Introduction of Microbiology and classification of bacteria 楊倍昌 成功大學 微生物暨免疫學研究所

Medical Microbiology 醫學微生物這門課包含的內容繁多, 千頭萬緒. 在 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. 醫學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. 上課內容在網頁上:http://myweb.ncku.edu.tw/~y1357/course/micro.html

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)

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你可以自己學吧!

如果你可以自己學，來課堂幹什麼?

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

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!

Leeuwenhoek 的困境 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

A short history of microbiology 由看見微生物到認定為致病原花了兩百年 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)

光是知道「它的存在」就有很大的威力 http://www.health.org.nz/tb.html By Robert Kock In Britain the decline of T.B. was continuous for over 100 years before the introduction of the vaccine.  The department of Health has, surprisingly, decided that routine BCG vaccination will be phased out in the 1990s. Actually, it comes back due to HIV. http://www.health.org.nz/tb.html

看清物質是建立生物學相關學門的基礎：Microbial classification and structure Description of bacteria (G+/G-) Yersinia pestis Bacillus sp.

看得見，看清楚才是細菌學發展的關鍵 By making photomicrobiographs, I can reveal the bacteria true to nature and free of subjective misinterpretation ---Rober Koch

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

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

Shapes Bacillus Coccus Coccobacillus Fusiform bacillus Spirillum Bacillus anthracis Bacillus Coccus Coccobacillus Fusiform bacillus Spirillum Spirochete Vibrio Staphylococcus aureus Pseudomonas aeruginosa Fusiform bacilli Spirillum volutans Vibrio cholerae Borrelia burgdorferi For lecture only BC Yang

Growth/Culture condition Plating technique of Robert Koch for single colony isolation Microbiol. Mol. Biol. Rev. 64:847-867 (2000) The majority of microbes persist attached to surfaces within a structured biofilm ecosystem and not as free floating organisms.

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

Visualization 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

看不見、叫不出名字就無法溝通 三種主要的分類方式 Phenotypic classification Morphology; Biotyping; Serotyping; phage typing; Antibiogram…. Analytic classification Cell wall fatty acid; lipid; protein; enzyme isomers….. Genotypic classification G/C ratio; DNA sequence compare; DNA/RNA hybridization, plasmid….

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

Tree of evolution (兩種概念)

分類學之外插曲 一八九六年，台灣安平爆發黑死病。年底，緒方正規應邀到台灣研究，在台北小南門外設立研究室。一八九七年初，他發現人、鼠均由老鼠身上的跳蚤傳染病菌。 一八九八年，巴斯德研究所的席蒙（Paul Louis Simond, 1858-1947）到印度孟買研究黑死病，以實驗證實了跳蚤是病菌傳播者。從此，黑死病又稱為鼠疫。

北里柴三郎 Robert Koch

科學發展2003年7月，367期76-79 一九○一年，北里與助手中川合寫的一篇文章說道：黑死病菌是桿菌，但是這種桿菌在腫大的淋巴結裡發現得最多，在肺、心、腦、脊髓中，牠們往往看來像球菌。在培養基生長的菌落，以球菌形為主。但是仔細觀察後，就會發現牠們是串接在一起的桿菌，而不是球菌團；在血清中培養的，呈典型桿菌狀。這種細菌是格蘭氏陽性菌。可是，葉赫森一開始就明白地報告：他發現的細菌是格蘭氏陰性的。 現在鼠疫菌的學名，既不是北里的「鼠疫桿菌」，也不是葉赫森的「巴斯德鼠疫菌」或勞森的「北里桿菌」， 而是「葉赫森屬鼠疫種」（Yersinia pestis），簡稱葉赫森菌。那麼，北里柴三郎發現的細菌究竟是打哪兒來的，他真的搞錯了嗎？根據細菌學者的意見，北里的第一篇正式報告大體而言是正確的，最引人矚目的問題出在他說「鼠疫菌有一些呈格蘭氏陽性反應」，不免讓人對於他的正確描述也看輕了。