Units of Measurement 1 µm = 10 -6 m = 10 -3 mm 1 nm = 10 -9 m = 10 -6 mm 1000 nm = 1 µm 0.001 µm = 1 nm
Simple Microscope A simple microscope has only one lens
Compound Microscope A compound microscope has two sets of lenses and is typically used in teaching and research laboratories. Parts of a student laboratory microscope
Optics : Magnification A specimen is magnified as light passes through the objective and ocular lenses. Total Magnification = objective lens X ocular lens (magnifications) The pathway of light and two stages of magnification of a compound microscope.
Optics: Resolution Resolution is the ability of the lenses to distinguish two points. A microscope with a resolving power of 0.2 um can distinguish between two points > 0.2 um. Resolution distinguishes magnified objects clearly.
Optics: Resolution Resolution can be increased by using immersion oil and shorter wavelengths of light.
Optics: Refraction 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.
Brightfield Microscopy Dark objects are visible against light background. Used to observe stained or unstained specimens. Most commonly used in laboratories.
Darkfield Microscopy Light objects are visible against a dark background. Light reflected off the specimen enters the objective lens. Used to screen for syphilis agent, Treponema pallidum.
Phase-contrast Microscopy Accentuates diffraction of light that passes through the specimen. Used to observe internal cellular detail of live specimens.
Differential Interference Contrast (DIC) Microscopy Accentuates diffraction of light that passes through the specimen. Uses two beams of light to produce more pronounced contrast.
Fluorescence Microscopy Cells are stained with fluorescent dyes called fluorochromes. Uses UV light as energy source. Fluorochromes absorb UV light and emit visible light Fluorescent staining of fresh sample of cheek scrapings
Confocal Microscopy Uses fluorochromes and laser light. The laser illuminates each plane in a specimen to produce a 3-D image.
Electron Microscopy Very high magnification of up to 100,000 X. Transmission electron microscope (TEM) View internal structures of cells Scanning electron microscope (SEM) Three-dimensional images
Transmission Electron Microscopy Transmission Electron Micrograph of (a) a virus and (b) a protozoan.
Scanning Electron Microscopy False-color Scanning Electron Micrograph of Paramecium sp.
Summary of Microscope Types
Optical and Electron Microscopy: A Comparison
TEM and SEM Compared Scanning Electron Microscope Transmission Electron Microscope
Scanning Probe Microscopy Scanning tunneling microscopy uses a metal probe to scan a specimen. Resolution 1/100 of an atom.
Scanning Probe Microscopy Atomic force microscopy uses a metal and diamond probe inserted into the specimen. Produces 3-D images.
Preparation of Specimens for Light Microscopy A thin film of a solution of microbes on a slide is a smear. A smear is usually fixed to attach the microbes to the slide and to kill the microbes.
Stains Positive stains Dye binds to the specimen Negative stains Dye does not bind to the specimen, but rather around the specimen.
Positive and Negative Stains Positive stains are basic dyes (cationic) that bind to negatively charged cells. Negative stains are acidic dyes (anionic) that bind the background.
Positive Stains Simple -One dye Differential -Two-different colored dyes Ex. Gram stain Special -Emphasize certain cell parts Ex. Capsule stain
Bacterial Stain Types
Simple Stain Use of a single basic dye is called a simple stain. A mordant may be used to hold the stain or coat the specimen to enlarge it.
Differential Stain: Gram Stain The Gram stain classifies bacteria into gram- positive and gram-negative. Gram-positive bacteria tend to be killed by penicillin and detergents. Gram-negative bacteria are more resistant to antibiotics.
Differential Stain: Gram Stain Color of Gram + cells Color of Gram – cells Primary stain: Crystal violet Purple Mordant: Iodine Purple Decolorizing agent: Alcohol-acetone PurpleColorless Counterstain: Safranin PurpleRed
Differential Stain: Gram Stain
Differential Stain: 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.
Culture of Microbes Five basic techniques Media Microbial growth