Microbiology Notes (Exam Prep)

Microbes, History, and Core Concepts

  • Microbes (Microorganisms) are organisms that are too small to be seen with unaided eye. They include living and non-living entities that still perform biological processes.

  • What makes something a living organism? Key characteristics typically include:

    • Responsiveness

    • Adaptability

    • Growth and reproduction

    • Note: Microbes sustain these traits rather than being inherently "evil".

Why Microbes Cause Diseases

  • To sustain the characteristics of living organisms, microbes must be able to:

    • Respond to environment (responsiveness)

    • Adapt to changing conditions (adaptability)

    • Grow and reproduce

  • Important framing: disease is not about malice but about biology and interaction with hosts.

Two Core Questions in Microbiology

  • Why is something happening? (causes, mechanisms)

  • How is something happening? (processes, pathways)

  • Example framing for disease: Why disease happened? How disease happened?

The Early Years of Microbiology and Taxonomy

  • Carolus Linnaeus developed a taxonomic system for naming plants and animals and grouping similar organisms.

  • Leeuwenhoek’s viewpoint: microorganisms were grouped into six categories:

    • Bacteria

    • Archaea

    • Fungi

    • Protozoa

    • Algae

    • Small multicellular animals

    • Note: these groupings foreshadow the diversity of microbes.

Prokaryotes: Bacteria and Archaea

  • Prokaryotic cells lack a true nucleus.

  • Size: much smaller than eukaryotes.

  • Habitat: found wherever moisture is sufficient; some from extreme environments.

  • Reproduction: primarily asexual.

  • Cell walls:

    • Bacteria: contain peptidoglycan; some lack cell walls.

    • Archaea: cell walls are composed of polymers other than peptidoglycan.

  • Visual cue: bacterial cells vs eukaryotic cheek cells show distinct internal organization.

Eukaryotes: Fungi, Protozoa, Algae, Parasites

  • Fungi: eukaryotic, membrane-bound nucleus; obtain food from other organisms; have cell walls.

    • Molds: multicellular; long filaments; reproduce sexually and asexually via spores.

    • Yeasts: unicellular; reproduce asexually by budding; may produce sexual spores.

  • Protozoa: single-celled eukaryotes; similar nutrient needs to animals; most reproduce asexually, some sexually; locomotion via:

    • Pseudopods

    • Cilia

    • Flagella

  • Algae: can be unicellular or multicellular; photosynthetic; simple reproductive structures; basis for many algae-derived products.

  • Parasites (Helminths): eukaryotic multicellular animals; live on or in a host and derive nutrients from the host.

Viruses and Prions

  • Viruses: particles that reproduce inside living host cells; hijack host machinery to produce viral RNA/DNA and coat proteins.

  • Prions: proteinaceous infectious particles; misfolded proteins that propagate without nucleic acids.

  • Viruses and prions are often termed non-living because they require a host cell to replicate.

The Golden Age of Microbiology

  • Scientists addressed four pivotal questions:

    1. Is spontaneous generation of microbial life possible?

    2. What causes fermentation?

    3. What causes disease?

    4. How can infection and disease be prevented?

Origins of Life and Scientific Method

  • Historical ideas about origins of life included:

    • Asexual reproduction

    • Sexual reproduction

    • Nonliving matter

  • Aristotle proposed spontaneous generation; teach that life could arise from nonliving matter.

  • Scientific approach asks testable hypotheses and controlled experimentation to support or refute hypotheses.

The Scientific Method in Microbiology

  • Framework for scientific research:

    • Observation

    • Question formulation

    • Hypothesis development

    • Experimentation with controls

    • Analysis of results

    • Conclusion: support or refutation of the hypothesis.

Redi’s Experiments (Biogenesis vs Spontaneous Generation)

  • Redi demonstrated that decaying meat kept away from flies did not develop maggots; meat exposed to flies did.

  • This challenged Aristotle’s spontaneous generation theory.

  • Acknowledgment: Redi’s experiment had limitations that later experiments addressed.

Pasteur’s Experiments (Fermentation and Microbial Life)

  • Swan-necked flask experiments showed:

    • Upright flasks: no microbial growth in infusion.

    • Tilted flasks: dust from the bend allowed microbes to enter and cause turbidity within a day.

  • Why Pasteur’s experiment was pivotal: it supported biogenesis and negated spontaneous generation in broth.

The Pasteur Investigation: Detailed Summary (Observations, Hypotheses, and Conclusions)

  • Observation: Fermenting grape juice contained yeasts and bacteria; fermentation could yield alcohol or acids depending on microbes.

  • Hypotheses tested:

    • I. Spontaneous fermentation occurs.

    • II. Air ferments grape juice.

    • III. Bacteria ferment grape juice into acids.

    • IV. Yeasts ferment grape juice into alcohol.

  • Experimental steps included heating to kill microbes, sealing flasks, inoculating with bacteria or yeasts, and observing fermentation outcomes.

  • Conclusions:

    • Only Hypothesis IV was fully supported: yeasts ferment grape juice into alcohol.

    • Bacteria can ferment juice into acids; air is not the sole fermenter; spontaneous generation of fermentation is not necessary.

Buchner’s Fermentation Experiments (Cell-Free Fermentation)

  • Demonstrated that fermentation does not require living cells.

  • Enzymes in yeast extracts (cell-free) could ferment sugars, leading to the field of biochemistry.

  • Implication: fermentation can occur via enzymes secreted by cells, not necessarily by intact living cells.

  • This undermined the concept of vitalism and highlighted biochemical catalysts.

Koch’s Experiments and Postulates

  • Koch introduced:

    • Simple staining techniques

    • Methods to estimate colony-forming units per milliliter (CFU/ml)

    • Use of Petri dishes and transfer techniques to isolate bacteria

    • Concept of bacteria as distinct species

  • Koch’s Postulates (pressing the link between microbe and disease):

    1. Presence of the microorganism in diseased individuals: must be consistently found in cases.

    2. Isolation and culture of the microorganism: must be grown in pure culture.

    3. Reproduction of the disease in healthy individuals: introduction of the cultured organism should cause disease in a healthy host.

    4. Re-isolation of the microorganism from the inoculated host: same organism should be re-isolated.

Exceptions to Koch’s Postulates

  • Practical limitations exist:

    1. The suspected agent may be absent from some disease cases or present in healthy hosts (opportunistic pathogens).

    2. Some microbes cannot be grown outside a host.

    3. Not all hosts are susceptible to the same microbe.

    4. Some agents do not grow in experimental animals.

Disinfection, Antisepsis, and Hygiene Milestones

  • Major advances in disease prevention arose from recognizing microbial causation of disease.

  • Six pivotal practitioners reshaped healthcare delivery and hygiene practices (names provided in lectures): Semmelweis, Lister, Nightingale, Snow, and others in vaccination and chemotherapy.

Handwashing, Antiseptics, and Medicine

  • Semmelweis: required medical students to wash hands in chlorinated lime water; improved patient survival.

  • Lister: antiseptic technique using carbolic acid (phenol) on wounds, incisions, and dressings.

  • Nightingale: cleanliness and antiseptic practices in nursing; hospital/public health policy reform.

  • Snow: mapped the cholera outbreak in London (1854); foundational for infection control and epidemiology.

Vaccination and Immunology

  • Jenner developed a vaccine against smallpox, validating vaccination and initiating immunology.

  • Ehrlich pursued “magic bullets” to destroy pathogens with minimal harm to humans, contributing to chemotherapy.

Bioremediation and Environmental Microbiology

  • Bioremediation: using microorganisms to detoxify polluted environments by altering conditions to stimulate growth and degradation of pollutants.

  • Biological treatment applies to wastewater, industrial waste, and solid waste.

  • Most environmental microbes are not pathogenic.

Serology, Immunology, and Chemotherapy

  • Serology: study of blood serum and antibodies/cells that fight infection.

  • Immunology: body’s defenses against specific pathogens.

  • Chemotherapy: chemically targeted therapies against microbes; key historical milestones include penicillin (Fleming) and sulfa drugs (Domagk).

Atoms, Elements, Isotopes, and Atomic Structure

  • Matter is anything that takes up space and has mass.

  • Atoms are the smallest chemical units of matter.

  • Elements are composed of a single type of atom.

  • Isotopes: atoms of the same element with different numbers of neutrons.

    • Example: Carbon isotopes discussed: ^{12} ext{C} (6 protons, 6 neutrons), ^{13} ext{C} (6 protons, 7 neutrons), ^{14} ext{C} (6 protons, 8 neutrons).

  • Neutron count difference leads to different isotopic masses and sometimes stability.

Atomic Parameters and Stability

  • Atomic number: number of protons, Z.

  • Mass number: sum of protons and neutrons, A.

  • Stability of electrons is influenced by the electron shell configuration.

  • Example: Carbon-12/13/14 illustrate isotopic variation within the same element.

Chemical Bonds and Bonding Principles

  • Valence: combining capacity of an atom.

  • Bond formation involves sharing or transferring valence electrons.

  • Stable outer shells typically contain eight electrons (octet rule): 8 electrons in outer shell.

Covalent Bonds and Electronegativity

  • Covalent bonds: sharing of a pair of electrons between two atoms.

  • Why share electrons? To achieve stable outer electron configurations.

  • Electronegativity: attraction of an atom for electrons; more electronegative atom pulls shared electrons more strongly, creating bond polarity.

  • Nonpolar covalent bonds: electrons shared equally due to similar electronegativities.

  • Polar covalent bonds: unequal sharing due to different electronegativities, enabling polarity and interactions like hydrogen bonding.

Nonpolar Covalent Bonds and Organic Compounds

  • Examples: Carbon forms four nonpolar covalent bonds with other atoms.

  • Organic compounds commonly contain carbon and hydrogen.

  • Diatomic molecules formed by covalent bonds: H2, O2, etc. (illustrative examples).

Polar Covalent Bonds and Hydrogen Bonding

  • Polar covalent bonds arise from electronegativity differences; hydrogen bonds are weaker than covalent bonds but essential for life.

  • Hydrogen bonding helps stabilize 3-D structures of large molecules.

Ionic Bonds

  • Occur when atoms with very different electronegativities come together.

  • Result in charged ions: cations (positive) and anions (negative).

  • Ionic bonds form crystals (salts) via electrostatic attraction; no electron sharing required.

  • Question posed in slides: which are stronger, ionic or covalent bonds? (Answer depends on context; covalent bonds in many contexts are very strong; ionic bonds can be strong in lattice structures but can be disrupted in aqueous environments.)

Hydrogen Bonds and Water

  • Hydrogen bonds are electrical attractions between partially charged hydrogen atoms and electronegative atoms in the same or different molecules.

  • Important for stabilizing biomolecules and solvent properties for life.

Chemical Reactions and Metabolism

  • Reactions involve making or breaking chemical bonds; involve reactants and products.

  • Biochemistry studies chemical reactions in living organisms.

  • Major reaction types:

    • Synthesis (anabolism): formation of larger, more complex molecules; often endothermic; dehydration synthesis; example: formation of triglycerides from glycerol and fatty acids with water release.

    • Decomposition (catabolism): breaking down molecules; often exothermic; hydrolysis.

    • Exchange reactions: involve breaking and forming covalent bonds with participation of multiple steps.

  • Metabolism: total sum of all chemical reactions in an organism.

Water, Acids, Bases, and pH

  • Water is the most abundant substance in organisms with polar covalent bonds and several key properties:

    • Cohesion and surface tension

    • Excellent solvent

    • Remains liquid across broad temperature ranges

    • High specific heat capacity (absorbs energy with little temperature change)

    • Participates in many chemical reactions

  • Acids and bases and pH:

    • Acids dissociate to produce H⁺ (and anions); bases bind H⁺ or release OH⁻.

    • pH is a measure of H⁺ concentration in solution.

    • Buffers resist drastic changes in pH.

The pH Scale and Buffers

  • pH scale ranges from 0 (extremely acidic) to 14 (extremely basic).

  • Examples of pH values (illustrative):

    • Battery acid (extremely acidic): 0

    • Hydrochloric acid: 1

    • Lemon juice: 2

    • Wine, tomatoes: 4

    • Pure water: 7

    • Seawater: around 8.5

    • Household ammonia: 11

    • Oven cleaner: 13

  • Buffers help maintain internal pH in biological systems.

Salts and Acid-Base Chemistry

  • Salt definition: a compound formed when the hydrogen of an acid is replaced by a metal.

  • Occurs when acids react with metals, alkalis, or metal carbonates. Examples:

    • Magnesium reacts with HCl to form MgCl₂ and H₂.

    • Sodium hydroxide (NaOH) reacts with nitric acid (HNO₃) to form NaNO₃ and H₂O.

Organic Macromolecules: Monomers, Polymers, and the Role of Carbon

  • Organic macromolecules are built from monomers linked into polymers.

  • Core elements: carbon and hydrogen are essential.

  • Monomers join to form polymers via dehydration synthesis; water is released in the process.

  • Polymers are large molecules made of repeated monomer units.

Functional Groups in Organic Molecules (Table 2.3 concept)

  • Hydroxyl (–OH): present in alcohols and sugars.

  • Ether (R–O–R): present in some lipids and polysaccharides.

  • Carbonyl groups:

    • Internal carbonyl (in ketones): R–CO–R'

    • Terminal carbonyl (in aldehydes): R–CHO

  • Carboxyl (–COOH): acidic group; involved in amino acids, fatty acids, etc.

  • Amino (–NH₂): amino groups found in amino acids and proteins.

  • Ester (–COO–R): found in fats and phospholipids.

  • Sulfhydryl (–SH): present in some amino acids (e.g., cysteine).

  • Phosphate (–PO₄): present in nucleotides and energy carriers (ATP).

  • These functional groups determine the class of compounds (e.g., alcohols, carbohydrates, amino acids, proteins, lipids, nucleotides, ATP).

Lipids: Structures and Roles

  • Lipids are hydrophobic (nonpolar) and do not form regular subunits.

  • Four groups: fats (triglycerides), phospholipids, waxes, steroids.

  • Fats (triglycerides): formed by dehydration synthesis of glycerol with three fatty acids via ester bonds; show saturated vs unsaturated fatty acids.

    • Triglyceride structure: glycerol + 3 fatty acids → fat; ester bonds connect them.

    • Saturated fatty acids have no double bonds; unsaturated have one or more double bonds (kinks).

  • Phospholipids form the core of cell membranes; phospholipid bilayer includes hydrophilic (polar) head and hydrophobic (nonpolar) tails; phospholipid nature drives membrane formation.

  • Waxes: long-chain fatty acids linked to long-chain alcohols; highly hydrophobic; completely insoluble in water.

  • Sterols (including cholesterol): a class of steroids; cholesterol is abundant in humans and interspersed in cell membranes; excessive cholesterol linked to atherosclerosis and heart disease. Apolipoproteins combine cholesterol with proteins to transport lipids in blood.

  • Hormones can be steroid-based; two main groups: adrenocortical hormones and sex hormones; derived from cholesterol; influence electrolyte balance and metabolic regulation.

Carbohydrates: Sugars and Their Roles

  • Carbohydrates are organic molecules with formula roughly

    • general formula:
      (CH2O)n

  • Functions:

    • Long-term energy storage (substrate for glycolysis)

    • Ready energy source

    • Backbone components of nucleic acids

    • Can be converted to amino acids

    • Glycocalyx formation around plasma membrane or cell wall

    • Involvement in cell-cell interactions

  • Types:

    • Monosaccharides (simple sugars): e.g., glucose (°), galactose, fructose

    • Disaccharides: e.g., sucrose (glucose + fructose), lactose (galactose + glucose)

    • Polysaccharides: glycogen (animal storage), starch (plant storage), cellulose (structural in plants)

  • Monosaccharide examples: glucose with ring structures, anomeric forms α and β (configurations).

Proteins: Building Blocks and Roles

  • Proteins are primarily built from carbon, hydrogen, oxygen, nitrogen, and sulfur.

  • Functions include:

    • Structural components and channels/receptors

    • Enzymatic catalysis

    • Regulation of gene expression

    • Transport, defense, and offense (antimicrobial activity, antibodies, etc.)

  • Amino acids: the monomers of proteins; there are 21 standard amino acids used in protein synthesis by most organisms.

  • Side chains (R groups) determine interactions and protein folding.

  • Peptide bond: covalent bond formed between amino acids.

  • Protein structure levels:

    • Primary: sequence of amino acids

    • Secondary: α-helix and β-pleated sheet formed by hydrogen bonds

    • Tertiary: three-dimensional folding and interactions

    • Quaternary: two or more polypeptides associating to form a functional protein

  • Fibrous vs Globular proteins:

    • Fibrous: long, parallel polypeptide chains; typically structural; e.g., keratin, collagen, silk

    • Globular: compact, folded, usually soluble; mainly enzymatic and regulatory roles; e.g., many enzymes and transport proteins

  • Visual note: alpha helices and beta-pleated sheets are stabilized by hydrogen bonds within the polypeptide backbone.

Nucleic Acids: DNA and RNA

  • DNA and RNA are genetic materials in organisms and many viruses; RNA can also act as an enzyme and participates in translation.

  • Nucleotides: basic units comprising a nitrogenous base, a sugar (deoxyribose in DNA, ribose in RNA), and a phosphate group.

  • Nitrogenous bases:

    • Purines: Adenine (A), Guanine (G)

    • Pyrimidines: Cytosine (C), Thymine (T) in DNA; Uracil (U) in RNA replaces T.

  • Base pairing:

    • A pairs with T via 2 hydrogen bonds in DNA (and A-U in RNA via 2 H-bonds)

    • G pairs with C via 3 hydrogen bonds

  • DNA structure:

    • Double-stranded, complementary strands

    • Antiparallel orientation: one strand 5'→3', the other 3'→5'

    • Sugar-phosphate backbones run in opposite directions

  • RNA structure and types:

    • mRNA: carries genetic information from DNA to ribosome

    • rRNA: part of ribosome, site of protein synthesis

    • tRNA: delivers amino acids to the ribosome during translation

ATP: The Energy Currency of the Cell

  • ATP structure: adenine base, ribose sugar, three phosphate groups (α, β, γ).

  • Energy release mechanism:

    • The terminal phosphate bond (between the β and γ phosphates) is hydrolyzed to yield ADP and inorganic phosphate (Pi):
      \text{ATP} \rightarrow \text{ADP} + \text{P}i + \Delta G{hyd}

    • This hydrolysis provides energy for cellular work.

  • Roles of ATP:

    • Transport: active transport via phosphorylation of membrane proteins

    • Mechanical work: phosphorylation of motor proteins (e.g., actin/myosin) for muscle contraction and vesicle movement

    • Chemical work: phosphorylation of reactants to drive biosynthetic reactions

  • After work, the phosphate is released as inorganic phosphate (Pi) and ATP can be recharged back to ATP in the cell.

Key Connections and Practical Implications

  • Biochemistry underpins microbiology: metabolism, energy transfer (ATP), and macromolecular structure determine how microbes function and respond to environments.

  • The cell membrane's phospholipid bilayer is essential for selective permeability and cellular homeostasis; cholesterol and sterols modulate membrane fluidity in eukaryotes.

  • Hydrogen bonding and water properties support protein folding, nucleic acid structure, and enzymatic activity.

  • Functional groups govern reactivity and the formation of macromolecules—underpinning synthetic biology and metabolic engineering.

  • Historical methods (Koсh, Pasteur, Redi, Buchner) illustrate the shift from vitalism to biochemistry-based understanding of life, disease, and fermentation.

Quick Reference: Key Equations and Concepts

  • Dehydration synthesis (condensation):
    \text{Monomer} + \text{Monomer} \rightarrow \text{Polymer} + \mathrm{H_2O}

  • Hydrolysis (decomposition):
    \text{Polymer} + \mathrm{H_2O} \rightarrow \text{Monomer} + \text{Monomer}

  • Nucleotide base pairing (DNA):

    • \text{A} \;\leftrightarrow\; \text{T} with 2 hydrogen bonds

    • \text{G} \;\leftrightarrow\; \text{C} with 3 hydrogen bonds

  • pH scale example values (illustrative):

    • Acidic: 0 \le \text{pH} < 7

    • Neutral: \text{pH} = 7

    • Basic: 7 < \text{pH} \le 14

  • Isotopes example:

    • ^{12}\text{C},\; ^{13}\text{C},\; ^{14}\text{C} with neutron counts differing by 0,1,2 respectively (for carbon), illustrating isotopes: Z=6 for all.

Connections to Real-World Relevance and Ethics

  • Bioremediation leverages microbial metabolism to detoxify polluted environments in a sustainable way.

  • Vaccination and chemotherapy have transformed public health, reducing morbidity and mortality from infectious diseases.

  • Understanding biochemical energy (ATP) informs medical therapies, drug design, and metabolic engineering.

  • Ethical considerations include safety in manipulating microbes, vaccine distribution, and equitable access to healthcare interventions.

Review Checklist for Exam Readiness

  • Define and distinguish microbes and their major groups (bacteria, archaea, fungi, protozoa, algae, parasites, viruses, prions).

  • Explain the differences between prokaryotes and eukaryotes, including cell wall composition.

  • Describe Pasteur’s and Redi’s experiments and their significance for biogenesis vs spontaneous generation.

  • State Koch’s postulates and discuss common exceptions.

  • Summarize the role of Semmelweis, Lister, Nightingale, and Snow in hygiene, antisepsis, and epidemiology.

  • Outline the functional groups of organic molecules and the classes of compounds formed from them.

  • Compare carbohydrates, lipids, proteins, and nucleic acids in terms of structure, monomers, and roles in cells.

  • Explain RNA vs DNA structure, base pairing, and the concept of anti-parallel strands.

  • Describe ATP structure and the three main cellular roles of ATP.

  • Understand the basics of chemical reactions in metabolism: synthesis, decomposition, and exchange reactions.

  • Recognize the properties of water and the importance of pH and buffers in biological systems.

  • Recall major historical milestones that connect chemistry to microbiology (e.g., enzyme catalysis, biochemistry, vaccination, antibiotics).

Extra things to know from HW

Antoni van Leeuwenhoek was the first person to visualize and describe microorganisms, which are now grouped into six categories: bacteria, archaea, fungi, protozoa, algae, and small multicellular animals.

Penicillin was discovered by Scottish physician and microbiologist Alexander Fleming in 1928.

Living microorganisms characterized by the absence of a nucleus are called prokaryotes. This group includes bacteria and archaea.

The experiments of scientists and the results of many years of observations form the foundation of scientific knowledge, leading to the development, refinement, or rejection of hypotheses and, subsequently, the formation of scientific theories and laws. This process is part of the scientific method, which involves systematic observation, measurement, experiment, and the formulation, testing, and modification of hypotheses.

The first nursing school was founded by Florence Nightingale.

No, protozoa are not called prokaryotes. The note states that protozoa are "single-celled eukaryotes," meaning they possess a membrane-bound nucleus. Prokaryotes, such as bacteria and archaea, lack a nucleus.

Yes, Louis Pasteur is widely considered one of the fathers of microbiology due to his groundbreaking work. His experiments, such as those with swan-necked flasks, disproved the theory of spontaneous generation and established the role of microbes in fermentation and disease. He also developed pasteurization and vaccines, significantly advancing the field.

Yes, a cell that contains a nucleus is called a eukaryotic cell. Eukaryotic cells possess a membrane-bound nucleus where their genetic material is stored.

No, Archaea are not fungi. According to the note, Archaea are prokaryotic, meaning they lack a nucleus, and are grouped with bacteria. Fungi, on the other hand, are eukaryotic, meaning they possess a membrane-bound nucleus.

Electrons do not significantly contribute to the mass of an atom. The mass of an atom primarily comes from its protons and neutrons, which are located in the nucleus. Electrons, while having mass, are approximately 1/1836 the mass of a proton, making their contribution negligible to the overall atomic mass.

Matter composed of a single type of atom is known as an element.

A stable atom typically has a full outermost (valence) electron shell. For most atoms, this means it has 8 electrons in its valence shell (known as the octet rule). However, for smaller atoms like hydrogen and helium, a full valence shell consists of 2 electrons.

In a chemical reaction, it is primarily the electrons in the outermost shell of an atom, known as valence electrons, that interact. These electrons are involved in forming, breaking, and rearranging chemical bonds between atoms.

The valence of an atom represents its combining power or capacity to form chemical bonds with other atoms. It is determined by the number of electrons in its outermost shell, known as valence electrons, which are involved in chemical interactions.

One important property of water is its polarity. Water molecules are polar, meaning they have a slight positive charge on the hydrogen atoms and a slight negative charge on the oxygen atom. This polarity allows water to form hydrogen bonds with other water molecules and with other polar substances, making it an excellent solvent for many compounds.

Fats, proteins, and complex carbohydrates are all synthesized by living organisms, specifically within their cells, through various metabolic pathways and enzymatic reactions.

Amino acids are the fundamental building blocks of proteins. Each amino acid universally consists of a central carbon atom (called the alpha-carbon) bonded to four main components: