透過您的圖書館登入
IP:18.224.68.177
  • 學位論文

雷射顯微摘取於細菌分離之研究

Bacterium Isolation Using Laser Capture Microdissection

指導教授 : 陳建銘
若您是本文的作者,可授權文章由華藝線上圖書館中協助推廣。

摘要


本研究中,我們將光纖探針式LCM系統架于一倒立式顯微鏡上,幷在顯微鏡的視野監視下進行細菌樣本的分離研究。細菌在微生物學中是十分重要的研究對象,由于無法用肉眼直接觀察其形態,因此其研究方法有獨特之處。此次先採用已分離、純化好的海洋菌Acinetobacter venetianus來做摘取、以驗證奈米級光纖探針式LCM系統可應用于海洋菌種的分離上。 此次實驗我們將LCM系統架設于顯微鏡上幷將欲摘取的杆菌做螢光染色後固定于玻片上。利用螢光使桿菌在顯微鏡中可清楚觀察。再使用顯微鏡對桿菌做觀察、定位的動作。摘取進行之前,需先將菌種樣本進行染色以利於觀察。實驗發現只有進行革蘭氏染色的細菌,在摘取後幷沒有辦法在顯微鏡的視野下進行觀察。因此將革蘭氏染色後的菌類樣本再進行DAPI螢光染色方便摘取後可以看出細菌樣本是否有被摘取成功。實驗結果發現在大小約為1.0μm 的球杆菌,在雷射探針距離薄膜小于1.0μm的情形下雷射需要觸發至少400ms才可以初步的將細菌摘取分離出

並列摘要


Bacterial cultures are very important study issues in microbiology. Human can’t observe bacterial cultures by naked eyes, thus, the way to study bacterial cultures is totally unique. In traditional, to study unknown bacterial cultures must purify the target bacterium from complex sample conditions. In nature, bacterial cultures are mixed and coexisted. There are among of 99% of the sea bacterial cultures can’t be isolated by culture. In this study, we use laser capture microdissection to isolated a sea bacterial culture, named acinetobacter venetianus which already had been well purified by culture, and to ensure that LCM system can be used in bacterial cultures isolation. we set up LCM system on a headstand microscope, and isolate bacterial cultures under microscope view. Before isolation, we dye bacterial samples easier to be observed. We find out bacterium has been Gram’s dyed can’t be observed on translated film after isolaed in microscope view. Thus, we add DAPI on Gram’s dyed sample and redo experience again. In this study, we discover that bacterial cultures about 1μm in size can be isolated and observed under the condition that the distance between laser probe and film less than 1.0μm and laser light must irradiate translate film more than 400ms

參考文獻


[2] 何淩峰, D. W. Armstrong, 毛細電泳在微生物分離的最新應用, "CHEMISTRY", Sept.2001 Vol.59, No, 3, p381-p390.
[3] R. F. Bonner, E. B. Michael, K. Cole, T. Pohida, R. Chuaqui, S. Goldstein and L. A. Liotta, "Laser capture microdissection: Molecular analysis of tissue," Science, 278, 1997, p1481-p1483.
[4] S. Curran, J. A. McKay, H. L. McLeod and G. I. Murray, "Laser capture microscopy," Molecular Pathology, 53, 2000, p64-p68.
[9] 顏畿府,利用近場光纖探針做奈米級雷射顯微摘取系統之開發,碩士論文,
[1] J. M. Becker, G. A. Caldwell, Eve Ann Zachgo “Biotechnology A Laboratory Course”,臺北市:藝軒圖書出版社,1994,p20-p60.

延伸閱讀