MB 251 Exam 1

Magnification

Ratio of image size to actual object size; indicates how much larger the specimen appears

Magnification equation

M = I/A

Scale bar

Visual reference showing the actual size of organisms or structures in a microscopy image

Resolution

Ability of a microscope to distinguish two close points as separate entities; determines image clarity

Resolving power

λ/2NA

λ

Wavelength of light; shorter wavelengths provide better resolution

NA

Numerical aperture; measure of a lens’s ability to collect/refract light

Electromagnetic waves

Waves of different frequencies that interact with materials through reflection, absorption, and transmission

Frequency

Number of wavelengths occurring within a specific time period

Refraction

Change in direction of light when it enters a new medium

Wavelength

Distance between corresponding points on a wave

Long wavelength

Produces low frequency

Short wavelength

Produces high frequency

Light microscopy

Microscopy using visible light and magnifying lenses

Visible light

Light with wavelengths of approximately 400–700 nm

Maximum light microscope magnification

Less than 1500×; most commonly 1000×

Maximum light microscope resolution

Approximately 200 nm

Objective lens

Lens closest to the specimen that provides magnification

Ocular lens

Eyepiece lens; typically provides 10× magnification

Total magnification

Objective magnification × ocular magnification

Working distance

Distance between the front of the objective lens and the cover glass/specimen when focused

Oil immersion

Technique using oil between the slide and 100× objective to improve resolution by preventing light refraction

Bright-field microscopy

Most common light microscopy; stained specimen appears against a bright background

Bright-field staining

Usually kills cells but increases contrast

Bright-field use

Examining bacterial morphology and external structures

Dark-field microscopy

Light enters at an angle and scatters off the specimen, producing a bright image on a dark background

Dark-field advantage

Requires no staining and can be used with live organisms

Dark-field use

Identifying spirochetes and examining morphology of eukaryotic microbes

Treponema pallidum

Spirochete bacterium that causes syphilis

Fluorescence microscopy

Uses naturally fluorescent specimens or fluorescent tags/stains

Fluorescent tag

Molecule attached to a specific protein, pathogen, or cell component for visualization

Direct fluorescent antibody (DFA) assay

Uses fluorescently tagged, pathogen-specific antibodies to detect pathogens

Confocal microscopy

Optical sectioning technique that scans thick specimens one layer at a time

Confocal microscopy advantage

Detects light from one specific plane, producing detailed images of structures such as biofilms

Phase-contrast microscopy

Uses refraction/interference to create high-contrast images of unstained live cells

Differential interference contrast (DIC)

Uses refraction and interference to produce high-contrast images without staining

Phase contrast/DIC advantage

Allows sharper observation of live cells without damaging chemical stains

Electron microscopy

Microscopy using an electron beam and magnetic lenses

Electron wavelength

Approximately 0.004 nm

Electron microscope magnification

Approximately 100,000×–1,000,000×

Electron microscope resolution

Approximately 0.1 nm

Transmission electron microscopy (TEM)

Electron beam passes through a thin specimen section to reveal internal details

TEM image density

Denser regions scatter more electrons and appear darker

TEM specimen preparation

Thin sectioning, freeze fracturing, or freeze etching

TEM use

Visualizing fine internal structures, including internal structures of viruses

Scanning electron microscopy (SEM)

Electron beam scans the specimen surface to produce detailed 3D surface images

SEM specimen preparation

Specimen is dried and coated with a thin metal layer, usually gold

SEM metal coating

Allows secondary electrons to be released and detected to create the image

Light microscopy vs. electron microscopy

Light microscopy uses visible light and has lower resolution; electron microscopy uses electrons and has much higher magnification/resolution

Staining

Use of dyes to increase contrast and improve visualization of microorganisms

Differential staining

Staining technique using multiple dyes to distinguish microorganisms or cell structures

Basic dye

Positively charged dye that binds to negatively charged cell structures

Acidic dye

Negatively charged dye repelled by negatively charged cell structures

Positive stain

Basic dye stains the cells directly

Negative stain

Acidic dye stains the background while cells remain unstained

Fluorescent dye

Dye that binds specific cell structures and requires activation by a specific excitation wavelength

Excitation wavelength

Specific wavelength of light that activates a fluorescent dye

Emission wavelength

Wavelength of light given off by an activated fluorescent dye

Simple staining

Uses one dye to highlight the overall shape and size of an organism

Gram stain

Most widely used differential stain; separates bacteria based on cell wall structure

Gram-positive bacteria

Stain purple during Gram staining

Gram-negative bacteria

Stain red during Gram staining

Crystal violet

Primary Gram stain that initially stains all cells purple

Iodine

Mordant that forms large complexes with crystal violet and makes the dye less soluble

Mordant

Substance that helps a dye adhere to a cell structure

Alcohol

Decolorizer that removes crystal violet from Gram-negative cells

Decolorization of Gram-positive cells

Thick peptidoglycan becomes dehydrated and traps crystal violet-iodine complexes

Decolorization of Gram-negative cells

Alcohol destroys/removes the outer membrane and the thin peptidoglycan cannot retain the dye

Safranin

Counterstain that stains Gram-negative cells red

Gram stain step 1

Add crystal violet; all cells become purple

Gram stain step 2

Add iodine; crystal violet-iodine complexes form

Gram stain step 3

Add alcohol; Gram-negative cells become colorless while Gram-positive cells remain purple

Gram stain step 4

Add safranin; Gram-negative cells become red while Gram-positive cells remain purple

Acid-fast stain

Differential stain specifically used to identify Mycobacteria

Mycobacteria

Acid-fast bacteria with mycolic acid-rich cell walls; include pathogens causing tuberculosis and leprosy

Mycolic acid

Waxy component of acid-fast cell walls that prevents uptake of standard dyes

Endospore stain

Specialized stain used to determine whether bacteria produce endospores

Endospore

Durable, resistant, dormant structure produced by some bacteria under poor survival conditions

Capsule stain

Staining technique used to visualize bacterial capsules using both positive and negative stains

Capsule

Gel-like, firmly attached polysaccharide protective coating surrounding some bacteria

Slime layer

Fluid, loosely attached glycocalyx layer

Capsule stain appearance

Dark background, stained cell, and clear/colorless capsule surrounding the cell

Flagella stain

Specialized stain using a mordant to coat flagella and build up dye so they can be seen

Flagella

Hair-like rotary protein filaments used for bacterial locomotion

Three domains of life

Bacteria, Archaea, and Eukarya

Bacteria

Domain containing prokaryotic organisms

Archaea

Domain of prokaryotes more closely related evolutionarily to eukaryotes

Eukarya

Domain containing eukaryotic organisms

Prokaryote

Organism lacking a membrane-bound nucleus and membrane-bound organelles

Bacterial morphology

Shape and arrangement of bacterial cells; useful for diagnosis

Cocci

Spherical/circular bacterial cells

Bacilli

Rod-shaped bacterial cells

Spirochetes

Spiral-shaped bacteria

Cell/plasma membrane

Selectively permeable barrier controlling nutrient/waste transport and containing many metabolic processes

Nucleoid

Region containing the bacterial chromosome and genetic material

Ribosomes

Structures responsible for protein synthesis

Bacterial cytoskeleton

Protein network that helps maintain bacterial shape and organize cellular processes

Cell wall

Structure that maintains bacterial shape and protects against osmotic stress

Inclusions

Granules or compartments containing stored organic/inorganic materials

Plasmid DNA

Extrachromosomal DNA that can replicate independently of the chromosome