Microscopy Kathy Huschle Northland Community and Technical College
Microscopy • microscope • produces enlarged images • studies the morphology (what it looks like) of a microorganism
Microscopy • microscopy includes • magnification • resolution • contrast
Magnification • magnification • a result of refraction or bending of light rays
Resolution • resolution • ability to distinguish detail • will allow you to observe one layer at a time
Contrast • contrast • visible shades in a specimen • contrast is often enhanced by staining
Light Microscopes • Bright-field Microscope • field of view is brightly illuminated with visible light • also known as compound microscope
Fluorescence Microscope • Fluorescence Microscope • use with fluorescent stains • emit light at a different wavelength • different wavelengths have different colors
Fluorescence Microscope • dyes can be chemically linked specific chemical targets • target can be visualized and quickly and positively identified • allows for rapid diagnosis Click icon to use a virtual fluorescence scope. Click “Try the Simulator”
Fluorescence Microscope Image of cell division taken by fluorescence microscope
Dark Field Microscope • Dark-field Microscope • enhance contrast without staining • most staining will kill the microorganism • allows for the viewing of live specimens • special condenseris used • focuses light at an angle • reflected off specimen Treponema pallidum image from Dark Field Microscope
Phase Contrast Microscope • Phase Contrast Microscope • contrast without staining • difference in density produces difference in light • brings direct and reflected light rays together to form an image of the specimen image of cell division taken by phase contrast microscope
Phase Contrast Microscope • allows for observation of living organisms Click icon to use a virtual phase contrast scope. Click “Try the Simulator”
Interference Microscope • Interference Microscope • two light waves combined • beam is split • one part through the specimen • one part around • recombined
Interference Microscope • specimen looks three dimensional
Confocal Scanning Laser Microscope • constructs a 3-dimensional image • specimen is stained with fluorescent dye
Confocal Scanning Laser Microscope • specimen is illuminated one plane at a time • computer processes the images into 2 and 3 dimensional images
Electron Microscopy • electron beam is used in the place of a beam of light • electromagnets control focus, illumination and magnification • higher magnification: up to 100,000X
Electron Microscopy • electron beam must be contained in a vacuum to eliminate disturbance from air molecules • large, bulky unit • specimen must be killed • special preparation process • problem with artifacts
Transmission Electron Microscope • TEM • used for observing layers of cells
Transmission Electron Microscope • 10,000 – 100,000 X magnification rubber particle magnified 100,000 times
Transmission Electron Microscope Click icon to use a virtual Transmission electron scope. Click “Try the Simulator”
Scanning Electron Microscope • SEM • used for surface features, not internal features • knocks electrons out of specimen surface • collected for image
Scanning Electron Microscope • SEM • produces a 3-D image • 1,000 – 10,000 X magnification image of table salt taken with an SEM
Scanning Electron Microscope Click icon to use a virtual Scanning electron scope. Click “Try the Simulator”
Contrast & Staining • stain is a salt with a negative and positive charged ion • color portion is a positive charge and will be attracted to the negative charged microbial cell Enterobacter
Contrast & Staining • stain • increases contrast between specimen and background • most microorganisms are colorless and difficult to see Unstained Halophile Stained Halophile
Contrast & Staining • simple stains • positive staining • cells are dark against light background • makes cell shapes and arrangements visible yeast stained with methylene blue stain
Contrast & Staining • negative staining • clear microorganisms against a dark background • better view of microbial shape due to lack of distortion • stain is not taken up by the microorganism • very useful with microorganisms with capsules negatively stained mixed bacteria culture
Differential Staining • differential staining • multiple stains • differentiate bacteria according to their reaction to the staining process • differentiation is based on differences in cell structure • Gram stain • acid fast stain
Differential Staining • Gram Stain • most widely used procedure • process uses a stain, wash, counterstain
Differential Staining • Gram Stain • Gram positive bacteria retain the purple stain • Gram negative bacteria appear pink after the counterstain Gram - Gram +
Differential Staining • acid-fast staining • waxy chemical in the cell wall allows the bacteria to retain the stain Cryptosporidum muris
Differential Staining • acid-fast staining • makes Mycobacterium (causative agent fortuberculosis)easy to recognize in sputum, amidst numerous organisms sputum sample containing M. tuberculosis
Special Stains • stains that color only certain parts of bacteria • endospore staining • reveals the presence of endospores • produced by few bacteria • Clostridium and Bacillus A is the microorganism, B indicates the endospore
Special Stains • flagella staining • adheres to an enlarges the flagella for ease of observation flagella
Bacteria Morphology:Shape • 3 basic shapes • coccus • bacilus • spiral spiral coccus bacillus
Bacteria Morphology:shape • coccus • primarily spherical in shape • can be oval, elongated or flattened on one side • plural: cocci Enterococcus sp.
Bacteria Morphology:shape • bacillus • rod shaped • plural: bacilli Bacillus anthracis
Bacteria Morphology:shape • spiral • one or more twists • never straight Treponema pallidum
Bacterial Morphology:Arrangement • single yeast cells
Bacterial Morphology:arrangement • diplococci or diplobacilli Neisseria gonorrhoeae
Bacterial Morphology:arrangement • chains of coccus or bacillus Streptococcus sp.
Bacterial Morphology:arrangement • clusters
