Microscopy: Light Microscopy, Staining, and Differential Techniques

Vocabulary flashcards covering key concepts from microscopy chapters on light microscopy, immersion oil, staining, and differential staining.

Compound light microscope

A microscope that uses visible light and two lenses (ocular and objective) to magnify specimens, with light passing through a condenser to illuminate the slide.

Ocular lens (eyepiece)

The lens at the top that magnifies the image formed by the objective; often 4x or 10x and adjustable for interpupillary distance.

Objective lens

Lenses closest to the specimen with varying magnifications (e.g., 4x, 10x, 40x, 100x).

Illuminator

The light source under the stage that provides illumination for observing the specimen.

Condenser

Lenses that direct and focus light onto the specimen.

Diaphragm

Controls the amount of light entering the condenser, acting like an iris.

Stage

Flat platform that holds the glass slide and moves in the X and Y directions.

Coarse adjustment knob

Large knob used for rough focusing; typically used at low magnification.

Fine adjustment knob

Small knob used for precise focusing; used after coarse adjustment.

Immersion oil

Oil with a refractive index close to glass; used with high-magnification objectives (e.g., 100x) to improve resolution.

Refractive index

Measure of how much light bends when passing between materials; affects focusing and image clarity.

Total magnification

Product of the objective magnification and the ocular magnification.

Resolution

Ability to distinguish two separate points; higher resolution with shorter wavelengths and higher numerical aperture.

Numerical aperture

Lens property that describes its light-gathering ability; higher NA improves resolution.

Visible light spectrum

Range of wavelengths visible to humans (about 380–700 nm); shorter wavelengths can improve resolution.

Bright field microscopy

Simple light microscopy where the specimen is darker on a bright background.

Dark field microscopy

Light is directed obliquely so only scattered light enters the objective; the specimen appears bright against a dark background.

Phase-contrast microscopy

Enhances contrast by using a phase plate to shift light waves and create constructive or destructive interference.

Interference (constructive/destructive)

When light waves align to reinforce each other (constructive) or cancel each other (destructive), enhancing image contrast in phase-contrast microscopy.

Fluorescence microscopy

Microscopy that uses fluorescence to visualize specimens, often with fluorochromes; requires excitation light and emission filtering.

Fluorochrome

Fluorescent dye that binds to cellular structures and emits light when excited.

GFP (green fluorescent protein)

A fluorescent protein used as a tag to visualize cells and proteins.

Ultraviolet (UV) light

Shorter-wavelength light used to excite fluorochromes, producing visible fluorescence with appropriate filters.

Fluorescence filter

Filter placed between specimen and eyepiece to block excitation light and pass emitted fluorescence.

Smear

Thin film of microbial culture spread on a slide in preparation for staining.

Fixation

Process of attaching the smear to the slide (via air drying and heat) to prevent loss during staining.

Inoculation loop

Tool used to transfer liquid culture to make a smear.

Needle (solid media smear)

Tool used to pick small amounts from solid media to create a smear.

Basic dye

Chromophore is positively charged; stains negatively charged surfaces of microbes.

Acid dye (acidic dye)

Chromophore is negatively charged; stains the background and leaves cells lighter.

Simple staining

Staining with a single basic dye to color the entire cell and reveal shape.

Differential staining

Staining that uses multiple dyes to distinguish types of cells or structures (e.g., Gram stain, acid-fast).

Gram stain

Differential stain that distinguishes Gram-positive (purple) from Gram-negative (pink) bacteria based on peptidoglycan thickness.

Crystal violet

Primary stain in Gram staining; stains all cells purple.

Iodine mordant

Mordant in Gram staining that forms a crystal violet–iodine complex to fix dye in cells.

Alcohol decolorization

Decolorizes Gram-negative cells by removing dye; Gram-positive cells retain the stain.

Safranin

Counterstain in Gram staining that colors Gram-negative cells pink.

Peptidoglycan

Cell wall polymer; thickness varies between Gram-positive (thicker) and Gram-negative (thinner), influencing stain retention.

Gram-positive

Bacteria with thick peptidoglycan walls that retain crystal violet–iodine complex and appear purple.

Gram-negative

Bacteria with thinner peptidoglycan walls that decolorize easily and appear pink after counterstaining.

Acid-fast stain

Differential stain for bacteria with waxy cell walls (e.g., Mycobacterium) that resist decolorization.

Mycobacterium

Genus with waxy cell walls rich in mycolic acids; includes M. tuberculosis and M. leprae.

Phase plate

Optical element in phase-contrast microscopy that shifts light phase to enhance contrast.