Microscopy and Cell Structure in General Microbiology

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Microscopy in Microbiology

What is a Microscope?

Microscopes are essential tools in microbiology, allowing visualization of objects and details too small for the naked eye. Different types of microscopes use light, electrons, or other probes to achieve magnification, and the choice of microscope depends on the specimen size and the details to be visualized.

Definition: An instrument used to view objects too small to be seen unaided.
Types: Light microscopes (LM), electron microscopes (SEM/TEM).
Application: Used for observing bacteria, viruses, blood cells, and cellular structures.

Scale of Microorganisms

Microorganisms vary greatly in size, and their visualization depends on the resolving power of the microscope used.

Red blood cell (~6 μm): Visible with light microscope (LM); bulk visible to unaided eye.
Bacteria (~2 μm): Visible with LM.
Viruses (<1 μm): Require electron microscopes (SEM/TEM).
Baseball bat (1 m): Visible with unaided eye (for scale comparison).

Types of Microscopes & Clinical Relevance

Unaided Eye: Can see colonies, fungi, helminths, parasites.
Light Microscopy (LM): Uses visible light; best for bacteria, protozoa, fungi, blood cells.
Electron Microscopy (SEM/TEM): Uses electrons for higher resolution; visualizes viruses, bacterial ultrastructure, and fine details.

Clinical Examples

Colony growth on agar (e.g., hemolysis on blood agar).
Worms (tapeworms, pinworms).
Mold colonies (Aspergillus).
Gram stain: Staphylococcus aureus (Gram+) vs. E. coli (Gram–).
Acid-fast stain: Mycobacterium tuberculosis.
Blood smear: Plasmodium (malaria).

Compound Light Microscope

Uses two lenses: objective and ocular.
Total magnification:

Resolution & Refraction

Resolution: Ability to distinguish two points as separate.
Shorter wavelengths (e.g., UV) provide greater resolution.
Oil immersion reduces refraction, improving resolution.

Light Microscopy Methods

Brightfield: Stained bacteria; routine use.
Phase-contrast: Living organisms, internal structures.
Fluorescence: Fluorochrome dyes (e.g., labeled antibodies).
Confocal: 3D reconstruction, biofilm studies.

Electron Microscopy

Uses electrons (shorter wavelength → higher resolution).
Visualizes viruses, bacterial ultrastructure, fine details.

Transmission Electron Microscope (TEM):

Electrons pass through thin sections.
Heavy metal stains for contrast.
Resolution ~10 nm.
Clinical examples: Viral ultrastructure (HIV, SARS-CoV-2), kidney biopsies, muscle biopsies, endospore visualization (Clostridium difficile).

Scanning Electron Microscope (SEM):

Electrons scan surface; 3D appearance.
Clinical examples: Viral spikes (SARS-CoV-2, Ebola virus), biofilm studies, contamination on catheters/prosthetics.

Staining Techniques

Simple stain: General morphology.
Negative stain: Preserves size/morphology (e.g., capsule visualization).
Differential stains:
- Gram stain: Clinical cornerstone (Gram+ vs. Gram–).
- Acid-fast stain: Mycobacterium diagnosis.
Special stains: Capsule stain (Streptococcus pneumoniae), endospore stain (Bacillus, Clostridium), flagella stain (motility structures).

Cell Structure: Prokaryotes vs. Eukaryotes

Cell Theory

Cells are the smallest living units.
They perform essential life processes.

Prokaryotes vs. Eukaryotes

Prokaryotes: No nucleus or organelles; DNA in nucleoid. Examples: Bacteria, Archaea. Seen with LM; pili/flagella best seen with SEM/TEM.
Eukaryotes: Nucleus, organelles (Golgi, ER, mitochondria). Seen with LM. Example: Malaria parasites (Plasmodium) in red blood cells.

Bacterial Size, Shape, and Arrangement

Shapes: Bacillus (rod), coccus (spherical), spiral.
Arrangements: Chains, clusters, tetrads.
Clinical tie-in: Staphylococcus aureus (clusters), Streptococcus pyogenes (chains).

Capsule

Outermost protective layer.
Seen with negative stains in LM or SEM.
Clinical tie-in: Capsule = virulence factor in Streptococcus pneumoniae.

Appendages of Prokaryotic Cells

Flagella: Too thin for LM unless stained; SEM/TEM reveal detail.
Fimbriae & Pili: Seen with SEM/TEM.
Clinical tie-in: Pili aid attachment in Neisseria gonorrhoeae; flagella aid motility in E. coli.

Bacterial Cell Wall

Gram-positive (thick peptidoglycan): Stains purple.
Gram-negative (thin PG + LPS outer membrane): Stains pink.
Clinical tie-in: Gram reaction guides antibiotic therapy; lipid A endotoxin from Gram– bacteria causes septic shock (E. coli, Salmonella).

Bacterial Movement

Swimming (flagella): Seen live under phase-contrast LM.
Twitching (pili): Seen with SEM.

Clinical Visibility Summary

Microscope	What It Shows	Clinical Uses	Examples
Unaided Eye	Colonies, large parasites	Colony morphology, parasite ID	Worms, mold (Aspergillus), lice
LM	Bacteria, protozoa, fungi, cells	Routine diagnosis	Gram stain (S. aureus, E. coli), AFB (M. tuberculosis), malaria, Candida
SEM	Surface features (3D)	Biofilm & pathogen surface studies	Viral spikes (SARS-CoV-2), pili (N. gonorrhoeae), biofilms on catheters
TEM	Internal ultrastructure	Viruses, pathology, ultrastructure	HIV imaging, glomerulonephritis biopsy, mitochondrial disease, spores (C. difficile)

Cellular Components and Functions

Extracellular Matrix and Plasma Membrane

The extracellular matrix is a sticky coat that holds cells together.
Each cell is surrounded by a flexible plasma membrane with projections and channels.

Cytoskeleton and Energy

The cytoskeleton is the structural framework of the cell and serves as tracks for transporting cargo.
All activity in the cell requires energy in the form of ATP, produced in mitochondria.

Protein Synthesis and Endomembrane System

Ribosomes build proteins by translating mRNA.
The endomembrane system includes the endoplasmic reticulum (ER), which is divided into rough (with ribosomes) and smooth (without ribosomes) types.
Proteins and lipids are processed and transported via vesicles to the Golgi apparatus for further modification and sorting.

Golgi Apparatus and Vesicles

Golgi apparatus modifies, sorts, and packages proteins and lipids into vesicles for transport.
Some vesicles fuse with the plasma membrane, releasing their contents outside the cell.

Additional Key Terms & Explanations

Phagocytosis: Process by which immune cells engulf and digest foreign particles, including bacteria.
Capsule: Thick outer layer of some bacteria; protects against phagocytosis and acts as a virulence factor.
Cytoskeleton: Maintains cell shape, supports organelles, and serves as tracks for vesicle transport.
ATP: Energy currency of the cell; produced in mitochondria.
mRNA: Carries instructions from DNA in the nucleus to ribosomes for protein synthesis.
Rough ER: Covered with ribosomes; site of protein synthesis.
Smooth ER: Lacks ribosomes; synthesizes lipids, detoxifies chemicals, stores calcium.
Golgi Apparatus: Modifies, sorts, and packages proteins and lipids into vesicles for transport.
Lysosomes: Contain digestive enzymes; break down worn-out organelles and cellular debris.
Plasma Membrane: Selective barrier regulating movement of substances into and out of the cell.

Gram Stain

Differentiates bacteria into Gram-positive (thick peptidoglycan → purple) and Gram-negative (thin peptidoglycan + outer membrane → pink).
Guides antibiotic therapy.

Flagella Movement

Flagella rotate 360 degrees, propelling bacteria forward.
Counterclockwise rotation causes the bacterium to "run" in a straight line; clockwise rotation causes the bacterium to "tumble" and change direction.

Taxis

Movement toward or away from a stimulus is called taxis.
Phototaxis: Stimulus is light.
Chemotaxis: Stimulus is a chemical.
Bacteria have receptors that signal the flagella to move toward or away from stimuli.

Practice & Review Questions

Order by size: Plasmid (20 nm) < Mastadenovirus (30 nm) < Gram-positive bacterium (6 μm) < Protozoan (1 μm) < Trematode (5 mm).
Convert: 0.25 μm (Chlamydia elementary body) = 250 nm.
Convert: 150 μm (Paramecium) = 0.15 mm.
Which microscope achieves highest magnification/resolution? — Electron microscope.
Which microscope observes surfaces of cells/viruses? — Scanning electron microscope.
Which stain is most useful for choosing antibiotics? — Gram stain.

Summary Table: Microscopy Methods and Clinical Applications

Method	Resolution	Best For	Clinical Example
Light Microscopy	~0.2 μm	Bacteria, protozoa, fungi, blood cells	Gram stain, malaria diagnosis
SEM	~10 nm	Surface features, 3D imaging	Viral spikes, biofilms
TEM	~1-2 nm	Internal ultrastructure	Viral structure, mitochondria

Additional info: These notes are structured to provide a comprehensive overview of microscopy and cell structure for introductory microbiology students, including definitions, clinical applications, and key comparisons between prokaryotic and eukaryotic cells.