Chapter 3 | Observing Microorganisms Through a Microscope

Micrometer (μm)

Unit of measurement equal to 10^-6 meters, used to measure most microorganisms

Nanometer (nm)

Unit of measurement equal to 10^-9 meters, used to measure viruses and fine structures

Simple microscope

A microscope with only one lens

Compound microscope

A microscope with multiple lenses

Total magnification

Objective lens magnification multiplied by ocular lens magnification

Resolution (resolving power)

The ability of a microscope to distinguish two close points as separate

Compound light microscope resolution

0.2 micrometers

Compound light microscope maximum magnification

1500 times

Brightfield illumination

Technique showing dark stained objects against a bright background

Darkfield microscopy

Technique using an opaque disk so only scattered light enters the lens, showing light objects against a dark background

Phase-contrast microscopy

Technique combining direct and diffracted light rays to observe living unstained organisms in detail

Differential interference contrast (DIC) microscopy

Technique using two light beams and prisms to create a colored three-dimensional image of living cells

Fluorescence microscopy

Technique using fluorochrome stains and ultraviolet light to make organisms appear bright against a dark background

Immunofluorescence (FA technique)

Diagnostic procedure based on fluorescence microscopy using fluorescent antibodies

Confocal microscopy

Technique using fluorescent dye and short-wavelength light to scan a specimen plane by plane for 3D reconstruction

Two-photon microscopy (TPM)

Technique using fluorochrome dye and long-wavelength light to study living cells up to 1 millimeter deep

Super-resolution light microscopy

Technique using two lasers to excite and quiet fluorescent molecules for nanometer-scale imaging

Scanning acoustic microscopy (SAM)

Technique using sound waves to study cells attached to surfaces such as biofilms

Electron microscope

A microscope that uses a beam of electrons instead of light, with electromagnets instead of glass lenses

Transmission electron microscope (TEM)

Electron microscope where electrons pass through thin specimen sections to reveal internal structures

TEM magnification range

10,000 to 10,000,000 times

TEM resolution

10 picometers

Scanning electron microscope (SEM)

Electron microscope where electrons scan the specimen surface to produce a three-dimensional image

SEM magnification range

1,000 to 500,000 times

SEM resolution

10 nanometers

Scanning tunneling microscopy (STM)

Scanned-probe technique producing three-dimensional images of a molecule's surface

Atomic force microscopy (AFM)

Scanned-probe technique producing three-dimensional images of a molecule's surface

Smear

A thin film of material containing microorganisms spread on a slide

Fixing

Using heat or alcohol to kill microorganisms and attach them to a slide

Staining

Coloring a microorganism with a dye to make structures more visible

Basic dye

A dye whose colored chromophore is a positive ion, used to stain negatively charged bacterial cells

Acidic dye

A dye whose colored chromophore is a negative ion, used to stain the background around cells

Negative staining

Staining the background instead of the cell, leaving the specimen unstained

Simple stain

Use of a single basic dye to reveal cell shape and structure

Mordant

A substance used to improve bonding between a stain and the specimen

Differential stain

A staining method that differentiates bacteria based on their reaction to the stain, such as Gram stain or acid-fast stain

Gram stain primary stain

Crystal violet (purple stain)

Gram stain mordant

Iodine

Gram stain decolorizer

Alcohol

Gram stain counterstain

Safranin (red stain)

Gram-positive bacteria

Bacteria with thick peptidoglycan cell walls that retain the purple stain after decolorization

Gram-negative bacteria

Bacteria with thin peptidoglycan cell walls and a lipopolysaccharide layer that lose the purple stain and appear pink

Acid-fast stain primary stain

Carbolfuchsin

Acid-fast stain decolorizer

Acid-alcohol

Acid-fast stain counterstain

Methylene blue

Acid-fast bacteria

Bacteria such as Mycobacterium and Nocardia with waxy cell walls that retain carbolfuchsin and appear red

Non-acid-fast bacteria

Bacteria that lose carbolfuchsin after decolorization and appear blue from the methylene blue counterstain

Special stain

A stain used to visualize specific structures such as capsules, endospores, or flagella

Capsule stain (negative staining)

Uses India ink or nigrosin to stain the background, making the capsule appear as a clear halo

Endospore stain primary stain

Malachite green, applied with heat

Endospore stain decolorizer

Water

Endospore stain counterstain

Safranin

Endospore appearance after staining

Green spores within red or pink cells

Flagella stain

Uses a mordant and carbolfuchsin to thicken flagella so they become visible under a light microscope

Refractive index

A measure of the light-bending ability of a medium

Immersion oil

Used with the oil immersion lens to prevent light refraction between the slide and the lens