University Study Notes: The Microbial World

The Human Microbiome and Microbial Presence

• Human-Bacterial Cargo: Humans carry a significant amount of bacteria, roughly equivalent to filling a large soup can.     * Mass: This accounts for between $3\,lb$ and $5\,lb$ of bacteria per person.     * Cell Counts: Bacterial cells in the human body outnumber human cells in a ratio of $10:1$.     * Body Composition: Despite their high numbers, bacteria account for only $1\%$ to $2\%$ of total body mass because they are significantly smaller than human cells.     * Waste Contribution: Bacteria make up approximately half of the human body's waste.     * National Institutes of Health (NIH) Human Microbiome Project: Coordinated by Lita Proctor, this project focuses on studying the communities of bacteria living on and inside the human body.

Belly Button Biodiversity Study

• Study Overview: Conducted in July 2011 at North Carolina State University, researchers sampled 60 belly buttons to catalog bacterial diversity.

• Findings:     * Total species found: $2{,}368$.     * New species: $1{,}458$ of the found species were potentially new to science.     * Variation: Individual belly buttons contained as few as $29$ species and as many as $107$.     * Average: Most belly buttons harbored approximately $67$ species.

• Identified Samples: Examples include Bacillus subtilis and Staphylococcus epidermidis.

Evolutionary Timeline of Life on Earth

• Earth's Origin: The probable origin of Earth was approximately $5 \times 10^9$ years ago.

• Bacteria and Archaea: These organisms have existed for more than $3.5 \times 10^9$ years.

• Eukaryotes: Appeared approximately $1.7 \times 10^9$ to $2 \times 10^9$ years ago.

• Simple Animals: Existed for about $600 \times 10^6$ years.

• Mammals: Existed for about $200 \times 10^6$ years.

• Ancestral Cell Type: All life stems from an ancestral cell type that preceded the divergence of Archaea, Bacteria, and Eukaryotes.

Microbial Role in Planetary Ecology

• Energy and Food Flow: Microbes are fundamental to Earth's ecosystems.

• Photosynthesis:     * Anoxygenic Photosynthesis: Bacteria performed photosynthesis without oxygen production early in history.     * Oxygenic Photosynthesis: The production of oxygen by microbes allowed for species diversification.     * Atmospheric Contribution: Photosynthetic microorganisms, including bacteria and algae, account for more than $70\%$ of Earth's photosynthesis and provide the majority of atmospheric oxygen.     * Sustainability: Without photosynthetic microorganisms, Earth's oxygen would be depleted in approximately $20$ years.

• Nitrogen Cycle: Nitrogen gas ($N_2$) makes up $78\%$ of the atmosphere. Microbes perform nitrogen fixation, which is essential for making nitrogen available to other life forms.

• Decomposition: Microbes break down waste products and remnants of dead organisms, a process that 'cleans up' the environment.

Historical Foundations of Microbiology

• Francesco Redi (1668):     * Experiment: Used meat in open jars, gauze-covered jars, and sealed jars to study the appearance of maggots.     * Conclusion: Disproved the theory of spontaneous generation by showing that maggots only appeared in the open jars where flies could land.

• Louis Pasteur (1861):     * Swan-Neck Flask Experiment: Boiled broth in flasks with curved necks. The curves prevented outside air/microbes from entering, keeping the broth sterile.     * Findings: When the neck was broken, bacteria reached the broth and growth occurred. This further disproved spontaneous generation and established that microorganisms are present in the air.

• The Germ Theory of Disease: Proved by Robert Koch in 1890 through the injection of pure cultures of Anthrax into animals.

National Collection of Type Cultures (NCTC) Milestones

• 1886: Theodore Escherich describes Bacterium coli commune (later Escherichia coli).

• 1887: Julius Petri invents the agar-coated glass dish (Petri dish).

• 1900: Almroth Wright isolates Salmonella enterica and persuades armed forces to produce $10 \times 10^6$ vaccine doses for WWI.

• 1915: Isolation of NCTC 1, a strain of Shigella flexneri from Private Ernest Cable, who died of dysentery.

• 1920: NCTC established.

• 1928: Alexander Fleming discovers Penicillin by accident. Between 1928 and 1948, he deposited 16 strains, including Haemophilus influenzae from his own nose.

• 1930s: Introduction of freeze-drying for sample shipment; development of the Kauffman-White classification for Salmonella.

• 1947: Edward Tatum and Joshua Lederberg produce the first genetic map of E. coli K12.

• 1953: Betty Constance Hobbs establishes Clostridium perfringens as a cause of food poisoning.

• 1977: Maxam-Gilbert and Sanger develop DNA sequencing; CDC researchers isolate Legionella pneumophila.

• 1982: Barry Marshall drinks Helicobacter pylori to prove it causes stomach ulcers.

• 1995: Complete genome sequence of Haemophilus influenzae is elucidated.

• 2014: NCTC and Wellcome Sanger Institute (WSI) launch a project to sequence $3{,}000$ bacterial strains.

Pathogens and Infectious Diseases

• Definition: A pathogen is any agent (bacteria, virus, fungus, protozoan, helminth) that causes disease.

• Stats: Nearly $2{,}000$ different microbes can cause disease.

• Leading Causes of Death (2008 Data):     * Worldwide: Lower-respiratory infections (3.5 million), Diarrheal diseases (2.5 million), HIV/AIDS (1.8 million), and Tuberculosis (1.3 million) are among the top 10.     * Comparison: Infectious diseases are the second leading cause of death worldwide ($16.2\%$ of all deaths), trailing only heart disease.

• Emerging Infectious Diseases (Since 1976):     * 1976: Ebola (Zaire); Legionnaires' Disease (USA); Cryptosporidiosis (USA).     * 1981: AIDS (USA).     * 1982: E. coli O157:H7 (USA).     * 1986: Bovine spongiform encephalopathy (UK).     * 1992: Vibrio cholerae O139 (India).     * 2003: SARS-CoV (Global threat).     * Recent Threats: Zika virus, MERS-CoV, H5N1 Influenza, MRSA, CRE, and C. difficile.

Specialized Areas and Scopes of Microbiology

• Microbiology Definition: The study of organisms too small to be seen with the naked eye (microbes, germs, viruses, agents, or "bugs").

• Main Branches:     * Medical Microbiology: Deals with microbes causing disease; focuses on virulence factors and inhibition mechanisms.     * Public Health Microbiology & Epidemiology: Monitors and controls disease spread (e.g., CDC and WHO).     * Immunology: Studies host tissue reactions to vaccines, blood transfusions, and allergies.

• Environmental Microbiology Subdisciplines:     * Aquatic Microbiology: Microbes in surface water.     * Soil Microbiology: Microbes in terrestrial parts of the planet.     * Geomicrobiology: Microbes in the Earth's crust.     * Astrobiology/Exobiology: Search for microbial life off-planet.

• Industrial Microbiology:     * Biotechnology: Application of living organisms to industrial processes.     * Bioremediation: Using microbes to clean up oil spills and pollutants.     * Pharmaceuticals: Production of antibiotics (e.g., from fungi and bacteria).

Biological Classification and Taxonomy

• Taxonomy: The science of classifying organisms based on similar characteristics.

• Three Domains:     1. Bacteria: Single-celled prokaryotes; cell walls have peptidoglycan; fatty acid-based membranes; sensitive to streptomycin; no introns.     2. Archaea: Single-celled prokaryotes; cell walls lack peptidoglycan; non-fatty acid lipid membranes; produce methane; often extremophiles; may contain introns.     3. Eukarya: Organisms with a true nucleus and membrane-bound organelles.

• Six Kingdoms: Bacteria, Archaea, Protist, Fungi, Plant, Animal.

• The Three-Domain System: Proposed by Dr. Carl Woese (late 1970s) based on differences in ribosomal RNA (rRNA).

Characteristics of Eukaryotic Microorganisms

• Algae:     * Found in fresh/sea water, rocks, and soil.     * Photosynthetic (contains Chlorophyll a).     * Rigid cell walls; move via complex flagella.

• Fungi:     * Includes molds (multicellular), mushrooms (macroscopic), and yeasts (microscopic unicellular).     * Degrade organic material for energy.     * Mostly terrestrial.

• Protozoa:     * Single-celled; shapes vary (e.g., Amoeba vs. Paramecium).     * No rigid cell wall; found in moist habitats.     * Derive energy from organic compounds.

• Helminths:     * Multicellular parasites (e.g., Taenia solium).     * Visible to the naked eye but included in microbiology due to microscopic eggs/larvae.

Non-Living Infectious Agents

• Viruses:     * Incapable of multiplying outside a host cell.     * Consist of a nucleic acid core (DNA or RNA) and a protein coat (capsid).     * Size: $10\,nm$ to $300\,nm$.

• Viroids:     * Composed only of a short strand of circular, single-stranded RNA.     * No protein coat; primarily infect plants.

• Prions:     * Abnormal forms of cellular proteins; consist only of protein (no DNA or RNA).

Microscopy and Visualization

• Discovery: Robert Hooke (1665) and Antony van Leeuwenhoek (1674), the "Father of the Microscope."

• Light Microscopy (Magnification up to $1000 \times$):     * Bright-field: Specimen is dark against a bright background.     * Dark-field: Bright specimen against a dark background; good for living, unstained cells.     * Phase-contrast: Uses refractive index differences to make dense material appear darker; ideal for intracellular structures.     * Differential Interference Contrast (DIC): Uses two prisms/beams to create vivid, 3D-appearing colored images.

• Fluorescence and Advanced Microscopy:     * Fluorescence Microscope: Uses UV light and fluorescent dyes to emit colored light.     * Confocal Microscope: Uses a laser beam and spatial filtering to capture sharp images of single planes in thick specimens.

• Electron Microscopy (Magnification up to $100{,}000{,}000 \times$):     * Transmission Electron Microscope (TEM): Transmits electrons through thin sections ($20-100\,nm$); views internal cell detail ($0.5\,nm$ resolution).     * Scanning Electron Microscope (SEM): Bombards metal-coated specimens with electrons to create 3D surface views.

• Scanning Probe Microscopy:     * Atomic Force Microscope (AFM): Uses a diamond/metal tip ($1-50\,nm$ radius) to scan surfaces at the atomic level.     * Scanning Tunneling Microscope (STM): Measures electrical current between a tip and a surface.

Staining Techniques

• Stain Definition: A substance that adheres to a cell to provide color and contrast.

• Basic Stains: Positively charged dyes (Crystal violet, Safranin, Methylene blue) that bind to negative cell components.

• Gram Stain: Differentiates bacteria by cell wall.     1. Crystal Violet: Primary stain (cells turn purple).     2. Iodine: Mordant (fixes the dye).     3. Alcohol: Decolorizer (Gram-positive stay purple; Gram-negative become colorless).     4. Safranin: Counterstain (Gram-negative turn pink).

• Acid-Fast Stain: Used for Mycobacterium species containing waxy mycolic acid in cell walls.

• Special Stains:     * Capsule Stain: Negative stain highlighting the protective capsule.     * Endospore Stain: Highlights dormant spores in Bacillus and Clostridium.     * Flagella Stain: Coats thin flagella to make them visible.