《实验室的工具研究微生物的方法.ppt》由会员分享，可在线阅读，更多相关《实验室的工具研究微生物的方法.ppt（83页珍藏版）》请在三一办公上搜索。
1、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 visu
2、alize（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）ass
3、ociations,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 intr
4、oduced 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（
5、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
6、）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 formChemical composition（组成
7、）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
8、 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 Gelid
9、ium（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
10、 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）
11、,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),
12、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 isol
13、ation,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 pos
14、sible.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 nut
15、rient 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 no
16、t 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 microo
17、rganisms.,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 an
18、alysis.,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 appe
19、arance 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 microor
20、gansims,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 magn
21、ifying 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 ocula
22、r,Totalmagnification,=,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）,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
23、 resoltion,Working of an oil immersion lens,PREPARING SPECIMENSFOR 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,whethe
24、r 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
25、,Drying,Fixation,Staining,Observing,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 nigrosi
26、n.(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 surrounding background,Types of microbiological stains,Positive s
27、tain,Negatively Staine,Negatively Stained Bacillus(芽孢杆菌),Simple Versus Differential Staining,Simple stain:requires only a single dye and an uncomplicated procedure.Differential stain:using two different colored dyes to distinguish between cell types or parts.,Simple stain,Gram staining（革兰氏染色）,Staini
28、ng with crystal violet（结晶紫）.Fixation with iodine（碘）stabilizes crystal violet staining.All bacteria remain purple（紫色）or blue.Extraction with alcohol（乙醇）or other solvent decolorizes.some bacteria(Gram negative)and not others(Gram positive).Counterstaining with safranin(蕃红).Gram positive bacteria are already stained with crystal violet and remain purple.Gram negative bacteria are stained pink.,The flow of Gram staining,Gram stain（革兰氏染色）,Endospore stain(芽孢染色),Capsule stain（荚膜染色）,Flagellar stain（鞭毛染色）,
