tortra mic chapter 3.ppt

Chapter 3 Observing Microorganisms Through a Microscope  Microbiology Culturing Microbes The Five “I’s Innoculation: Producing a pure culture Isolation: Colony on media, one kind of microbe, pure culture Incubation: growing microbes...

Chapter 3 Observing Microorganisms Through a Microscope  Microbiology Culturing Microbes The Five “I’s Innoculation: Producing a pure culture Isolation: Colony on media, one kind of microbe, pure culture Incubation: growing microbes under proper conditions Inspection: Observation of characteristics (data) Identification: use of data, correaltion, to ID organism to exact species Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Units of Measurement Table 3.1 1 µm micrometer = 10-6 m = 10-3 mm 1 nm nanometer = 10-9 m = 10-6 mm 1000 nm = 1 µm 0.001 µm = 1 nm Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Microscopy: The Instruments  A simple microscope has only one lens. Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Microscopy: The Instruments In a compound microscope the image from the objective lens is magnified again by the ocular lens. Total magnification = objective lens  ocular lens Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Figure 3.1b Microscopy: The Instruments Resolution is the ability of the lenses to distinguish two points. A microscope with a resolving power of 0.4 nm can distinguish between two points ≥ 0.4 nm. Shorter wavelengths of light provide greater resolution Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Microscopy: The Instruments Light in microscopes either reflected, refracted or absorbed. Refractive index 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. Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Figure 3.3  Animation: Light Microscopy Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Phase-Contrast Microscopy Allows examination of living organisms and internal cell structures Brings together two sets of light rays, direct rays, and diffracted rays to form an image. Accentuates diffraction of the light that passes through a specimen. Direct and reflected light rays are combined at the eye. Increasing contrast Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Darkfield Microscopy Light objects are visible against a dark background. Opaque disk  placed in condenser, Only light reflected off the specimen enters the objective lens, making specimen bright, background dark. (useful for observing living cells, particularly spirochetes) Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Light Microscopy Bright-field microscope: dark image against a brighter background. Light reflected off the specimen does not enter the objective lens Dark-field microscope: only refracted light enters objective, making specimen bright, background dark (useful for observing living cells, particularly spirochetes) Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Fluorescence Microscopy Uses UV light. Fluorescent substances absorb UV light and emit visible light. Cells may be stained with fluorescent dyes (fluorochromes). Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Figure 3.6b  Electron Microscopy Uses electrons instead of light The shorter wavelength of electrons gives greater resolution Used for images too small to be seen with light microscopes, such as viruses Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Electron Microscopy  Two types: Transmission (TEM): electrons pass through thin section of specimen.  Light passes through specimen, then an electromagnetic lens, to a screen or film.  2 dimensional structure  Specimens may be stained with heavy metal salts. Magnifies objects 10,000 to 10,000,000x; resolution of 10 pm. Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Scanning (SEM): Scanning (SEM): image produced by electrons which are emitted from the surface of an object.  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 3 dimensional structure. Magnifies objects 1,000 to 500,000x; resolution of 10 nm Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Staining Staining: coloring microorganisms with a dye that emphasizes certain structures Smear: a thin film of a material containing microorganisms spread over a slide. Fixation: process by which internal and external structures of cells are preserved Heat-fixation - fix an air-dried thin film (smear) by passing through flame. A smear is usually Heat-fixed to attach the microbes to the slide and to kill the microbes Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Animation: Microscopy and Staining: Overview Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Staining (continued) Dyes: have chromophore groups (give color) and bind to cells by ionic, covalent, or hydrophobic bonding Ionic stains  Basic dyes ,the chromophore is a cation  Acid dyes, the chromophore is an anion Hydrophobic - Sudan black, stains lipids Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Staining (continued) Stains consist of a positive and negative ion, one of which is colored (chromophore) In a basic dye, the chromophore is a cation In an acidic dye, the chromophore is an anion Staining the background instead of the cell is called negative staining Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Simple Stains Simple stain: use of a single basic dye Highlights the entire microorganism to visualize cell shapes and structures A mordant may be used to hold the stain or coat the specimen to enlarge it Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Stains Simple Stain: uses one staining agent Differential Stain: divides bacteria into separate groups based on staining properties Gram stain most important staining procedure Dr. Christian Gram, 1884 Gram positive - traps crystal violet-iodine complex, due to thick layer of peptidoglycan in cell wall Gram negative - lipids in cell wall are dissolved by the ethanol, allowing crystal violet-iodine complex to escape, must add counterstain to colorless cells Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Differential Stains: Gram Stain Color of Color of Gram + cells Gram – cells Primary stain: Purple Purple Crystal violet Mordant: Purple Purple Iodine Decolorizing agent: Purple Colorless Alcohol-acetone Counterstain: Purple Red Safranin Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Differential Stains: Gram Stain Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Figure 3.11b  Stains (continued) Acid-fast stain: used to stain Mycobacterium, which have high mycolic acid content (waxy) Must use steam heat to force carbolfuchsin stain into cells Acid-alcohol decolorizer removes stain from non-acid fast cells counterstain with methylene blue Negative/Capsular stain: reveals capsule layer around cells mix bacteria with nigrosin, spread on clean slide, dry useful to observe Klebsiella pneumonia Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Differential Stains: Acid-Fast Stain Cells that retain a basic stain in the presence of acid-alcohol are called acid-fast. Non–acid-fast cells lose the basic stain when rinsed with acid-alcohol, and are usually counterstained (with a different color basic stain) to see them. Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Figure 3.12  Acid-Fast Stain Color of Color of Blank Acid-Fast Non-Acid-Fast Primary Stain: Red Red Carbolfuchsin Decolorizing Agent: Red Colorless Acid-Alcohol Counterstain: Red Blue Methylene Blue Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Stains (continued) Spore stain: used to stain endospores of Clostridium and Bacillus endospores do not readily take up dye, but once it penetrates the stain is not easily decolorized heat smear over steam, rinse with water counterstain with safranin Endospores - Green; Vegetative cells - Red Flagellar stain: flagella of bacteria are coated with tannic acid or potassium alum and stained with basic fuchsin (Gray Method) Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Special Stains . Heat is required to drive a stain into endospores. Flagella staining requires a mordant to make the flagella wide enough to see. Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings Figure 3.13a-c  Negative Staining for Capsules Capsules are a gelatinous covering that do not accept most dyes Suspension of India ink or nigrosine contrasts the background with the capsule, which appears as a halo around the cell. Negative staining is useful for capsules Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Endospore Staining Endospores are resistant, dormant structures inside some cells that cannot be stained by ordinary methods Primary stain: malachite green, usually with heat Decolorize cells: water Counterstain: safranin Spores appear green within red or pink cells. Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings  Flagella Staining Flagella are structures of locomotion Uses a mordant and carbolfuchsin to thicken appearance of flagella, making them visible under the light microscope Copyright © 2004 Pearson Education, Inc., publishing as Benjamin Cummings