Microscopy and Staining Techniques Flashcards

Vocabulary flashcards covering microscopic instruments, principles of microscopy, electron and scanned-probe techniques, and microbial staining methods based on Chapter 3.

Simple Microscope

A microscope containing only one lens, similar to a magnifying glass but with a higher magnification lens.

Light Microscopy

The use of any kind of microscope that utilizes visible light to observe specimens.

Total Magnification

In a compound microscope, the overall magnification calculated as $\text{objective lens magnification} \times \text{ocular lens magnification}$.

Resolution

The ability of lenses to distinguish two points, or to distinguish fine detail and structure.

Brightfield Illumination

Microscopy in which dark objects are visible against a bright background, and light reflected off the specimen does not enter the objective lens.

Darkfield Microscopy

Microscopy using a condenser with an opaque disk to block direct light, allowing only light reflected off the specimen to enter the objective lens to show bright objects against a dark background.

Treponema pallidum

The very slender spirochete bacterium that causes syphilis, which can be viewed using darkfield microscopy.

Phase-Contrast Microscopy

Microscopy that combines direct light rays and diffracted light rays to examine living organisms and internal cell structures without fixation or staining.

Differential Interference Contrast (DIC) Microscopy

Microscopy similar to phase-contrast that uses two light beams and prisms to split light, providing higher contrast, color, and a three-dimensional appearance.

Fluorescence Microscopy

Microscopy using short-wavelength UV light to excite substances or fluorochromes, which then emit longer-wavelength visible light.

Auramine O

A fluorochrome dye used in fluorescence microscopy that specifically stains Mycobacterium tuberculosis.

Fluorescent-Antibody (FA) Technique

An immunofluorescence method where pathogen-specific antibodies tagged with a fluorochrome adhere to target microbes for rapid and specific detection.

Confocal Microscopy

Microscopy that uses fluorochrome dyes and short-wavelength blue light to excite a single plane of a specimen, forming clear 2D planes that a computer combines into a 3D image.

Two-Photon Microscopy

Microscopy using fluorochrome dyes and two photons of long-wavelength red light to excite dyes, allowing real-time tracking of living cells up to $1\,mm$ deep.

Super-Resolution Light Microscopy

Microscopy using two laser beams—one to excite fluorescent molecules and one to cancel fluorescence except within $1\,nm$—scanned nanometer by nanometer to build an image.

Scanning Acoustic Microscopy (SAM)

Microscopy measuring reflected sound waves to study cells attached to surfaces, such as cancer cells, arterial plaque, and bacterial biofilms, with a resolution of $1\,\mu m$.

Transmission Electron Microscopy (TEM)

Electron microscopy where electrons pass through ultrathin specimen sections, offering magnifications of $10,000\times$ to $10,000,000\times$ and a resolution limit of $0.2\,nm$.

Scanning Electron Microscopy (SEM)

Electron microscopy where an electron gun scans secondary electrons across a specimen surface, providing 3D images at magnifications of $1000\times$ to $500,000\times$ and a resolution limit of $0.5\,nm$.

Scanned Probe Microscopy

Techniques that use physical probes to map specimen surfaces at atomic detail with electric current without modifying the specimen.

Scanning Tunneling Microscopy (STM)

A type of scanned-probe microscopy that uses a tungsten probe to scan surfaces with electric current, resolving details down to atomic scale, such as DNA molecules.

Atomic Force Microscopy (AFM)

A type of scanned-probe microscopy that uses a metal-and-diamond probe placed onto a specimen to record movement and generate 3D images in near-atomic detail.

Staining

The process of coloring microorganisms with a dye to emphasize specific cellular structures.

Smear

A thin film of material containing microorganisms spread over a microscope slide.

Fixing

The procedure of passing a smear through a flame or covering it with methanol for $1\,min$ to attach microbes to the slide, kill them, and preserve their structures.

Chromophore

The colored ion (either a positive cation or negative anion) present in a stain.

Basic Dye

A stain whose chromophore is a cation (e.g., crystal violet, methylene blue, safranin) that adheres directly to negatively charged bacterial cells.

Acidic Dye

A stain whose chromophore is an anion (e.g., eosin, acid fuchsin, nigrosin) that is repelled by bacterial cells and colors the background instead.

Negative Staining

A staining procedure using acidic dyes that colors the background rather than the microbial cell.

Simple Stain

The use of a single basic dye to highlight an entire microorganism so its shape and structures can be visualized.

Mordant

A chemical additive used to hold a stain in place or to coat a specimen to enlarge it.

Gram Stain

A differential stain classifying bacteria into gram-positive (purple, thick peptidoglycan wall) or gram-negative (pink/red, thin peptidoglycan wall and outer membrane).

Acid-Fast Stain

A differential stain that binds to bacteria with waxy cell walls that resist acid-alcohol decolorization, used to identify Mycobacterium and Nocardia.

Capsule

A gelatinous outer covering on certain bacteria that repels most dyes, visualized as a halo around cells using negative staining with India ink or nigrosin.

Schaeffer-Fulton Endospore Stain

A special staining technique using malachite green with heat as the primary stain, water as a decolorizer, and safranin as a counterstain to reveal green spores within pink/red cells.

Flagella Stain

A special staining procedure using a mordant and carbolfuchsin to build up the diameter of slender flagella so they become visible under a light microscope.