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Chapter 3: Observing Microorganisms Through a Microscope

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Chapter 3: Observing Microorganisms Through a Microscope Microscopy: The technology of making very small things visible to the naked eye. Units of Measurement: The metric system is Chapter 3: Observing used to measure microorganisms. Microorganisms Through a Metric system: Microscope u Basic unit of length: Meter. u All units are related to each other by factors of 10. u Prefixes are used to indicate the relationship of a unit to the basic unit (e.g.: meter). Metric Units of Length and U.S. Instruments of Microscopy: Equivalents: 1. Simple Microscopes: Metric Relationship to U.S. uOnly have one lens, similar to a magnifying glass. Unit basic unit (meter) Equivalent uLeeuwenhoeck’s simple microscopes allowed kilometer (km) 1 km = 1000 m 1 mile = 1.61 km him to magnify images from 100 to 300 X. meter (m) 1 m = 39.37 in uThey were so difficult to focus, he built a new one for decimeter (dm) 1 dm = 0.1 m = 10-1 m 1 dm = 3.94 in each specimen, a total of 419. centimeter (cm) 1 cm = 0.01 m = 10-2 m 2.54 cm = 1 in uHe did not share his techniques with other scientists. millimeter (mm) 1 mm = 0.001 m = 10-3 m Even today, his source of lighting is unknown. micrometer (um) 1 um = 0.000001 m = 10-6 m uHis daughter donated 100 of his microscopes to the Royal Society shortly before his death in 1723. nanometer (nm) 1 nm = 0.000000001 m = 10-9 m picometer (pm) 1 pm = 0.000000000001 m = 10-12 m Instruments of Microscopy: Instruments of Microscopy: 2. Compound Light (CL) Microscopy 2. Compound Light Microscopy History of CL Microscopes: u Have several lenses: uFirst developed by Zaccharias Janssen, Dutch 1.Light originates from an illuminator and passes spectacle maker in 1600. through condenser lenses, which direct light onto the specimen. u Poor quality 2. Light then enters the objective lenses, which magnify u Could not see bacteria the image. These are the closest lenses to the specimen: u Joseph Jackson Lister (Lister’s father) developed uScanning objective lens: 4 X improved compound light microscope in 1830s. uLow power objective lens: 10 X uHigh power objective lens: 40-45 X u Basis for modern microscopes uOil immersion lens: 95-100 X uUse visible light as a source of illumination. 3. The image of the specimen is magnified once again by the ocular lens or eyepiece (10 X). 1 Instruments of Microscopy: Instruments of Microscopy: 2. Compound Light Microscopy 2. Compound Light Microscopy u Resolution (Resolving power): Ability of u Total magnification: Obtained by multiplying microscope to see two items as separate and objective lens power by ocular lens power. discrete units. (Condenser lenses do not magnify image). u The smaller the distance between objects at which they can be distinguished as separate, the greater the Lens Magnification Ocular Mag. Total Mag. resolving power. Scanning 4 X 10 X = 40 X u Light must pass between two objects in order for them to be seen as separate. Low power 10 X 10 X = 100 X u Depends on light wavelength. If wavelength is too High power 45 X 10 X = 450 X long to pass between objects, they will appear as one. uWhite light has a relatively long wavelength (550 nm), and Oil immersion 100X 10 X = 1000 X cannot resolve structures less than 220 nm (0.2 um) apart. Highest possible magnification with CL microscope is u Ultraviolet (UV) light has a shorter wavelength (100 to 400 nm), and can resolve distances as small as 110 nm . about 2000 X. Instruments of Microscopy: Instruments of Microscopy: 2. Compound Light Microscopy 3. DarkfieldMicroscopy u Refraction: Bending of light as it passes from u Useful to examine live or unstained specimens. one medium to another of different density. u Light sensitive organisms u Index of refraction: A measure of the speed at which light passes through a material. u Specimens that lack contrast with their background. u Can be changed by staining, which increases contrast uSpirochetes which cause syphilis. between specimen and surrounding medium. u Darkfield condenser with opaque disc blocks u When two substances have a different index of light that would enter objective lens directly: refraction, the light will bend as it passes from one material to another. u Light reflects off specimen at an angle. u As light passes through a glass slide, air, and the u Only light reflected by specimen enters objective lens. objective lens, it bends each time, causing loss of light u No direct background light. and a blurred image. u Immersion oil has the same index of refraction as u Image : Light specimen against dark glass slide, preventing light loss from refraction. background. Instruments of Microscopy: Instruments of Microscopy: 4. Phase Contrast Microscopy 5. Fluorescence Microscopy u Useful to examine live specimens: uFluorescence: Ability of substances to absorb short wavelengths of light (ultraviolet light) and u Doesn’t require fixing or staining, which usually kill emit them at a longer wavelength. and/or distort microorganisms. u Natural Fluorescence: Some microorganisms u Permits detailedexamination of internal fluoresce naturally under UV light (Pseudomonas). structures. u Fluorochrome: Fluorescent dye. uAcridine orange: Binds to nucleic acids, colors cells orange, u Special objective lenses and condenser with ring green, or yellow depending on light source. shaped diaphragm accentuate small differences in u Immunofluorescence: Fluorescent antibodies can be refractive indexes of internal structures. used to detect specific antigens. Very useful for the u Image: Direct rays and reflected light rays come rapid diagnosis of specific diseases (e.g.: syphilis). together, forming an image with many shades of uImage: Luminescent bright object against a dark gray to black. background. 2 Instruments of Microscopy: Instruments of Microscopy: Limitations of light microscopy: 6. Electron Microscopy u Magnification: Up to 2000 X. u Electron microscopes were first developed in 1932, and became widely available in 1940s. u Resolving Power: Up to 0.2 um. u Use a beam of electrons instead of a beam of Because of the limits of magnification and light. u Wavelength of electron beam is about 100,000 times resolving power, viruses and most internal smaller than visible light. structures of cells cannot be seen with a light u Used to examine structures too small to be resolved microscope. with a light microscope. u Two types of electron microscope: A. Transmission Electron Microscope (TEM) B. Scanning Electron Microscope (SEM) Instruments of Microscopy: Instruments of Microscopy: 6. Electron Microscopy 6. Electron Microscopy A. Transmission Electron Microscope (TEM) B. Scanning Electron Microscope (SEM) u Gives excellent view ofinternal structures. u Gives excellent view ofexternal surface. u Magnification: 100,000 X or more. u Magnification: 10,000 X or more. u Resolving power: 20 nm or better. u Resolving power: 2.5 nm or better. u Three dimensional image. u Two dimensionalimage. u More recent invention than TEM. Used mainly to u Drawbacks of TEM: observe the surfaces of cells and viruses. u Due to limited penetrating power of electrons, can only u Specimens are covered with a layer of heavy metal view very thin slices (70-90 nm) of specimen. (gold or palladium). u Must slice, fix, dehydrate, and view specimen under a u A narrow beam of electrons (primary electron beam) vacuum. Staining may be used to enhance image is swept across specimen surface. contrast. u Electrons on the specimen surface are knocked out, u Treatments kill specimen and may cause shrinkage and creating a secondary electron beam which is collected and amplified to produce an image. distortion of cells (artifacts). 7. Scanning Tunneling Microscopy and Preparation of Specimens for Light Atomic Force Microscopy (AFM) Microscopy u Developed in the 1980s. 1. Smear: Spread a thin film of material containing u Used to observe structure and surface of microorganisms over slide surface. Allow to air biological molecules and silicon computer chips. dry. A. Scanning Tunneling Microscope (STM) 2. Fixing: Process that kills microorganisms and Uses a thin metal probe that scans the surface of a specimen. attaches them to a microscope slide. Fixing B. Atomic Force Microscopy (AFM) preserves and minimizes distortion of cells. Uses a diamond and metal probe that scans Two main methods of fixation: surface of specimen. u Heat fixation: Pass over Bunsen burner flame several Advantages of both microscopes: times. u Higher resolving power than electron microscopes u Chemical fixation: Cover with methanol for 1 minute. u No special specimen preparation required 3 Preparation of Specimens for Light Preparation of Specimens for Light Microscopy Microscopy 3. Staining: Coloring microorganisms with a dye A. Basic dyes : that emphasizes certain structures. Before staining u Chromophor is in positive ions. a sample, it must be fixed. u Most commonly used dyes. Stains are salts composed of a positive ion u Bacteria are slightly negatively charged at pH (cation) and a negative ion (anion). 7, therefore they stain with basic dyes. The colored ion is called the chromophore . u Examples: Two types of dyes: uCrystal violet A. Basic dyes u Methylene blue B. Acidic dyes u Saffranin Preparation of Specimens for Light Preparation of Specimens for Microscopy Microscopy 1. Simple Stains B. Acidic dyes: u Aqueous or alcohol solution of a single basic u Color is in negative ions. dye. uStain the background: negative staining. u Primary purpose is to stain entire microorganism u Bacteria do not stain with acidic dyes. to view cell shape and basic structures. u Procedure: u Used to observe cell shape, size, and capsules. u Stain is applied for a certain time, and then washed off. u Minimal distortion because heat fixing is not u Slide is dried and examined. necessary an dye is not taken up by cells.
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