Post on 28-Dec-2015
Honors Microbiology: Chapter 3 Microscopy and Staining
I. Principles of Microscopy
• A. Properties of light: wavelength and resolution– Wavelength – length of a light ray– Resolution – ability to see two items as separate
and discrete– Resolving power (RP) numerical measure of the
resolution of the lens– Numerical aperture (NA) – widest cone of light
that can enter a lens
• B. Properties of light: light and objects– Reflection: when light strikes an object and
bounces back, giving the object color
– Transmission: passage of light through an object
– Absorption: when light rays are taken up by an object
– Refraction: bending of light as it passes from one medium to another of different density
– Diffraction: when light waves are broken up into bands of different wavelengths as they pass through a small opening
Light Travel
• Reflection
• Transmission
• Absorption
II. Light Microscopy
• A. Basic feature: uses visible light to make specimens observable
• B. Compound light microscope: has more than one lens
Compound Light Microscopy• Condenser
• Iris diaphragm
• Objective lenses
• Ocular lens(es)
• Stage
• Focusing knobs
• Total Magnification
Brightfield Microscopy
• C. Darkfield microscopy – has a condenser that prevents light from being transmitted through the specimen. It causes light to reflect off the specimen at an angle, showing a light object against a dark background.
Darkfield Microscopy
• D. Phase- contrast microscopy: has a condenser that accentuates small differences in the refractive index of various structures within the cell, causing parts of the cell to display different degrees of brightness.
Phase-contrast Microscopy
• E. Differential interference contrast microscopy (Nomarski) – uses differences in refractive index to visualize structures, producing a nearly 3-D image
Nomarski microscopy
• F. Fluorescence microscopy: ultraviolet light is used to excite molecules so they release light of different colors.
Fluorescence microscopy
III. Electron Microscopy
• A. Basic features: uses a beam of electrons rather than light, and electromagnets, rather than glass lenses, to produce an image
Electron Microscopy (EM)
• Transmission (TEM)
• Scanning (SEM)
• Scanning Tunneling
• B. Transmission electron microscope (TEM): Gives an excellent view of the internal
structure of microbes, magnifying objects up to 500,000x. Very thin slices of specimens are used.
Transmission Electron Microscopy
• C. Scanning electron microscope (SEM): used to create images of the surfaces of specimens, magnifying objects up to 50,000x
• D. Scanning tunneling microscopy (STM): used to create 3-D images and movies of individual molecules and atoms.
Scanning Electron Microscopy
IV. Techniques of light microscopy
• A. Preparation of specimens for the light microscope:– 1. Wet mounts: a drop of medium containing the
organisms is placed on a slide, and living microbes can be observed.
– 2. Smears – microbes from a loopful of medium are spread onto the surface of a glass slide and heat-killed, so that killed microbes can be observed.
• B. Principles of staining:– 1. Simple stains – make use of a single dye and
reveals basic cell shapes and arrangements– 2. Differential stains – uses two or more dyes and
distinguishes between two kinds of organisms or two different parts of an organism
• C. Gram Stain – a differential stain. Groups that can be distinguished with the Gram stain:– 1. gram-positive, which stain violet– 2. gram-negative, which stain pink– 3. gram-nonreactive, which do not stain, or stain
poorly– 4. gram-variable, which stain unevenly
Gram Stain
• Technique
• Significance– Cell wall anatomy
– Diagnosis
• D. Ziehl-Neelsen acid-fast stain: used to detect tuberculosis, and leprosy-causing organisms
Acid fast Staining
• E. Special Staining Procedures:– 1. Negative staining– 2. Flagellar staining– 3. Endospore staining– 4. Capsular staining
Negative Staining
Flagellar Staining
Endospore Staining
Capsular Staining