水產養殖通常利用抗生素的使用抑制致病菌。然而，長期使用抗生素會導致
抗藥菌的產生，對生物及人類會造成威脅。因此，利用噬菌體來控制環境中的致病菌及抗藥菌。本研究中水樣採集的地點為台南 CLOZ 牡蠣養殖區和雲林 SCKF
錦鯉場。於 CLOZ 水樣及牡蠣體組織中分離出弧菌噬菌體，而 SCKF中分離出抗藥菌噬菌體。呈現小圓溶菌斑的弧菌噬菌體的透過 Top ager 濃度調降，使得溶菌
斑變大易於觀察。於電子顯微鏡分類中，該弧菌噬菌體與淡水抗藥噬菌體屬於Podoviridae 和 Myroviridae Family。萃取出的弧菌噬菌體與抗藥菌噬菌體的
genome ，於不同限制酶作用下皆能被HindIII切出不同大小之DNA片段。經NCBI已知相似大小之噬菌體染色體，經限制酶圖譜分出不同片段大小下比對。弧菌噬
菌體ϕ20 可能分屬於 Podoviridae 的 Picovirinae。而淡水抗藥菌噬菌體ϕ70 則可能為 Myroviridae的 Mu-like viruses 或 unclassified Myoviridae。在不同效價噬菌體於不同菌齡的感染中，在效價(103 PFU/ml)進行感染，弧菌噬菌體裂解宿主的情形較不明顯，而以效價(107
PFU/ml)進行感染於不同菌齡的於不同的宿主生長期感
染噬菌體ϕA 和ϕ20，ϕA於不同菌齡感染皆可於感染後兩個小時後溶裂使OD達最低，ϕ20可於感染後在log phase進行裂解宿主，淡水抗藥菌噬菌體ϕ70則在不同菌齡宿主生長階段感染後六個小時，進行溶裂使OD達最低。

Abstract
The use of antibiotics in aquaculture may cause development of antibiotic resistance among pathogens infecting cultured animals and humans. Therefore, the phages were isolated from the culture environment that can infect the pathogen and resistant bacteria. In this study, there were vibriophage and antibacterial phage isolated
from CLOZ andSCKF. The small and circle plaque of vibriophage could become
striking by decrease in top agar percentage. The electron micrographs of vibriophage
and antibacterial phage belonged to the Podoviridae and Myroviridae family. The
phages genome could be cut by HidinIII. The different size fragments were compared
and matched to similar genome size of phages from NCBI. For the result, vibriophage may belong to the Picovirinae in Podoviridae. The antibacterial phage would be classified into either Mu-like viruses or unclassified Myoviridae. In the infecting test with (103 PFU/ml), the vibriophage lysing the host cell was not evident. Then, infecting with ϕA, ϕ20 and ϕ70 107 PFU/ml), the ϕA could lyse the cell and test the lowest OD after two hours by infecting. ϕ20 lysed the cell at exponential phase and antibacterial phage ϕ70 could lysed the host cell at different ages after six hours by infecting. A could lyse the cell and test the lowest OD after two hours by infecting. ϕ20 lysed the cell at exponential phase and antibacterial phage ϕ70 could lysed the host cell at different ages after six hours by infecting.

謝誌…………………………………………………………I
摘要…………………………………………………………II
Abstract……………………………………………………III
目錄……………………………………………………………IV
表目錄……………………………………………… VIII
圖目錄…………………………………………… IX

壹、前言……………………………………………………………1
一、台灣的養殖現況及發展………………………………1
二、牡蠣養殖現況…………………………………………2
三、觀賞錦鯉養殖現況……………………………………2
四、弧菌致病菌對養殖之影響…………………………………………3
五、抗生素使用對養殖之影響…………………………………………5
六、細菌抗藥性的檢測…………………………………………………5
1. 紙錠瓊脂擴散試驗法……………………………………………6
2.稀釋試驗法………………………………………………6
七、噬菌體………………………………………7
1. 噬菌體發現………………………………………………7
2. 噬菌體之外觀及分類………………………………7
3. 噬菌體的生活史…………………………………………………9
八、噬菌體的療法…………………………………………………10
九、噬菌體療法之應用……………………………………………11
1. 噬菌體療法人體應用………………………………11
2. 噬菌體療法動物實驗……………………………………………11
3. 噬菌體療法養殖生物實驗………………………………………11
十、研究目的………………………………………………………13
貳、材料和方法…………………………………………………………14
一、樣品採集…………………………………………………………14
二、細菌藥物敏感性測試……………………………………………… 14
三、特殊培養基篩選與分類…………………………………………… 16
四、革蘭氏染色…………………………………………………… 17
五、宿主細菌的培養…………………………………………………… 17
六、噬菌體的分離(isolation)……………………………………………18
七、噬菌體效價之測定(titer)……………………………………………18
八、純化單株噬菌體…………………………………………………… 18
九、菌種和噬菌體之保存………………………………………………19
十、噬菌體之增殖(amplification)………………………………………19
十一、大量噬菌體液製備………………………………………………19
十二、不同濃度上層膠對溶菌斑大小影響……………………………20
十三、噬菌體之吸附能力………………………………………………20
十四、噬菌體宿主專一性………………………………………………20
十五、電子顯微鏡之前處理……………………………………………21
十六、噬菌體DNA之萃取………………………………………………21
十七、洋菜凝膠電泳製備與分析(agarose gel electrophoresis)…………22
十八、限制酶切割(Restriction enzyme digestion)………………………23
十九、細菌的生長曲線與細菌的裂解(bacteriolysis)曲線………………23
1. 細菌的生長曲線…………………………………………………23
2. 細菌裂解(bacteriolysis)曲線……………………………………23
二十、噬菌體與宿主細菌對溫度之敏感性……………………………24
二十一、不同效價噬菌體感染不同菌齡之影響……………………… 24
参、結果………………………………………………………………………25
一、CLOZ抗藥菌株分析…………………………………………………25
二、SCKF抗藥菌株分析………………………………………………26
三、CLOZ海水噬菌體之分離…………………………………………27
四、SCKF淡水噬菌體之分離…………………………………………28
五、噬菌體在MSWYE和ASW吸附能力………………………………28
六、上層膠(top agar)的濃度對噬菌體溶菌斑大小之影響……………29
七、噬菌體ϕ20和ϕ70負染電顯圖………………………………………. 30
八、弧菌及弧菌噬菌體ϕA和ϕP對溫度的敏感性……………………… 31
九、噬菌體宿主專一性………………………………………………… 32
十、宿主成長曲線與噬菌體裂解曲線………………………………… 32
十一、噬菌體(103 PFU/ml)感染不同菌齡的宿主……………………33
十二、噬菌體(107 PFU/ml)感染不同菌齡的宿主……………………… 35
十三、噬菌體限制酶 (restriction enzyme) 圖譜分析…………………38
肆、討論………………………………………………………………………40
一、抗藥菌比例和特性分析……………………………………………40
二、噬菌體分類………………………………………………………… 42
三、上層膠對噬菌體溶菌斑(plaque)大小………………………………43
四、噬菌體宿主範圍…………………………………………………… 44
五、不同環境條件下噬菌體的吸附力(adsorption)…………………… 45
六、不同溫度對噬菌體和宿主之敏感性……………………………… 46
七、不同效價噬菌體感染不同宿主菌齡之影響……………………… 47
八、噬菌體限制酶(restriction enzyme)圖譜分析……………………… 50
九、未來展望……………………………………………………………52
伍、參考文獻…………………………………………………………………53
陸、表………………………………………………………………………… 59
柒、圖………………………………………………………………………… 75
附錄A…………………………………………………………………………97
附錄B…………………………………………………………………………98
附錄C…………………………………………………………………………99
附錄D…………………………………………………………………………100
附錄E…………………………………………………………………………101

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