Principles and Techniques of Light Microscopy

Compound light microscope

Uses visible light to illuminate specimens.

Bright-field microscopy

Visualizes specimens based on contrast differences.

Phase-contrast microscopy

Enhances contrast without staining live samples.

Dark-field microscopy

Illuminates specimen from the sides, creating contrast.

Fluorescence microscopy

Visualizes specimens that emit light when illuminated.

Total magnification

Objective magnification multiplied by ocular magnification.

Maximum magnification

Light microscope can achieve ~2,000x magnification.

Resolution

Ability to distinguish two adjacent objects clearly.

Limit of resolution

Approximately 0.2 µm for light microscopes.

Contrast

Difference in light intensity between image and background.

Staining

Improves contrast by binding dyes to cellular materials.

Common stains

Examples include methylene blue, safranin, crystal violet.

Differential stains

Separate bacteria into groups based on staining properties.

Gram stain

Widely used differential stain in microbiology.

Gram-positive bacteria

Appear purple after Gram staining procedure.

Gram-negative bacteria

Appear red (pink) after Gram staining procedure.

Crystal violet

First stain used in Gram staining protocol.

Iodine solution

Used to fix crystal violet in Gram staining.

Alcohol decolorization

Differentiates Gram-positive from Gram-negative cells.

Counterstain

Safranin used to visualize Gram-negative cells.

Phase ring

Part of objective lens that amplifies refractive index.

Autofluorescence

Natural fluorescence of cells without additional staining.

DAPI

Fluorescent dye that binds to AT-rich regions.

Differential interference contrast microscopy

Uses polarized light for 3D cell imaging.

Confocal scanning laser microscopy

Generates 3D images using laser and computer.

Resolution of CSLM

Achieves a resolution of 0.1 µm.

Transmission electron microscopy

Uses electrons for high-resolution imaging.

Resolution of TEM

Achieves resolution of 0.2 nm.

Scanning electron microscopy

Produces images by scanning with electron beam.

Magnification range of SEM

Magnifies specimens from 15✕ to 100,000✕.

Resolution of SEM

Achieves resolution of approximately 10 nm.

Coccus

Spherical or ovoid cell shape.

Rod (bacillus)

Cylindrical cell shape.

Spirillum

Spiral-shaped cell morphology.

Spirochete

Flexible spiral-shaped bacteria.

Filamentous bacteria

Bacteria with long, thread-like structures.

Budding bacteria

Bacteria that reproduce by budding off.

Gliding motility

Non-flagellated movement along surfaces.

Prokaryotic cell size range

Typically 0.2 µm to >700 µm in diameter.

Cultured bacillus size

0.5-4.0 µm wide and <15 µm long.

Epulopiscium fishelsoni

Large prokaryote, 600 µm long.

Thiomargarita namibiensis

Prokaryote, 400-750 µm wide.

Cell morphology

Shape of cells influencing various functions.

Selective forces on morphology

Factors influencing cell shape and function.

Optimization for nutrient uptake

Small cells enhance surface-to-volume ratio.

Surface-to-volume ratio

Higher in small cells, enhances nutrient exchange.

Nutrient exchange

Greater per unit volume in small cells.

Growth rates

Small cells typically grow faster than larger cells.

Lower limit of cell size

Cells <0.15 µm diameter are rare.

Cytoplasmic membrane

Thin barrier separating cytoplasm from environment.

Selective permeability

Membrane allows specific metabolite concentration.

Phospholipid bilayer

Basic structure of cellular membranes.

Hydrophobic fatty acids

Point inward, forming a hydrophobic environment.

Hydrophilic components

Expose to external environment or cytoplasm.

Integral membrane proteins

Firmly embedded proteins within the membrane.

Peripheral membrane proteins

Anchored proteins interacting with membrane surface.

Membrane stabilization

Mg2+ and Ca2+ stabilize phospholipid interactions.

Transport proteins

Facilitate movement of molecules across membranes.

Proton motive force

Energy source for active transport processes.

Active transport

Moves solutes against concentration gradient.

Carrier-mediated transport

Saturation effect observed in nutrient transport.

Simple diffusion

Passive movement of molecules across membranes.

Simple transport

One of three active transport systems.

Group translocation

Transport system modifying substrate during transport.

ABC system

ATP binding cassette transport requiring energy.

Transport events

Include uniport, symport, and antiport mechanisms.

Uniporters

Transport one molecule in one direction.

Symporters

Co-transport two molecules in the same direction.

Antiporters

Transport one molecule in opposite direction.

Lac permease

E. coli symporter using proton motive force.

Phosphotransferase system

Group translocation system in E. coli.

Group Translocation

Transport method modifying substance during membrane crossing.

Phosphoenolpyruvate

Energy-rich glycolysis intermediate for transport.

ABC Systems

Transport systems utilizing ATP in prokaryotes.

Substrate Specificity

High affinity for specific organic and inorganic compounds.

Periplasmic-binding Proteins

Proteins aiding transport in Gram-negative bacteria.

Substrate-binding Proteins

Proteins anchored to surface in Gram-positive bacteria.

Efflux Systems

Mechanisms for pumping out substances like antibiotics.

Peptidoglycan

Rigid layer providing strength to bacterial cell walls.

N-acetylglucosamine

Component of peptidoglycan structure in bacteria.

N-acetylmuramic Acid

Another component of bacterial peptidoglycan.

Amino Acids in Peptidoglycan

Include L-alanine, D-alanine, D-glutamic acid.

Diaminopimelic Acid

Structural analog of L-lysine in peptidoglycan.

Gram-positive Bacteria

Bacteria with thick peptidoglycan layers (15+ layers).

Gram-negative Bacteria

Bacteria with thin peptidoglycan and outer membrane.

Interbridge

Amino acid connections in peptidoglycan layers.

Teichoic Acids

Acidic substances in Gram-positive cell walls.

Lipoteichoic Acids

Teichoic acids bound to membrane lipids.

Osmotic Protection

Survival mechanism for wall-less prokaryotes.

Mycoplasmas

Pathogenic bacteria lacking cell walls.

Thermoplasma

Archaea with lipoglycans for membrane rigidity.

Lipopolysaccharide (LPS)

Major component of Gram-negative outer membrane.

Endotoxin

Toxic component of LPS, specifically Lipid A.

Porin Proteins

Proteins forming channels in Gram-negative membranes.

Porins

Channels for hydrophilic low-molecular-weight substances.

Periplasm

Space between cytoplasmic and outer membranes, ~15 nm.

Peptidoglycan

Polymer forming bacterial cell wall structure.

Pseudomurein

Polysaccharide similar to peptidoglycan in Archaea.

S-Layers

Outer layer of interlocking protein or glycoprotein.

Capsule

Tight polysaccharide layer excluding small particles.