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