chapter实验室的工具研究微生物的方法.ppt

Tools of the Laboratory（实验 ） Chapter 3 Main Themes Methods of Culturing（培养） Microorganisms（微生物） The Microscope（显微镜） Over the past 30 years,nearly 20 new infectious（传染性的） agents（介质） have appeared suddenly and unexpectedly（意外的）. Chapter Overview A driving force of microbiology has been to find ways to visualize（显现） and handle （处置 ）microorganisms; Microbes are managed and characterized （概括特征）by implementing the Five I,S; The microscope is a powerful tool for magnifying（放大） and resolving cells and their parts; Methods of Culturing Microorganisms Problems: Most habitats（栖息地） harbor（居住） microbes in complex（复杂的） associations ,so it is often necessary to separate the species from one another; To maintain and keep track of （了解）such small reserarch subjects,mircrobiologists（微生物学家） usually have to grow them under artificial conditions; They are invisible and widely distributes（分布广泛） ,and undesirable（意外的） ones can be introduced into an experiment and cause misleading results. If you want to study a specified（特定 地） microorganism ,you should do experiments step by setp - according to the Five I,S. INOCULATION:PRODUCING A CULTURE To cultivate（培养） microorganisms ,one introduces（引入） a tiny sample into a container（容器） of nutrient（营养物） medium,which provides an environment in which they multiply（繁殖）.This process is called inoculation(接种). ISOLATION:SEPARATE ONE SPECIES FROM ANOTHER If an individual bacterial cell is separated from other cells and provided adequate（ 充足的） space on a nutrient surface,it will grow into a discrete（分立的） mound（ 堆积物） of cells called a colony(菌落). Isolation Technique Methods for isolating bacteria The spreading tool MEDIA:PROVIDING NUTRIENTS IN THE LABORATORY A medium（培养基） is a liquid or gel（凝胶） designed to support the growth of microorganisms or cells . Types of Media Physical form Chemical composition（组成） Functional type(table 3.1) Physical States of Media Liquid media are defined as water-based that do not solidify（固化） at temperatures above freezing and that tend to flow freely when the container is tilted（ 倾斜）. Sample liquid media Semilolid media They contain some an amount of solidifying agent that thickens them but does not produce a firm substrate（基础）. Sample semisolid media Solid media Solid media provide a firm surface on which cells can form discrete colonies and are advantageous（适合的） for isolating and culturing bacteria and fungi（真菌）. Solid media Agar(琼脂) Isolated from the red alga Gelidium（石花 菜属 ）; It is solid at room temperature; Onced liquefied（液化）,agar does not resolidify until it cools to 42； Semisolid media(0.3-0.5%)、solid media (0.5-2%). Nutrient agar（营养琼脂） is a microbiological growth medium commonly used for the routine（常规的） cultivation of non-fastidious（苛求的） bacteria. Nutrient agar typically contains (w/v): 0.5% peptone （蛋白胨） 0.3% beef extract （牛肉膏） 1.5% agar （琼脂） pH adjusted to neutral at 25 . Nutrient broth does not contain agar. Peptone （蛋白胨） Any water soluble（可溶的） product, a mixture of polypeptides（多肽） and amino acids（氨基酸） formed by the partial hydrolysis（水解作用） of protein（蛋白质）. Peptone（蛋白胨） Chemical Content of Media Chemical content of media are various(多 种多样的); They are classified into synthetic（合成的 ） ingredients and non-synthetic（非合成 的） ingredients（成分）; Natural media and synthetic media; Natural comounds(天然化合物) Peptone（蛋白胨） Beef extract（牛肉膏） Potato（土豆） Bread（面包） Synthetic compounds(合成化合物) Nacl kH2PO4 Mgso4 Media Undefined media(天然培养基) all natural resources Chemical defined media all chemical compounds Semidefined media(半组合培养基) natural resources +chemical compounds Media to Suit Every Functions Microbiologists have many types of media at their disposal(实验); Media are used for primary isolation,to maintain cultures in the lab,to detemine chemical and growth characteristics（特征 ） ,and for other functions; General media（基础培养基）、enriched meida（加富培养基）、selective and differential media（选择分化培养基）、 Basal(general) media Basal media(基础培养基) are designed to grow as broad a spectrum(范围) of microbes as possible. Peptone （蛋白胨） Beef extract（牛肉膏） Nacl（氯化钠） Agar（琼脂） Water （水） Enriched media(加富培养基) An enriched media contains complex(复杂 的) orgainc（有机物） substances such as blood ,serum（血清），hemoglbin （血色 素）,or special growth factors that certain species must have in order to grow. When a media is prepared which have a nutrient constituent（成份） which encourage the growth of particular bacteria or microbe is called enriched media, such as blood agar（血琼脂） and chocolate agar(巧克力琼脂). Examples of enriched media Selective Media(选择性培养基) A selective media contains one or more agents that inhibit(禁止) the growth of A microbe but not B and therby encourages,or selcets B microbe and allow it to grow. Comparison of Selective media and general media Example of selective media Differential media(鉴别培养基) Differential media grow several types of microorganisms and are desigened to display（展示） visible（可见的） differences among those microorganisms. EMB meida（伊红美蓝琼脂培养基 ） Media that differentiate characteristics A reducing medium contains a substance that absorbs oxygen（氧气） Of slowing the penetration（渗透） of oxygen in a medium. Transport （运输） mediaare used to maintain and preserve specimens that have to be held for a period of time before analysis. Carbohydrate fermentation in broths（肉汤） INCUBATION,INSPECTION,AND IDENTIFECATION The usual temperatures used in lab fall between 20 C and 40 C. If you incubate in the liquid meida,you may see cloudiness（混浊 ）,sediment （沉淀 ）and colour. A common manifestation(例证) of growth on solid media is the appearance of colonies（菌落）. Liquid media Various conditions of cultures A mixed culture is a container that holds two or more identified,easily differentiated （不同的） species of microorganism. Inspection Identification Biochemistry（生化） and molecular tests Maintenance and disposal of Cultures Disposed microorgansims Sterilization Burning Boiling The Microscope:Window on an Invisible realm Microscope Magnification Showing detail The pathway of light and the stages in magnification of a compound microscope Effects of magnification The parts of a laboratory microscope Principle of Light Microscopy The magnifying power of the objectives alone usually ranges from 4to 100,and the power of the ocular（视觉的） alone ranges from 10to 20.The total power of magnification of the final image formed by the combined lenses is a product of the separate powers of th two lens（透镜）: Power of objective Usual power of ocular Total magnification = Resolution（分辨力） Resolution is th capacity of an optical system to distinguish or separate two adjacent objects or points from one another. Resolving（分辨率） conditionresolving Naked eyes0.2mm optical microscope 0.2m electron microscope 0.2nm R.p.=Wavelength of light in nm（波长 ） / 2*Numerical aperture (数值孔径) Numerical aperture =n*sin a/2 n:折射率 Effect of wavelength on resoltion Working of an oil immersion lens PREPARING SPECIMENS FOR OPTICAL MICROSCOPES The condition of the specimen,either in a living or preserved(保存) state; The aims of the examiner,whether to observe overall structure,identify the micororganism,or see movment； The type of microsopy abailable,whether is is bright-fiele(暗视野),phase-contrast（相 差）,or fluorescence（荧光）. Preparing specimens（标本） Fresh,Living Preparations Fixed（固定）,Stained （染色） Smears Simple（简单） Versus Differential（鉴别） Staining Slide(载玻片) Cover glass(盖玻片) The making of smear（血涂片） The flow of optical microscope （显微镜观察流程） Spreading a thin film Drying Fixation StainingObserving Dark-Field Microscope (暗视野显微镜光路图) Negative Versue Positive Staining Positive stain:the dys actually sticks to the specimen and gives it color; Negative stain:For bright field microscopy, negative staining is typically performed using a black ink fluid such as nigrosin.(苯胺黑) The specimen, such as a wet bacterial culture spread on a glass slide, is mixed with the negative stain and allowed to dry. When viewed with the microscope the bacterial cells, and perhaps their spores, appear light against the dark su