、腸炎弧菌Vp1137 經冷加飢餓誘導進入VBNC 過程穿透式電子顯微
鏡觀察結果……………………………………………………………… 72
圖十三、腸炎弧菌Vp1137 經冷加飢餓誘導不同天數後菌體長度之變化 … 73
圖十四、腸炎弧菌Vp1137 經冷加飢餓誘導不同天數後菌體細胞壁厚度之變
化 ……………………………………………………………………… 74
圖十五、腸炎弧菌Vp1137 經冷加飢餓誘導不同天數後以50 oC 熱處理結果
………………………………………………………………………… 75
圖十六、腸炎弧菌Vp1137 經冷加飢餓誘導不同天數後以0.03% H2O2 處理結
果 ………………………………………………………………………… 76
圖十七、腸炎弧菌Vp1137 經冷加飢餓誘導不同天數後以2% 膽鹽處理結果
………………………………………………………………………….. 77
圖十八、腸炎弧菌Vp1137 經冷加飢餓誘導不同天數後以PBS-0.2% NaCl
處理結果 ……………………………………………………………… 78
附圖目錄
附圖一、VayTek Image 軟體使用介面 ………………………………………… 34

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