教案课件微生物chapter2-s

1、MicrobiologyChapter TwoThe methods for studying microbiology 2.1 General laboratory techniques studing microorganisms2.2 The microscope 2.3 Methods of isolating and culturing microorganisms2.1 General laboratory techniques studing microorganismsThe Six IsInoculation(接种)Incubation(培养)Isolation(分离)Ins

2、pection (检查)Information gathering (信息收集）Identification(鉴定)Figure 3.1 An overview of some general laboratory techniques carried out by microbiologists（Foundations-8th，P60）2.2 The microscope 1.The basic plan of an optical microscope2. Major types of optical microscopes3.3. Other microscopes4.4. Prepar

3、ing Specimens for Optical MicroscopesSimple microscopes: contains a single magnifying lens and a few working parts.Compound (two-lens) microscope: contains two magnifying lens, illuminating lamp, condenserLight sourceFocus knobs1.The basic plan of an optical microscope In a compound microscope the i

4、mage from the objective lens is magnified again by the ocular lens. Total magnification =objective lens ocular lensMagnification ability to enlarge objects Chapter 2 Resolution (分辨率)is the ability of the lenses to distinguish two points. Shorter wavelengths () of light provide greater resolution.R.P

5、. = -Wavelength of light in nm2xNumerical apertureResolving power ability to show detail Numerical aperture: a mathematical constant that describes the relative efficiency of a lens in bending light rays. (range from 0.1 to 1.25 in oil immersion)R.P. = -Wavelength of light in nm2xNumerical apertureR

6、.P. = - = 200 nm500 nm2 1.25For oil immersion lens using a blue-green light: (or 0.2m)Refractive index (n) is the light-bending ability of a medium.The light may bend in air so much that it misses the small high-magnification lens.Immersion oil is used to keep light from bending as it passes through

7、 the specimend=1.250.5x500 nm=0.2md = -0.5 n sin Chapter 2Effect of magnificationComparison of objects that would not be resolvable versus those that would be resolvable under oil immersion at 1000 x magnification.Good resolution means being able to observe an objective clearlyDark objects are visib

8、le against a bright background.Light reflected off the specimen does not enter the objective lens.2. Major types of optical microscopes The Bright-Field MicroscopeParameciumThe Dark-Field Microscope Living, unstained cells and organisms can be observedLight objects are visible against a dark backgro

9、und.Light reflected off the specimen enters the objective lens.ParameciumAccentuates diffraction of the light that passes through a specimen.Useful for studying living cellsPhase-Contrast MicroscopyParamecium15光线透过标本后发生折射，偏离了原来的光路，同时被延迟了1/4（波长）。经过相板后再被延迟1/4（波长），达1/2。两束光合轴后干涉加强，振幅增大或降低，提高反差。 Accentua

10、tes diffraction of the light that passes through a specimen; uses two beams of light. Useful for studying a live, unstained specimen Differential Interference Contrast Microscopy微分干涉相差显微镜微分干涉相差显微镜 Uses UV light. Fluorescent substances absorb UV light and emit visible light. Cells may be stained with

11、 fluorescent dyes (fluorochromes). An essential tool in medical microbiology and microbial ecology.Fluorescence Microscopy共聚焦显微镜（Confocal Laser Scanning Microscope）a) E.coli stained with fluorescent antibodies. The green material is debris;b)Paramecium tetraurelia conjugating; acridine-orange fluore

12、scence;c) Protozoan stained with fluorescent antibodies to show the kinetoplast;d) Fluorescent staining on a fresh sample of cheek scrapings from the oral cavityConfocal microscopic images Uses electrons instead of light. The shorter wavelength of electrons gives greater resolution.(0.5 nm.100,000 t

13、imes shorter than the waves of visible light)3 . Other microscopesFigure 3.12 Comparison of light and electron microscopes (Foundations-8th, P67)Ultrathin sections of specimens.Light passes through specimen, then an electromagnetic lens, to a screen or film.Specimens may be stained with heavy metal

14、salts.Transmission Electron Microscopy (TEM)ultramicrotome An electron gun produces a beam of electrons that scans the surface of a whole specimen. Secondary electrons emitted from the specimen produce the image. Scanning Electron Microscopy (SEM)1000-10,000; resolution 20 nm超分辨率技术（super-resolution

15、technique)Resolving power:1-50 nm Scanning tunneling microscopy uses a metal probe to scan a specimen. Resolution 1/100 of an atom.Scanning-Probe Microscopy扫描探针显微术扫描探针显微术Atomic force microscopy (原子力显微术原子力显微术)uses a metal and diamond probe inserted into the specimen.Produces 3-D images.28 single CO m

16、oleculesA nanographene molecule world-atomic-microscope-chemical-bonds.html(Foundations-8th, P65)4. Preparing Specimens for OpticalMicroscopesFresh, Living Preparationswet mount : a drop or two of the culture placed on a slide and overlaid with a cover glasshanging drop slideA thin film of a solutio

17、n of microbes on a slide is a smear(涂片).A smear is usually fixed to attach the microbes to the slide and to kill the microbes. Heat fixation or chemical fixationStaining (染色) is any procedure that applies colored chemicals called dyes to specimens.Basic dyes have a positive charge, acidic dyes have

18、a negative charge. Fixed, Stained SStainPositive Stain(正染色正染色): dye sticks to the specimen Negative Stain (负染色负染色): Staining the background instead of the cell Simple Stain(单染单染): use of a single basic or acid dye Differential Stain(鉴别染色鉴别染色): use two different-colored d

19、yes to distinguish between cell types or parts. Such as Gram stain, acid-fast stain and endospore stainSpecial Stain: use to emphasize certain cell parts that are not revealed by conventional staining method. such as capsule and flagellar stainsFigure 3.15 Staining reactions of dyes. Basic dyes are

20、positively charged and react with negatively charged cell areas. methylene blue crystal violet, and safranin.(b) Acidic dyes are negatively charged and react with positively charged cell areas. nigrosin India ink(c) Acidic dyes can be used with negative staining to create a background around a cell.

21、Because bacteria contain large amounts of negatively charged substances, they stain readily with basic dyesTypes of stainsFlagellar stain of Listeria monocytogenesDifferential Stains: Gram StainThe Gram stain classifies bacteria into gram-positive and gram-negative, developed in 1884 by the Danish p

22、hysician Christian Gram.2.3. Methods of isolating and culturing microorganisms Pure culture: a population of cells arising from a single cell.Pure culture techniques were developed by Koch. Colony : a visible growth or cluster of microoganisms on a solid medium. Each colony represents a pure culture

23、. A colony consists of one speciespipettebeakerBunsenspreaderInoculating loop1. Isolation technique Chapter 2P61, NP63Common methods to isolate a colony: streak plate (平板划平板划线法线法), Loop dilution（接种环稀释）（接种环稀释）and spread plate（平（平板涂布法）板涂布法） Aseptic transfer of a cultureAseptic technique is one of the

24、first methods learned by the novice microbiologist2. Media The Foundations of CulturingLiquid mediaSemisolid mediaPhysical States of MediaSolid media agar is a complex polysaccharide isolated from red algae solid at room temp, liquefies at boiling (100oC), does not resolidify until it cools to 42oC

25、provides framework to hold moisture & nutrients not digestible for most microbesAgar stripsSolid media that reversible to liquidsSynthetic media: with a chemically defined composition, contain pure chemical nutrients. Complex (nonsynthetic) media: even one component of a given medium is not chem

26、ically definable. Substances include extracts from animal, plant tissues, blood, serum, meat extracts, infusions, milk, soybean digests, and peptone.Chemical Content of MediaChocolate agar(cooked blood) Functional type of mediaGeneral-purpose media are designed to grow a broad spectrum of microbes t

27、hat do not have special growth requirementsEnriched medium contains complex organic substances (blood, serum, hemoglobin, or special growth factors) that certain species must be provided in order to growBlood agar platebacteria from the human throatNeisseria Comparison of selective and differential

28、media with general-purpose media.A selective medium contains one or more agents that inhibit the growth of a certain microbe or microbes.Differential media grow several types of microorganisms but are designed to bring out visible differences among those microorganisms.Mannitol salt agar (with 7.5%N

29、aCl) is used to isolate members of the genus Staphylococcus.Example of selective and differential mediaCHROMagar OrientationTM uses color-forming reactions to distinguish at least 7 species and permits rapid identification and treatment. Some microbial groups (viruses, rickettsias, and a few bacteri

30、a) will only grow on live cells or animalsA single medium can be classified in more than one category depending on the ingredients it containsOther media Reducing medium, Transport media, Carbohydrate fermentation media, Assay media, Enumeration media1.Do “Multiple Choice Quiz” in2. /sites/ /student

31、_view0/chapter3/multiple_choice_quiz.html3.2. Review Questions 1, 2, 3 (Brock, P46)4.3. Watch video “microbiology_Gram strain”5.Deadlines: Sep. 30Chapter 2Discussion 2Critical Thinking Questions 3-5 (Foundations-8th, P88):3. a. What kind of medium might you make to selectively grow a bacterium that

32、lives in the ocean? b. One that lives in the human stomach? c. What characteristic of dyes makes them useful in differential media? d. Why are intestinal bacteria able to grow on media containing bile?4. Go back to page 6 and observe the six micrographs in figure 1.3. See if you can tell what kind o

33、f microscope was used to make the photograph based on magnification and appearance.5. Could the Gram stain be used to diagnose the flu? Why or why not?Can you design an experiment to study those Uncultured microbes (Foundations-8th, P75) in terms of the Six I s(we will discuss these questions all to

34、gether, please prepare to answer them. ) Negative staining is useful for capsules. Heat is required to drive a stain (malachite green孔雀绿) into endospores. Flagella staining requires a mordant (tannic acid单宁酸, and potassium alum明矾钾) to make the flagella wide enough to see.Special Stains Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast. Nonacid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with a different color basic stain) to see them.Differential Stains: Acid-Fast Stain(抗