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Ch 4


4 General Principles of Microscopy 1. Wavelength of radiation 2. Magnification 3. Resolution 4. Contrast
Wavelength Smaller wavelengths of radiation result in enhanced microscopy
Magnification Is the apparent increase in size of object
Resolution *Is the ability to distinguish objects that are close together *Smaller wavelengths = increased resolution *Resolution distance = 0.61 x wavelength/numerical aperture
Contrast Differences in intensity between two objects or between an object and its background
Resolution increased resolving power
Bright-Field Microscopes - Simple Contain a single magnifying lens – similar to magnifying glass & Leeuwenhoek’s microscopes
Bright-Field Microscopes - Compound *Series of lenses for magnification * Light passes through specimen into objective lens * Oil immersion lens increases resolution *1 or 2 ocular lenses * Total magnification = obj len × ocul lens * Most have condenser lens
Dark-Field Microscopes * Best for observing pale objects * Only light rays scattered by specimen enter objective lens * Specimen appears light against dark background * Increases contrast/enables observation of more details * Useful for examining small or colorless cells
Phase Microscope Used to examine living organisms or specimens that would be damaged/altered by attaching them to slides or staining 2 types: Phase-contrast microscope (good 4 cilia & flagella) Diff interference contrast microscope (creates a 3D shadowed appearance)
Fluorescence Microscopes * Direct UV light source at specimen * Specimen radiates energy back as a longer, visible wavelength (fluorescent) * UV light increases resolution/contrast * Used in immunofluorescence to identify pathogens and to make visible a variety of proteins
Confocal Microscopes Use fluorescent dyes Use UV lasers to illuminate fluorescent chemicals in a single plane Resolution is increased because emitted light passes through pinhole aperture Computer constructs 3-D image from individual digitized images
Electron Microscopy * Transmission electron microscopes TEM (2D images of internal cell structures) * Scanning electron microscopes SEM (3D images of cell surface features)
Probe Microscopy 100,000,000× 2 types Scanning tunneling microscopes STM Measures electron flow (tunneling current) to&frm the probe&spec surf Resolution of 0.01nm Atomic force microscopes AFM Pass probe on the spec surf viruses, bacteria, proteins, amino acids.
Principles of Staining Dyes used stains are usually salts tht are composed of a + charged cation & a – charged anion Acidic dyes stain alkaline structures (neg stains) Basic dyes stain acidic structures (simple stains) Most cells/ bacteria are neg charged
Chromophore colored portion of the dye
Differential Stains Use more than one dye Used to distinguish between diff cells, chemicals, structures Common differ stains: Gram Acid-fast Endospore Histological Bacillus & Clostridium produce endospores *Highly resistant to desiccation, heat, chemicals
Special Stains Simple stains used to identify specific microbial structures Special stains include: * Negative stains: are negatively charged and repelled by a negatively charged bacterium *Flagellar stains *Fluorescent stains
Taxonomy consists of classification, nomenclature, and identification
Carolus Linnaeus His system classified organisms based on common characteristics- 1st name ALWAYS cap Grouped organisms that can successfully interbreed into categories called species - 2nd name ALWAYS lc Used binomial nomenclature (latin) Eg; Streptococcus pneumoniae
5 taxonomy kingdoms Animalia, Plantae, Fungi, Protista, and Prokaryotae
3 domains as determined by ribosomal nucleotide sequences: Eukarya, Bacteria, and Archaea
Taxonomic and Identifying Characteristics Physical characteristics Biochemical tests Serological tests (Blood) Phage typing (Virus) Analysis of nucleic acids
MicroScan All bacteria on 1 sheet for testing - quicker results
Phage Typing Bacteriophages (phages) are viruses that infect bacterial cells Phages are specific for the hosts they infect
Analysis of Nucleic Acids Nucleic acid sequence can be used to classify and identify microbes Prokaryotic taxonomy now includes the G + C content of an organism's DNA
Dichotomous keys Series of paired statements where only one of two "either/or" choices applies to any particular organism Key directs user to another pair of statements or provides name of organism
Which of the following is smallest? a. Decimeter B. Millimeter C. Nanometer D. Micrometer Nanometer
A nanometer is __________ that a micrometer. a. 10 times larger b. 10 times smaller c. 1000 times larger d. 1000 times smaller 1000 times smaller
Resolution is best described as the __________. a. ability to view something that is small. b. ability to magnify a specimen c. ability to distinguish between 2 adjacent objects d. difference between 2 waves of electromagnetic radiation Ability to distinguish between 2 adjacent objects
Curved glass lenses __________ light. a. refract b. bend c. magnify d. both a and b both a and b
Which of the following factors is important in making an image appear larger? a. thickness of the lens b. curvature of the lens c. speed of the light passing through the lens d. all of the above all of the above
Which of the following is different between light microscopy and transmission electron microscopy? a. magnification b. resolution c. wavelengths d. all of the above all of the above
Which of the following types of microscopes produces a three-dimensional image with a shadowed appearance? a. simple microscope b. differential interference contrast microscope c. fluorescence microscope d. transmission electron microscope b
Which of the following microscopes combines the greatest magnification with the best resolution? a. confocal microscope b. phase-contrast microscope c. dark-field microscope d. bright-field microscope confocal microscope
Negative stains such as eosin are also called __________. a. capsule stains b. endospore stains c. simple stains d. acid-fast stains capsule stains
In the binomial system of nomenclature, which term is always in lowercase letters? a. kingdom b. domain c. genus d. specific epithet specific epithet
different objective 4x scanning 10X low power 40X high dry 100X oil immersion
If an object magnifies 40X, and each binocular lens magnifies 15X, the total magnification of the object being viewed is ___________. 600x
Immersion oil __________ the numerical aperture, which ___________ resolution because _________ light rays are involved. increases, increases, more
What refers to differences in intensity between two objects? contrast
What are Koch's Postulates? A series of steps that must be taken to prove the cause of any infectious disease
The type of fixation developed by Koch for bacteria is heat fixation
Catonic chromophore said such as methylene blue ionically bond to Negatively charged chemicals such as DNA and proteins
Created by: Cpvander