Study of Bacterial Endospore Germination under Acoustic Stimulation with an Integrated Microfluidic Platform

• Other Authors: Liu, Sili (author.)
• Format: Others
• Language: English; Chinese
• Published: 2016
• Online Access: http://repository.lib.cuhk.edu.hk/en/item/cuhk-1292435

桿菌屬和梭菌屬的細菌在惡劣環境外部壓力下可以形成脫水的芽孢，以確保遺傳物質的安全及傳遞。環境因素如營養物和水，以及各種實驗室控制的非營養類化學物質可以促使休眠的芽孢萌發。在萌發過程中，休眠芽孢所具有的的極端抗性都會失去。細菌的化學通訊已被廣泛研究，之前的研究中也發現芽孢在萌發過程中有同步現象發生，但並沒能夠識別其化學介質。而細菌之間物理通訊仍有很大的研究空間。在本論文中，我們利用微流體平台研究聲刺激下的細菌芽孢萌發，以此來研究聲音作為通訊介質在芽孢萌發同步中所起到的作用。 === 首先，我們使用鋱-2,6-吡啶二羧酸（Tb-DPA）熒光法，相差顯微鏡法，光密度測量法，和ATP生物發光法來監測在傳統壓電發聲器作用下的大量芽孢懸液的萌發過程。為了更好的提高聲音能量的傳輸效率，以及整合檢測方法，在本論文中，我們設計製造了結合壓電薄膜發聲器傳及電導傳感器的微流體芯片。我們採用β型聚偏氟乙烯（β-PVDF）作為整合於聚二甲基矽氧烷（PDMS）微流控芯片中的壓電發聲器。因為它的聲阻與PDMS和水最為接近，從而保證最大的能量傳遞。壓電發聲器被設計成環形結構，中心的透明區域提供了相差顯微觀測和未來熒光光譜觀測的光路窗口。 PVDF壓電薄膜的振動處於彎曲模態，從而液體環境不會影響到發聲器的聲壓級（Sound pressure level）。這個性質對於該芯片在生物應有中是極為有利的，因為大部分生物實驗需要在液體環境中進行。芯片上層PVDF-PDMS部分同下層石英玻璃基地的粘合是可逆的，使得我們可以更容易的徹底清洗芯片從而可以重複利用芯片。通過使用該芯片，我們不僅可以檢測在聲音刺激下芽孢菌群的發芽，而且可以實時現場監測單個芽孢的萌發。同時，我們還集合了一個環形插指形電導電極於芯片中，提供芽孢萌發中離子釋放的定性測量。 === 實驗結果表明：1）5千赫茲聲波刺激能夠促進L-丙氨酸誘導下的萎縮芽孢桿菌芽孢的發芽，但對枯草芽孢桿菌芽孢沒有效果; 2）聲波刺激對L-丙氨酸誘導發芽的影響與頻率相關：5千赫茲的促進作用最顯著，而20千赫茲則抑制芽孢萌發; 3）5千赫茲聲波刺激能夠促進L-丙氨酸誘導的萎縮芽孢桿菌芽孢萌發過程中ATP的合成; 4）5千赫茲聲波對芽孢萌發的促進作用同萌髮劑種類相關：L-谷氨酸，D-葡萄糖，D-果糖，鉀離子（AGFK）組成的混合萌髮劑，鈣-2,6-吡啶二羧酸（Ca-DPA）萌髮劑，和十二胺萌髮劑分別誘導的芽孢萌發並不受5千赫茲聲波刺激; 5）通過觀察單個芽孢的萌發，聲波刺激不影響萌發過程中水的流入。 === 本論文對微流體工程和微生物學都有新的貢獻。我們所開發的微流體芯片不僅適合於該項目，同時在其他生物實驗上也有應用潛力。聲波刺激對芽孢萌發作用上的新發現有助於人們進一步研究芽孢萌發機制以及聲波在細菌通訊中的作用。 === Endospores are highly dehydrated cells formed under external stress by certain bacteria species, such as Bacillus and Clostridium, in order to survive the harsh environment. Germination of dormant endospores can be triggered by environmental cues such as nutrients and water, as well as a variety of laboratory-controlled non-nutrient-based chemicals, during which the dormancy and extreme resistance of endospores are concomitantly lost. This process has been extensively studied from the molecular biological, biochemical, and electrochemical perspectives. Chemical communication of bacteria has been widely studied, but researchers failed to identify the chemical mediator during germination process although synchronization behaviors have been observed among spores. In this thesis, we initiated a new field of study that investigates the role of the acoustic wave in the germination process of bacterial endospore of Bacillus as a possible channel for germination synchronization by taking a novel approach to study the acoustic wave stimulation effect on endospore germination. === First, germinating endospores in a microtiter plate were exposed to audible sound wave generated by an array of piezoelectric transducers. Germination kinetics was measured by terbium-dipicolinate (Tb-DPA) fluorescence assay, optical density measurement, phase contrast microscopy and ATP bioluminescence assay. To integrate stimulation and detection methods with better acoustic energy transmission, in this thesis, a microfluidic platform coupling both piezoelectric microspeaker and conductance sensor was designed and fabricated. We employed polyvinylidene fluoride (PVDF) in β-phase for PDMS-based microfluidic application because it has a closer acoustic impedance to PDMS and water to ensure maximum energy transfer. The transparent window at the center of ring-shaped microspeaker provided an optical path to perform in situ microscopic and future spectroscopic measurements. The PVDF film was driven at bending mode in which the sound pressure level (SPL) is not compromised with water fulfilled inside the chamber. This property makes this device an excellent candidate for biological application because the liquid environment is mostly required in such experiment. The upper PVDF-PDMS layer is reversibly bonded with quartz glass substrate such that the device can be clean easily and thus reusable. By using this device, not only the germination in a population, but also in single spore can be studied with real-time monitoring. An integrated conductance electrode was able to provide an additional qualitative measurement of germination kinetics. === Results revealed that 1) 5 kHz stimulation is able to promote the L-alanine induced germination of Bacillus atrophaeus spore but not Bacillus subtilis spore; 2) The effect of acoustic wave stimulation on L-alanine induced germination is frequency dependent that 5 kHz gives the most significant promotion effect, while 20 kHz inhibited the germination; 3) 5 kHz stimulation is able to promote the ATP synthesis during L-alanine induced germination of Bacillus atrophaeus spore; 4) The promotion effect of 5 kHz stimulation on spore germination is germinant dependent that mixture of L-asparagine, D-glucose, D-fructose, and K+ (AGFK), Ca-DPA, and dodecylamine induced germination are not stimulated by 5 kHz sound treatment; 5) Observed by single spore germination kinetics, the acoustic wave stimulation does not affect the water influx process. === This thesis combines both novel works on engineering and science. The microfluidic platform developed here is not only suitable for this project but also has potentials for other biological applications. The novel finding in the acoustic wave stimulation on spore germination sheds light on germination mechanism and cell-wave interaction. It has initiated a new field of study that investigating the role of the acoustic wave in the spore germination and possibility of acoustic wave communication during other microbial activities. === Liu, Sili. === Thesis Ph.D. Chinese University of Hong Kong 2016. === Includes bibliographical references (leaves ). === Abstracts also in Chinese. === Title from PDF title page (viewed on …). === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only. === Detailed summary in vernacular field only.