臺灣博碩士論文加值系統

細菌培養在微生物學上是個基本且重要的技術，而晶片上微小化生物反應室是為了改善大尺度生物反應室需要消耗大量的試劑與人力的問題，我們設計了一個兩間隔隔室的微流體生物反應室，並且利用它成功的完成了序列稀釋的實驗，我們的晶片尺度在奈升等級，利用電腦可自動化操作實驗並且長時間的觀測、培養微生物。由於晶片內的高面積體積比使得生物反應室不只可培養浮游生物，更能有效的成長生物膜，為了得到浮游微生物不受生物膜影響下的生長狀況，我利用介面活性劑Tween20抑制生物膜的成長並且量測出純浮游生物在反應室內的生長狀況。

Bacterial culture is a basic technique in both fundamental and applied microbiology.The excessive reagent consumption and laborious maintenance of bulk bioreactors for microbial culture have prompted the development of miniaturized on-chip bioreactors.With the minimal choice of two compartments (N=2) and discrete time,periodic dilution steps,we realize a microfluidic bioreactor that mimics macroscopic serial dilution transfer culture.This device supports automated, long-term microbial cultures with a nanoliter-scale working volume and real-time monitoring of microbial populations at single-cell resolution. The use of surfactant Tween20 suppresses the biofilm growth and enables the pure planktonic growth.

一、緒論 1
1-1研究動機 1
1-2文獻回顧 2
1-2-1細菌培養 2
1-2-2恆化器 3
1-2-3微型恆化器 4
1-3微流體晶片介紹 6
1-3-1光罩 6
1-3-2模仁 7
1-3-3閥門 7
1-3-4蠕動汞浦與圓形汞浦 8
1-3-5生物反應器 9
二、 微流體晶片製作與系統架設 10
2-1微流體晶片製作 10
2-1-1微流體晶片設計 10
2-1-2模仁製作 12
2-1-3晶片製作 13
2-1-4 1/4稀釋 16
2-2系統架設 17
2-2-1硬體系統 17
2-2-2 Laview控制系統 18

2-2-3溫度控制系統及光學顯微鏡 19
三、 實驗結果 20
3-1 大腸桿菌樣品準備 20
3-2實驗前晶片表面處理及系統參數 20
3-3培養結果 22
3-4 M9營養液與LB營養液之大腸桿菌培養比較 30
3-5加藥實驗 33
四、結論 34
附錄 35
中英對照表 40
參考文獻 41

 

1.J. Monod, "The Growth of Bacterial Cultures," Annu. Rev. Microbiol. 3, 371 (1949).

2.J. Monod, "La technique de culture continue.Théorie et applications," Ann. Inst. Pasteur. 79, 390 (1950).

3.A. Novick and L. Szilard, "Description of Chemostat," Science 112, 715 (1950).

4.H. L. Smith and P. Waltman, "The theory of chemostat: Dynamics of microbial competition," Cambridge University Press. (1995).

5.D. B. Weibel et al., "Microfabrication meets microbiology," Nat. Rev. Microbiol. 5, 209 (2007).

6.H. M. Hegab, A. El Mekawy, and T. Stakeborg, "Review of microfluidic microbioreactor technology for high-throughput submerged microbiological cultivation," Biomicrofluidics 7, 21502 (2013).

7.A. Groisman et al., "A microfluidic chemostat for experiments with bacterial and yeast cells," Nat. Methods 2, 685 (2005).

8.J. R. Moffitt et al., "The single-cell chemostat: an agarose-based, microfluidic device for high-throughput, single-cell studies of bacteria and bacterial communities," Lab Chip 12, 1487 (2012).

9.Z. Long et al., "Microfluidic chemostat for measuring single cell dynamics in bacteria," Lab Chip 13, 947 (2013).

10.F. K. Balagadde, L. C. You, C. L. Hansen, F. H. Arnold, and S. R. Quake, "Long-Term Monitoring of Bacteria Undergoing Programmed Population Control in a Microchemostat," Science 309, 137 (2005).

11.F. K. Balagadde et al., "A synthetic Escherichia coli predator–prey ecosystem," Mol. Syst. Biol. 4, 187 (2008).

12.J. Park, J. Wu, M. Polymenis, and A. Han, "Microchemostat array with small-volume fraction replenishment for steady-state microbial culture," Lab Chip 13, 4217 (2013).

13.J. W. Costerton, P. S. Stewart, and E. P. Greenberg, "Bacterial Biofilms:A Common Cause of Persistent Infections," Science 284, 1318 (1999).

14.T. Danino, O. Mondragon-Palomino, L. Tsimring, and J. Hasty, "A synchronized quorum of genetic clocks," Nature 463, 326 (2010).

15.R. A. Kellogg, R. Gomez-Sjoberg, A. A. Leyrat, and S. Tay, "High-throughput microfluidic single-cell analysis pipeline for studies of signaling dynamics," Nat. Protocols 9, 1713 (2014).

16.Guo-Yue Gu, Yi-Wei Lee, Chih-Chung Chiang, and Ya-Tang Yang, "A nanoliter microfluidic serial dilution bioreactor," Biomicrofluidics 9, 044126 (2015).

17.R. Mohan et al., "A multiplexed microfluidic platform for rapid antibiotic susceptibility testing," Biosens. Bioelectron. 49, 118 (2013).

18.D. S. Clark and H. W. Blanch, "Biochemical Engineering, 2nd ed," CRC Press, Boca Raton (1996).

19.Chih-Chung Chiang, "A minimal nanoliter microfluidic chemostat," Master Thesis, National Tsing Hua University (2014).

20.B. Okumus, S. Yildiz, and E. Toprak, "Fluidic and microfluidic tools for quantitative systems biology," Curr. Opin. Biotechnol. 25, 30 (2014).

21.R. Kishony et al., "Evolutionary paths to antibiotic resistance under dynamically sustained drug selection," Nat. Genetics 44, 101 (2012).

22.R. Kishony et al., " Building a morbidostat: an automated continuous-culture device for studying bacterial drug resistance under dynamically sustained drug inhibition," Nat. Protocols 8, 555 (2013).