溶藻弧菌是一種擁有兩種鞭毛馬達系統的海洋類細菌，兩種鞭毛馬達分別是單一長在菌體極端且由鈉離子驅動的鞭毛（極端鞭毛）和多條分佈在全身的氫離子驅動鞭毛（側邊鞭毛）。極端鞭毛在每個生長周期都會生長，而側邊鞭毛的生長則仰賴特定環境的誘發。當細菌長在瓊脂平板上時，菌體會抑制分裂，進入群游的型態，長出數量較多的側邊鞭毛。相對於在液體裡游動的細菌，群游細菌展現了對於環境的不同反應。然而並沒有一個適當的方法來控制在瓊脂平板上的群游細菌實驗。在本篇論文裡，藉由微米玻璃毛細管在瓊脂平板上刻畫微通道，我們發展了一套實驗方法來移動群游細菌。實驗涵蓋了三個方法：1. 利用不同種類的微通道來引導細菌；2. 製造能限制細菌的環境；3. 對細菌作化學藥物的測試。經由以上三種方法，可以在瓊脂平板上設計適當的實驗環境來研究群游細菌。 在生物物理的研究上，為了研究單一細菌如何感應外在的化學物質梯度並作出反應，化學趨向性是一個強而有力的研究模型。大部分對於化學趨向性的研究在於細菌在液體環境裡的游動，鮮少著重在群游細菌的反應。傳統上使用大毛細管以及近代微流道的化學趨向性實驗非常難應用在群游細菌的環境裡。在本篇論文裡，我們發展了一套「瓊脂平板上的群游細菌控制實驗」來研究群游細菌的化學趨向性。在瓊脂平板上生長的群游細菌可以被引導至特定的地方，體驗特定的吸引或排斥物質，並且可以觀測單一細菌的反應。在瓊脂平板上，只有側邊鞭毛的溶藻弧菌對於苯酚展現了兩種有趣的化學趨向性反應：1. 對於不同濃度的苯酚展現了兩種不同化學鍵結濃度的特徵反應。2. 在靠近與遠離苯酚時，兩者的速度大小差距甚遠。這兩個新發現對於群游細菌的化學趨向性研究提供了一個嶄新的方向。 ;Vibrio alginolyticus is a marine bacterium with dual flagellar motor systems, single polar flagellum and lateral flagella. Polar flagella expression is encoded in each division cycle and the lateral flagella expression is environmental induced. While the cells growing on agar plate, they elongate without division into swarming phase and express high density lateral flagella. Swarming cells shows environmental sensing and different responses to swimming cells. However, there is no experimental assay suitable for well controlled swarm cell experiments due to the special two dimensional semi-solid agar surface environments. In this thesis, we develop a technique to manipulate swarm cells by drawing “channel” on the agar plate with a glass micro-pipette manipulator system. We demonstrate three manipulation methods on agar plates. 1. Guide cells in different type of channels. 2. Create local constrains to trap cells. 3. Local chemical treatments on cells. Combining these powerful methods, one can design desired experiments based on this “Lab on the agar plates” assay.
In biophysical research, bacterial chemotaxis represents a powerful model system to understand how single-cell organisms sense and respond to external chemical gradients. Most of the chemotaxis researches focus on the cells swimming in aqueous environment. However, very little is known regarding the swarm cell chemotaxis. Traditional chemotaxis capillary assay and modern microfuidic assay are very difficult to apply to the swarm cell chemotaxis in situ. In this thesis, we use “Lab on the agar plates” assay to perform chemotaxis experiments on the swarm cells in situ. Swarming cells developed on the swarm colony can be guided to specific locations to experience particular attractant or repellent with designed gradient for high resolution single cell observation. Vibrio alginolyticus swarm cells with only lateral flagellar shows two interesting chemotaxis response to the repellent phenol. First, there concentration response shows two binding concentration. Second, the cells shows different speed of moving toward and away from the phenol. These two new finding shed new directions of understanding the swarm cells chemotaxis.
