日常生活中的微生物，大二以上，40人 A Daily Intake of Microbes 微生物對歷史與文化變遷過程中的影響 深入了解日常生活中存在的微生物，不僅僅是會致病而已 認識台灣現有微生物產業的應用及發展 藉由實際操作去了解微生物在日常生活中的應用 跨領域、實作 授課老師：黃一修、辛致煒、楊倍昌 聯絡人：黃一修

楊倍昌 醫學院微生物暨免疫學研究所 醫學、科技與社會研究中心 日常生活中的微生物 Introduction: Microbiology as science 經驗主義：世界的真理存在於人自己的知覺 楊倍昌 醫學院微生物暨免疫學研究所 醫學、科技與社會研究中心

Murray PR et al. 編寫的 Medical Microbiology 所寫的序言裡 說: 學生最好能 " Thinking like a physician", 要常常問七個問 題: Who, Where, When, Why, Which, What and How. 微生物的內容不只是應用醫學的一項, 它也是一門自然科學. 它也有科學思辨的趣味, 知識不只是知道名詞而已. 學生還可 以 “ Thinking like a scientist”. 要常常問古早人是怎麼想的? 問題的困難在哪裡? 有沒有比目前的解釋更好的答案? 知識依自己所長加以斟酌.

知識之所在! 1. 貴族城堡 2. 公共圖書館 (~2000) 3. 大學(~1000yr) 4. 網域(~10yr)

一個有用的網址 http://www.scienceprofonline.com/ Science Prof Online (SPO) is a free science education website that provides fully-developed Virtual Science Classrooms, science-related PowerPoints, articles and images. The site is designed to be a helpful resource for students, educators, and anyone interested in learning about science. The SPO Virtual Classrooms offer many educational resources, including practice test questions, review questions, lecture PowerPoints, video tutorials, sample assignments and course syllabi. New materials are continually being developed, so check back frequently, or follow us on Facebook (Science Prof Online) or Twitter (ScienceProfSPO) for updates. Many SPO PowerPoints are available in a variety of formats, such as fully editable PowerPoint files, as well as uneditable versions in smaller file sizes, such as PowerPoint Shows and Portable Document Format (.pdf), for ease of printing. Images used on this resource, and on the SPO website are, wherever possible, credited and linked to their source. Any words underlined and appearing in blue are links that can be clicked on for more information. PowerPoints must be viewed in slide show mode to use the hyperlinks directly. Several helpful links to fun and interactive learning tools are included throughout the PPT and on the Smart Links slide, near the end of each presentation. You must be in slide show mode to utilize hyperlinks and animations. This digital resource is licensed under Creative Commons Attribution-ShareAlike 3.0: http://creativecommons.org/licenses/by-sa/3.0/ Welcome to Science Prof Online PowerPoint Resources! This PowerPoint Presentation comes from the Virtual Cell Biology Classroom of Science Prof Online, and, as such, is licensed under Creative Commons Attribution-ShareAlike 3.0.; meaning you can download, share and alter any of this presentation, but you can’t sell it or repackage and sell any part of it. Please credit Science Prof Online as the source of this presentation. Please abide by credited image copyrights. Thank you for using this resource.

http://www.scienceprofonline.com/

你可以自己學吧!

如果你可以自己學，來課堂幹什麼? 並不是說你不必來上課!

Microbiology as science 的要件 微 - 生物 工具 因果基礎 質性分析 量化分析

Microbiology as science 的要件 微 - 生物 工具 因果基礎 質性分析 量化分析

Scale of life：當你看不見的時候，你怎麼辦？ Viroid ~400bp Paramecium Bacterial Cell HIV virion Prion 27-30 kDa In mm3 20-50,000 0.01-50 0.00001-0.01 Unicellular algae Photosynthetic bacteria Pox group

Microbiology as science 的要件 微 - 生物 工具 因果基礎 質性分析 量化分析

Discovery of microbes Antony van Leeuwenhoek My work, which I've done for a long time, was not pursued in order to gain the praise I now enjoy, but chiefly from a craving after knowledge, which I notice resides in me more than in most other men. And therewithal, whenever I found out anything remarkable, I have thought it my duty to put down my discovery on paper, so that all ingenious people might be informed thereof. Antony van Leeuwenhoek. (1632-1723) Letter of June 12, 1716 他說了一種新態度。 只是看見新東西不能稱之為知識! 1632-1723 Robert Hooke 1635-?,

Anton van Leeuwenhoek, the Dutchman first observed bacteria through his single-lens microscope in 1674. He made his own simple lenses to try to satisfy his curiosity about living things. He observed the bacteria in suspension of white material he obtained from his teeth – dental plaque. Story about Leeuvenhoek’s microscope http://delftschoolmicrobiology.weblog.tudelft.nl/2015/06/23/finding-more-van-leeuwenhoek-microscopes/

Antony van Leeuwenhoek 回應英國皇家學院刊物Philosophical Transactions給他的建議(可能是他在英國的通訊人Oldenburg)信 我被許多人要求，應該要將那些透過我新發明的顯微鏡所看見的記錄下來。通常我都拒絕了。首先，因為我沒有書寫風格，或畫圖的能力，正確的來表達我的想法；其次，因為我沒有接受語言或藝術教育，而只學會做生意；第三，我不喜歡跟引發爭議或被別人監督審查。然而，在Dr. Reg de Graaf的請求下，我已經放棄了以上的想法。我給了他我的紀錄，關於我看到的黴菌，蜜蜂的刺和四肢，還有蝨子的刺。… 因此，那些可能會注意這些事的先生們，請記住，我的觀察和想法純粹是出自於自發的衝動和好奇心。 Translated in Dobell C. 1958 Antony van Leeuwenhoek and his little animals, p. 41. New York, NY: Russell and Russell.

DIY 的年代 http://www.funsci.com/fun3_en/antoni/vlen.htm 資料來源：

Cell theory Micrographia; 1664 It takes 190 years

A short history of microbiology 由看見微生物到認定為致病原花了近兩百年 On spontaneous generation: 1668, Francesco Redi (with cover, against); 1749, John Needham (simple boiling, for); 1765, Lazzaro Spallanzani (extensive boiling, against) 1673: Anton van Leeuwenhoek (microbes) 1678: Robert Hooke (cell structure) 1861: Louies Pasteur (gooseneck bottle; against) 1870: Joseph Lister (1% carbolic acid, phenol in surgical procedure) 1881: Robert Koch (Koch’s postulate) 1892: Dmitrii Iwanowski (Tobacco Mosaic Virus) 1909: Paul Ehrlich (chemotherapy, arsenicals against spirochetes) 1915: Frederick W. Twort (bacteriophage) 1940: O.T. Avery (DNA as transforming factor)

知道了小小小生物之後, 科學家能想出什麼研究? 由1683到1861是: 198年的空白 為什麼是空白？

Microbiology as science 的要件 微 - 生物 工具 因果基礎 質性分析 量化分析

看似不相干的問題 生命由那裡來？ 腐草生螢、腐肉生蛆！ 濕紙化為菌。 九尾金毛狐狸轉生成妲己。 孫悟空由石頭中蹦出來。 Thomas Moffet (1553-1604) 認為蜂是由腐爛的小牛自然產生。 Jan Baptista van Helmont (1577-1644) 認為池塘的爛泥巴會變成青蛙，盆子內擺進臭汗衫加上一些麥穀就可以生出老鼠。

歷史上第一個有對照組的生物實驗 (1668年) Esperienze intorno alla generazione degl' Insetti (p. 187) where Francesco Redi published a description of the experiment in 1668. Francesco Redi (1626 –1697) http://ihm.nlm.nih.gov/images/B21503

Levine, Russell and Evers, Chris Levine, Russell and Evers, Chris. The Slow Death of Spontaneous Generation (1668-1859). http://www.accessexcellence.org/AB/BC/Spontaneous_Generation.html pc.maricopa.edu Redi 描述他的實驗：我在四個廣口瓶內各放進一隻蛇、一些河魚、四隻Aron 河的小河鰻、以及一塊牛肉。我小心的瓶口用紙和線封綁緊密。我還另外找來四個廣口瓶也各放入相同的東西，但是讓它們瓶口打開著。不久之後，開著口的瓶子內的魚肉就長了很多蛆，三個星期後我就看到很多的蒼蠅飛繞在瓶子。但是在瓶口密封的瓶子內，我看不到一隻蛆。

兩百年的思索 1668 Francesco Redi 證明只要隔絕蒼蠅、腐肉就不會生蛆。 1745 John Needham 以玻璃瓶裝雞湯，煮沸，再冷卻之後，微生物仍然產生。懷疑微生物可能自然產生。 1768 Lazzaro Spallanzani 重做Needham的實驗。在抽掉空氣後，微生物就不會生成。 1859 Louis Pasteur 以鵝頸瓶證明微生物也不會自然生成。 1877 John Tyndall 以接續加熱法殺死耐熱性(產孢)的細菌。

鵝頸瓶：1859年 傾斜

Pasteur's own drawings of the swan-necked flasks L. Pasteur at "scientific evening" of the Sorbonne University in Paris " And I wait, I watch, I question it!-begging it to recommence for me the beautiful spectacle of the first creation. But it is dumb, dumb since these experiments were begun several years ago; it is dumb because I have kept it from the only thing the human mind does not know how to produce: from the germs which float in the air, from Life, for life is a germ and a germ is life. 我等待，我凝視，我質問它！乞求它為我重現美麗的創造奇觀。但是它毫無聲息，自從幾年前實驗開始時就無言無聲。它是不會有聲息的，因為我隔離了唯一人不知道如何產生的東西：隔離了漂浮在空氣中的微生物，隔離了生命。因為生命是微生物，微生物是生命。(物質理由的前奏曲)

鵝頸瓶實驗具有宗教一般的神秘氣質。 Jonathan M. Metzl & Joel D. Howell (2004) Making history: Lessons from the great moments series of pharmaceutical advertisements. Academic Medicine 79, (11): 1027-1032.

1859年我們學到了什麼？ 腐肉不會自己生蛆。 生命不會自然發生，自生論是錯的。 這樣而已嗎？ 不是！ 應該注意對照組！ 應該設計對照組！

Microbiology as science 的要件 微 - 生物 工具 因果基礎 質性分析 量化分析

生物科學中，有對照組並不一定萬事就解決了! In 1844, Semmelweis was appointed assistant lecturer in the First Obstetric Division of the Vienna Lying-In Hospital, the division in which medical students received their training. He was appalled by the division's high mortality rate from puerperal fever — 16% of all women giving birth in the years 1841–1843. In contrast, in the Second Division, where midwives or midwifery students did the deliveries, the mortality rate from the fever was much lower, at about 2%. Semmelweis also noted that puerperal sepsis was rare in women who gave birth before arriving at the hospital. Ignac Semmelweis a native of Hungary http://general-anaesthesia.com/images/ignaz-semmelweis.html Sinclair WJ. Semmelweis: his life and his doctrine. Manchester: Manchester University Press,1909.

It is always hard for anyone to admit they are wrong It is always hard for anyone to admit they are wrong. "Okay, so I was wrong about the Cubs winning the pennant." Not so hard to admit. But if the item is central to your profession, and admitting it not only questions your judgment but implicates you, although at the time unknowingly, in the many deaths your error has caused, that is a little harder to swallow. Obviously, many would, and did, deny the error rather than face the upset. The Doctors' Plague by Sherwin B. Nuland Semmelweis wrote a series of “open letters” to his former professors, accusing them — rightly, as it turned out — of being “medical Neros” and “murderers”. (錯誤的策略)

Some key factors Improvement of optical instruments Establishment of pure culture system Staining technique

Big person in microbiology Robert Koch,1843-1910, Germany

看清物質是建立生物學相關學門的基礎：Communication and classification By making photomicrobiographs, I can reveal the bacteria true to nature and free of subjective misinterpretation. ---Robert Koch 我很清楚，我的攝影作品仍然非常不完美，但我絕對肯定，關於生物的一張壞照片仍然比一張誤導或可能不正確的繪圖要好上一百倍。

Privy Health Officer Berlin W, 15, June 30, 1904 Dear Sir, I still remember very well the first time I took an oil system into my hands and myself witnessed the dramatic progress achieved by the optical workshop of Carl Zeiss under the direction of Professor Abbe's ingenious advisory board. Every time I subsequently used oil systems, I thought with admiration and gratitude of the magnificent gift made by the Zeiss optical workshop to everyone working with a microscope. After all, I owe a large percentage of the success I have had the privilege of achieving for science to your excellent microscopes which were of enormous benefit to me in the expedition to South Africa which I have just completed. Respectfully yours, R. Koch http://www.zeiss.com/C12567A100537AB9/allBySubject/DAF3D66784516F2CC1256B45003DA810

科霍1884年，結核病的病原學 因惡性炭疽死亡的動物血中發現了許多規則、桿狀、無色、不動的 桿狀體。 在體外，觀察這些桿狀體會生長、形成孢子，並且從孢子長出桿狀 體而確認它們具有生命，屬於低等植物類。 如果將惡性炭疽動物中含桿狀體的少量血液接種到另一隻動物身上 ，後者將死於惡性炭疽病，而且其血液中含有炭疽桿菌。 但是，光這樣並不能證明炭疽病是經由此桿菌傳染的，因為血液中 還有其它的物資(血液腐敗物質)也跟著桿菌被接種進去。為了區分 由血液中的桿菌或者是其他物質的感染造成惡性炭疽病，這桿菌必 須自血液中單獨分離出來，然後再進行接種試驗。接著，科霍仔細 描述在固體培養基(明膠、煮過的馬鈴薯)上培養細菌的技術。透過 體外培養，將桿菌完全分離後，在顯微鏡下已看不到血液中的其他 物質。繼代培養二、三十次之後，桿菌就不再混雜任何患病體內疾 病的副產物。 當純粹的桿菌接種到健康的個體，它可以誘發此動物罹患致命的惡 性炭疽病，而在發病動物的血液中亦可發現不可計數的炭疽桿菌。 Milestones in Microbiology 1546-1940,

An obvious true is never easy to be recognized and accepted Koch’s demonstrations in the Institute of plant pathology; University of Breslau (15 Nov. 1876) Now leave everything as it is, and go to Koch, This man has made a magnificent discovery ----Julius Cohnheim 1817-1891 Whole business seemed quite improbable; Anything I couldn’t see with a dry lens wasn’t worth looking at. (But, why?) ----Rudolf Virchow 1821-1902

Morphological study of bacteria Instruments: Light microscope: 0.2 mm Phase-contrast; dark field Electron microscope: 0.0001 mm TEM; SEM Near-field microscope: atom-level

光是知道「它的存在」就有很大的威力 Tetanus Tetanus toxin: Patient number in Canada After antitoxin vaccine 1941-1995 Tetanus Tetanus toxin:

Microbiological stains

Morphological study Unstained Fix and stained Simple stain: General shape, motility, spore, intracellular granules Fix and stained Simple stain: Positive: methylene blue, methyl violet, basic fuchsin, silver… Negative: acidic dye for background… Gram’s stain: (by Hans C. Gram, 1884) base on the resistance to decoloration with acetone, alcohol or aniline oil after stained with a triphenyl methane dye. (+: purple; -:red) Acid fast stain reaction: (Ziehl-Neelsen method) cell wall consists of lipids and forms a special anatomical disposition of the lipids. Immunostaining: labeled antibodies For lecture only

叫不出名字就無法溝通 Phenotypic classification Analytic classification (composition) Genotypic classification (gene constitute)

Gram stain (cell wall structure) Mole percent G+C in the genome The prokaryotes and eukaryotic protists were more difficult to classify. They are much simpler, and there are few obvious features. Classifications based upon features such as these were not natural systems (Bergey's Manual of Determinative Bacteriology ) Gram stain (cell wall structure) Mole percent G+C in the genome Growth temperature Ability to form heat stable spores Electron acceptors for respiration Photosynthetic ability Motility Cell shape Ability to use various carbon and nitrogen sources Special nutritional requirements For lecture only

簡易二分法 epidermidis Simple Stain Cocci Bacilli Gram Stain Gram Negative positive Mannitol Salt yellow pink Staphylococcus aureus negative bacilli Gram positive bacilli MacConkey’s No color change Salmonella pullorum Pink colonies E. coli Enterobacter aerogenes Acid Fast stain Acid Fast Mycobacterium tuberculosis Not acid fast Endospore stain Forms endospores Bacillus subtilus

Classification: Tree of evolution

Microbiology as science 的要件 微 - 生物 工具 因果基礎 質性分析 量化分析

Finding a solid medium What can be a replacement? Agar's first use discovered by Frau Franny Eilshemius (a physician's wife) whose husband told Robert Koch, who is credited with the discovery! Dr. Walther and Fanny Hess Agar is derived from Gracilaria (Gelidium species) a bright red sea vegetable with the botanical name of Gleidium purpurascens.  Agar due to its high gelling properties is considered the queen of gelling agents.

Counting the viable cells: Dilution (兩種物理特性) 科學的第一步在於數字化: 定量

Growth measurement Cell count: microscopic observation; flow cytometer (direct) Colony formation: Measure the living cell (direct) Biomass determination: dry weight; essential cell component (indirect) Turbidity (indirect)

1, 2, 4, 8, 16, 32, 64, 128, 256, …. Doubling time Escherichia coli:12.5 min Vibrio cholerae : 13 min (can kill a man within 12 h) Mycobacterium tuberculosis : 24 h (develop symptom after months)

Growth and survival Lag phase: adaptation to the environment Exponential logarithmic growth: machine in full rum Stationary phase: nutrition exhausted, toxin increased Decline: cell die (steady biomass) or lysis (decrease biomass) Dormant as spore, non-viable state

Thermophiles 70o-110o Mesophiles 10o-50o

Thermophils visible in hot springs when single-celled photosynthetic bacteria (cyanobacteria) form dense layers of biomass called bacterial mats. contained "environmentally friendly" enzymes that were stable under high temperatures and could be used in place of more dangerous chemicals that have been concocted by modern industry. Bacteria and Archaea live in hot springs heated by geothermal reactions deep in the Earth.  Some springs reach temperatures of 80 C (177F).  Taq polymerase Distinct membrane

1861 Louis Pasteur introduced the terms aerobic and anaerobic in describing the growth of yeast at the expense of sugar in the presence or absence of oxygen. Reference: 1.  Pasteur, L. "Animalcules infusoires vivant sans gaz oxygene libre et determinant des fermentations." Compt. Rend. Acad. Sci. (Paris) 52:344-347, 1861 1995. Craig Venter, Hamilton Smith, Claire Fraser, and colleagues at TIGR elucidate the first complete genome sequence of a microorganism - Haemophilus influenza.