Microbiology Class 2

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If a bacterium is 1 um and your index finger= 6.5 cm, how many bacteria can you place end to end on your finger? 6.5cm=65mm=65000 um/1um=65000 bacteria

UNITS OF MEASUREMENT

•Microorganisms are measured in micrometers (um) and nanometers (um)

•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

MICROSCOPY : THE INSTRUMENTS

•Simple microscope - has only one lens

•Used by van Leeuwenhoek in the 17th century

•Compound microscope – has a series of microscopes

LIGHT MICROSCOPY

•Any kind of microscope that uses visible light to observe specimens

•Types of light microscopy

•Compound light microscopy

•Darkfield microscopy

•Phase-contrast microscopy

•Differential interference contrast (DIC) microscopy

•Fluorescence microscopy

•Confocal microscopy

COMPUND LIGHT MICROSCOPY

•Image from the objective lens is magnified again by the ocular lens

•Total magnification = objective lens x ocular lens

•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 at least 0.4 nm apart

•smaller wavelengths of light provide greater resolution (ex UV light=better resolution)

•Refraction is a measure of the light-bending ability of a medium

•Light may refract after passing through a specimen to an extent that it does not pass through the objective lens

•Immersion oil is used to keep light from refracting

•Brightfield illumination

•Dark objects are visible against a bright background

•Light passes through the specimen and then is collected by an objective lens

•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

•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

•Differential Interference Contrast (DIC) Microscopy

•Similar to phase-contrast

•Uses two light beams and prisms to split light beams, giving more contrast and color to the specimen

•Fluorescence Microscopy

•Uses UV (short wavelength) light

•Fluorescent substances UV light and emitting longer wavelengths?? (visible) light

•Cells may be stained with fluorescent dyes (fluorochromes) if they do not naturally fluoresce

•Confocal Microscopy

•Cells are stained with fluorochrome dyes

•Short-wavelength (blue) light is used to excite a single plane of a specimen

•Each plane in a specimen is illuminated and a three-dimensional image is constructed with a computer

ELECTRON MICROSCOPY

•Uses electrons instead of light

•Operates under vacuum

•Uses electronegativity instead of glass lenses

•The shorter wavelength of electrons gives greater resolution

•Used for structures too small to be seen with light microscopes, such as viruses

TRANSMISSION ELECTRON MICROSCOPY

•A beam of electrons passes through ultra thin sections of a specimen, then through an electromagnetic lens, then focused on a projector lens

•Specimens may be stained with heavy-metal salts for contra

•Magnifies objects 10000x->100000x; resolution of 10 pm (pico)

•Useful for examining different layers of a specimen

SCANNING ELECTRON MICROSCOPY

•An electron gun produces a beam of electrons that scans the surface of an entire specimen

•Secondary electrons emitted from the specimen produce a three-dimensional image

•Magnifies objects 1000x to 10000x; resolution of 10 nm

•Useful for studying surface structures

STAINING

•staining: coloring microorganisms with a dye that emphasizes certain structures (for contrast)

•smear: a thin film of a material containing microorganisms spread over a slide

•Microorganisms are fixed to the slide, which kills the microorganisms

•Stains consist of a positive and negative ion, one of which is colored (chromophore)

•In a base dye, the chromophore is a cation (+ charged)

•In an acid dye, the chromophore is an anion (- charged)

•Staining the background instead of the cell is called negative staining

•simple stain: use of a single (1) 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

•Differential Stains: Used to distinguish between bacteria

acid fast

•Gram stain

•Developed by Hans Christian Gram in 1884

•Classifies bacteria into gram +

or gram -

•Gram-positive bacteria have thick peptidoglycan cell walls

•Gram-negative bacteria have thin peptidoglycan cell walls and a layer of ___________________

special stains

•Used to distinguish parts of microorganisms

•Capsule stain

•Endospore stain

•Flagella stain

•Capsules are a gelatinous covering that do not accept most dyes

•Suspension of India ink or nigrosin contrasts the background with the capsule, which appears as a halo around the cell

•Flagella are structures of locomotion

•Uses a mordant and carbolfuchsion