快速檢定抗生素對細菌生物膜敏感性之新技術
The purpose of this study is to set up a quick, easy and economical way to evaluate the ability of different concentration of various antibiotics to penetrate biofilm and establish the antimicrobial susceptibility patterns of various antibiotics. The susceptibility of five antibiotics upon sessile cultures of Bacillus subtlis ATCC 6633, Escherichia coli XL, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Sarcina lutea ATCC 9341 were measured ATCC 27853, and S. aureus ATCC 29213 proved to be very difficult to eradicate, with only Gentamicin proving to effective at achievable drug concentrations, but the S. lutea ATCC 9341 biofilm was the most susceptible to the Penicillin. The results demonstrated that for biofilms of the same organisms, several hundred to thousand times the concentration of a certain antibiotic were often required for the antibiotic to be effect, while other antibiotics were found to be effective at the MICs. The concentration of antibiotic to penetrate the biofilm is proportional to the thickness of biofilm. Indeed, our research have already indicated that the use of MIC values to indicate antibiotics effectiveness is misleading, because MIC values can not represent the actual effect of anticbiotics on microbiologicals that have developed biofilm. The antimicrobial susceptibility patterns of antibiotics to various bacterial biofilm are different. The susceptibility of the mixed biofilm depends on the physical and biological change of biofilm. Our biofilm device offers a new technology for the rational evaluation of antibiotics effective against microbial biofilms and for the screening of new effective antibiotic drugs.此研究之目的是要建立一套操作簡便、快速且費用低廉之生物膜厚度產生方法，藉此探討不同生物膜厚度對抗生素抗菌之影響，進而完成抗生素對生物模之抗菌圖譜。本實驗將測試五種抗生素Bacillus subtilis ATCC6633，Escherichia coli XL，Pseudomonas aeruginosa ATCC27853，Staphylococcus aureus ATCC29213和Sarcina lutea ATCC9341之抗菌圖譜。試驗結果顯示Gentamicin對前四株試菌有較佳之穿透力，對S.lutea ATCC 9341則是Penicillin。實驗結果證明最小抑制濃度值確實無法實際有效地表達對已形成生物膜菌體之抗菌效果，要完全去除生物膜之抗生素濃度是為最小抑制濃度之數百倍到數千倍，而且抗生素用藥濃度隨著生物膜厚度增加而成比例增加。不同抗生素對不同菌株生物膜有不同之抗菌圖譜，混合菌株生物膜是否會促進或抑制抗生素之穿透力，端視其生物膜結構有無改變。本實驗方法可以做為一種快速檢定抗生素對細菌生物膜敏感性之新技術，同時亦可有效地篩選新的抗生素藥物對生物膜的抗菌效應。
Ethidium Bromide 對p53 基因缺陷老鼠致癌影響
p53 蛋白是一種轉錄調控因子，其在抑制癌症的形成扮演很重要的角色。已知的報告顯示它能影響下游特定蛋白表現進而調控細胞週期或促使細胞進行凋亡作用。如此作用機制對於可能癌化的細胞在生物體造成傷害之前能被先行消滅，因此正常的p53 蛋白存在與否，對癌症的產生有很大的影響。 根據上述結果，我們想分析不同表現量的p53 是否對於致癌藥劑-EtBr所引發的癌化細胞有所影響。所以我們設計實驗，將具有正常表現量p53 蛋白的老鼠(p53+/+)與僅能表現少量p53 蛋白的老鼠(p53+/-)，均塗以等劑量之致癌藥劑EtBr於其大腿皮膚上，觀察其致癌情形的差異。實驗結果發現 : 在塗EtBr 的14 隻p53+/-老鼠中，有3 隻出現了組織病變的症狀，包括脾臟腫大，淋巴組織長出腫瘤，而其他對照組(一)塗EtBr 的p53+/+老鼠，(二)不塗EtBr 的p53+/+老鼠或(三)不塗EtBr 的p53+/-老鼠都沒有產生異常的症狀。因此根據上述實驗結果進行推論，發現經EtBr 的刺激後，p53+/-的老鼠的確較其他的老鼠更容易得到癌化細胞。;p53 is a transcription factor that plays an important role in suppression of tumorgenesis. Previous reports showed it regulates cell cycle and enhances cell apoptosis by effecting specific proteins expression. By this way, the problematic tumor cell can be deleted to protect organism from tumor damage. According above conclusion, we are interested to know whether p53 plays a role involved the carcinogens-EtBr induced tumorgenesis. We compared the differences of tumorgenesis between mice express normal (p53+/+) or low (p53+/-) expression level of p53 proteins when they are smeared equal amount of EtBr on skin of thigh. In here, our results show three of fourteen p53+/- mice had pathological changes, containing spleen enlargement and lymphoma. However, other three comparisons, smear p53+/+ mice with EtBr (1), without EtBr (2) or p53+/- without EtBr(3), are normal. Therefore, We suggest that p53+/- mice are easily to obtain tumor than p53+/+ mice after EtBr treatment.
酒精對斑馬魚胚胎眼睛發育的影響
長期以來，臨床研究發現會影響人類胚胎的正常發育，造成胚胎畸形、成長遲緩等現象，稱為胎兒酒精症候群（fetal alcohol syndrome），但是其病理機制仍不清楚。最近的研究發現斑馬魚胚胎對於酒精有非常靈敏的反應，出現明顯的發育異常現象，例如心臟膨大、眼睛縮小、骨頭變形等現象，與人類胎兒酒精症候群相似，顯示斑馬魚非常適合作為研究胎兒酒精症候群的模式動物。在本實驗中，我們針對酒精影響視網膜神經發育的現象進行探討，並且也利用基因晶片篩選出胚胎發育時期受酒精影的的基因轉錄子，藉以了解酒雞造成胚胎畸形發育的機制。實驗結果顯示在眼睛發育過程中，1.5% 酒精浸泡會抑制視網膜神經細胞的分層（Lamination），而進一步利用RNA定位雜交的方式以眼睛的標示基因rx1、pax6b、six3b、alpha-crystallin、rho、neuroＤ、huC觀察他們的表現，發現酒精會減少視網膜神經細胞的分化，造成眼睛發育的缺陷。在基因晶片的分析中，我們發現酒精處理會抑制許多控制眼睛發育的基因調控子的表現，包括了crx、rybp、irx4a、optx2、rx1、brn3b1 與 vsx1等，此外約有16%的眼睛相關基因液受到酒精的抑制，遠高於其他基因受抑制的比例，顯示眼睛是酒精傷害最嚴重的區域之一。我們從實驗結果得知酒精並未對胚胎發育初期眼睛的發育造成明顯的影響，但是隨後由於控制細胞分化的幾個重要基因受到酒精抑制作用，造成視網膜神經細胞的分化停止，引起眼睛的異常發育的情況。It was known that prenatal alcohol exposure may cause serious birth defects and developmental disabilities. The molecular mechanism of this fetal alcohol syndrome still remains unclear. Here we used zebrafish embryo as a model to investigate the toxic effect of alcohol in retinal development. The histochemical analysis revealed that the cell lamination was prohibited by alcohol incubation. It appears that the retinal cell differentiation was inhibited. As revealed by whole mount RNA in situ hybridization, it appears that the transcription of a number of retinal-related regulatory genes, including rx1, pax6b, six3b, alpha-crystallin, rho, neuroD and huC, were all inhibited in zebrafish embryo by alcohol exposure. The transcriptional profile of alcohol-exposed embryos was also compared with normal embryos by microarray analysis at different stages. It appears that 16% of retinal-related genes were all repressed by 1.5% alcohol incubation, including several retina-related transcriptional factors, including crx, rybp, irx4a, optx2, rx1, brn3b1and vsx1. Our results suggest that alcohol did not interfere the early development of the eye, but has inhibited the final cell differentiation of retina cells. This study helps us understand the molecular mechanism of alcohol-mediated retinal malformation.
解開蔗糖水解的秘密
本研究利用偏振片、量角器為刻度盤、雷射光為光源，及照度計為偵測器，組裝一個簡易且可靠的旋光度計。我們利用單位時間旋光度的變化量當作反應速率，來測量蔗糖的水解速率，同時求出蔗糖水解反應的反應級數、速率常數(k)。利用糖類的旋光度具有加成性之特性，找出不同混合比例時的旋光度，追蹤實際蔗糖水解的每個狀態，找出最後平衡狀態，同時將蔗糖水解平衡結果顯示，旋光度與濃度有線性關係，而蔗糖水解反應對蔗糖而言為一級反應。接著，我們在蔗糖水溶液中加入不同種類的酸，探討催化劑的種類與蔗糖水解反應速率的關係。 In this research, in order to measure the optical rotation accurately without expensive equipments or complex process, we assembled a polarimeter by ourselves. With simple materials which can be found in ordinary senior high school laboratories, including a calibrated scale, a simple Luxmeter, a laser as the photo source, and other side devices. The Polarimeter ended up operating fluently and accurately. We put the laser under a tube, which has two pieces of polar screens on the top of it and on the bottom of it, ,and put a luxmeter just above the tube. When we slowly rotate the polar screen on the top, the figure shown on the luxmeter changes. By numerical analysis, we can get information about the hydrolysis of polarized substance. Secondary, we measured the optical rotation of glucose, fructose, malt sugar, galactose, and sucrose to get their specific rotation. Then we measured the optical rotation of sucrose every five minutes. By doing this, we could keep track of the hydrolysis rate of sucrose, figure out the order of reaction, and the rate constant (k) and the equilibrium constant (K). Thirdly, we used different kinds of acids into sucrose solution as the catalyst, and observed the effect. The result showed that hydrochloric acid is a better catalyst to this reaction than sulfuric acid and nitric acid. The polarimeter of this research can be used in science education of junior and senior high school. By teaching students to assemble and operate the self-made polarimeter, students can know better about optical rotation and polarized substance. Also, the interest in this experiement will add to students’ motivation to do science research.
台灣珍稀水生蕨類槐葉蘋形態、生活史及生存環境的研究
槐葉蘋(Salvinia natnas)生長於台灣低海拔淡水濕地，目前已列為嚴重瀕臨滅絕的台灣原生物種，為不具有根的植物，是世界珍稀的漂浮型水生蕨類。本研究是探討槐葉蘋形態、生活史及生存環境因子，實驗發現可藉由成熟浮水葉外部形態特徵來區別槐葉蘋與外來種之人厭槐葉蘋(Salvinia molesta)；槐葉蘋成熟浮水葉呈橢圓形，葉上毛被物是叢生且分岔，人厭槐葉蘋成熟浮水葉呈雙耳形，葉上毛被物則像打蛋器。當兩物種共存於同一個環境空間時，人厭槐葉蘋以平均11.6 cm2/week 的生長率將槐葉蘋完全取而代之，顯示人厭槐葉蘋之入侵對槐葉蘋生存影響之深遠。經由兩年的槐葉蘋物候觀察，發現3~11 月為抽芽成長期、3~12 月為成熟繁殖期、12 月~隔年2 月為冬枯期及孢子囊果出現期，12 月~隔年5 月為孢子囊果成熟開裂期。其繁衍策略可分為無性繁殖（頂芽及側芽生長）及有性生殖（異配子體交配）。探討環境因子（光照度、氣溫、濕度、水質、水溫、pH 值）分析結果，適合槐葉蘋生存環境的條件為(1)陽光間接照射（半遮蔭，遮蔭度58.33%）、(2)乾淨未受污染的水質(pH 6.5~8)、(3)通風性良好。生長環境符合以上條件即可達到移地保育的目的。Salvinia natans, a floating fern without roots, grows in low elevation fresh water wetlands of Taiwan, and is a critically endangered precious Taiwanese native species. This research investigates the life form, life history, and living environment of Salvinia natans. Our experiments show that we can differentiate Salvinia natans and Salvinia molesta, two easily mixed up species. The shape of matured floating leaves of Salvinia natans is elliptical and smaller, while it is twin-ear shape and larger for Salvinia molesta. Also, they can be distinguished by their leaf hairs. The hairs of Salvinia natans are tufted and separated at the tips, while the hairs of Salvinia molesta form an ‘eggbeater’ shape at the tip. When these two species lived together, Salvinia molesta grew in a rate of 11.6 cm2/week and will replace all Salvinia natans eventually. This shows the profound impact of invasion of Salvinia molesta. From the data of 2-year phenology observation, we concluded that budding took place from Mar. to Nov., growing and reproducing from Mar. to Dec., decaying from Dec. to Feb. (sporocarps were born in this period), and sporocarps matured from Dec. to May. There are two reproduction strategies: sexual reproduction (intergametophytic mating), and asexual propagation (by terminal and axillary growth). After investigating the environment factors (illuminance, air temperature, water temperature, humidity, pH), we found that ex situ conservation for Salvinia natans requires 1) indirect sunshine, 2) unpolluted water (pH 6.5 ~8), and 3) good ventilation.
調幅超聲波解調高指向可聽音之研究
可聽聲有向四周擴散繞射特性，而超聲波具有指向性，改以超聲波載送可聽音訊號後，其載波與旁頻帶均在超聲波範圍，實驗中人耳卻可聽到高度指向性聲音，且調幅解調後的可聽聲衰減率比純超聲波來的低。那為什麼超聲波會解調可聽音？我們以非線性的數學轉換概念，成功以數學推導解釋實驗中所聽到的可聽聲，是由旁頻經由非線性轉換而來的。為了證實空氣中的超聲波有非線性現象，以發射40KHz單頻訊號，除了接收到40KHz訊號外還可接收80KHz訊號，而80KHz訊號振幅，會隨著發射強度而遞增，也會隨著傳輸距離增加至穩定狀態，這所我們從文獻中的非線性理論所吻合。接下來進行調幅超聲波實驗，我們經理論計算旁頻帶強度為頻率響應與調變率乘積的一半，而解調可聽聲的強度為調變率、頻率響應與非線性係數三者乘積，我們也由實驗數據證實理論計算結果，在實驗中，換能器在40KHz有最佳的頻率響應，其非線性係數與所載送可聽聲頻率高低約略成正相關，並且與換能器距離遞增而越遠而增加。此外在提高高指向可聽音輸出功率方面，除製作專屬的放大器、運用方波取代正弦波來載波、配合陣列換能器輸出；在改善音質方面，利用等化器調整訊源頻譜分佈，降低低頻振幅，增強高頻振幅，讓各頻率的原始訊號都能有適當的調變，達到最佳音質。The audible sound has the characteristics of spreading and diffracting. And ultrasonic is directive. We modulate sound into ultrasonic signal. The carrier and sideband are ultrasonic frequency bands. But in the experiment, human can hear highly directive sound. In terms of attenuation rate, AM demodulation sound is lower than pure ultrasonic wave. Why can human hear the directive sound? By using the nonlinear mathematical transform, we managed to explain the audible sound which is transformed from sideband with nonlinear effect in the experiment. In order to confirm that nonlinear phenomena in the air ultrasonic, we launch 40KHz single tone ultrasonic signal. Besides the 40KHz signal, we also received 80KHz signal. The amplitude of 80KHz signal will increase with the emission intensity, and also with the transmission distance to increase its stability. These are consistent with nonlinear theory in the literature. Next we began AM ultrasonic experiment. We calculated the sideband intensity that is the product of frequency response and modulation index. The demodulation sound intensity is the product of modulation index, frequency response, and nonlinear coefficient. We also proved the calculated consequence through the experiment. In the experiment, the ultrasonic transducer has a best frequency response in 40KHz. The nonlinear coefficient has positive correlation with the modulation frequency, and increases transmission distance. To boost the power of directive audible sound, we made an amplifier, using square wave to replace sine wave of carrier, and in conjunction with array transducer output. To improve the sound quality, We use the spectrum-Equalizer to adjust the frequency distribution of the origin signal. The EQ reduces the low-frequency amplitude, and boost high-frequency amplitude, which enables every frequency of the original signal to be properly modulated, achieving the best sound quality.