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The Fundamental Building Blocks
of Life: Cells and Microorganisms
Introduction to Microbiology and Microscopes
Cells and Cell Theory
Cells are the fundamental component of life and cells give rise to other cells. Cell theory has three tenets: Living organisms are made up of one or more cells. Living cells arise from pre-existing cells. Cells are the fundamental unit of structure and function of living organisms.
Spontaneous Generation vs. Biogenesis
Spontaneous generation was the theory that life arises from nonliving matter. Biogenesis is the generation of living matter from other living matter. Francesco Redi's experiment refuted the idea of spontaneous generation of maggots on rotting meat.
Koch's Postulates
Koch's four postulates summarized his method for determining whether a particular microorganism was the cause of a particular disease:
The suspected pathogen must be found in every case of disease and not be found in healthy individuals. The suspected pathogen can be isolated and grown in pure culture. A healthy test subject infected with the suspected pathogen must develop the same signs and symptoms of disease as seen in postulate 1. The pathogen must be re-isolated from the new host and must be identical to the pathogen from postulate 2.
Chemotherapy
Chemotherapy refers to the use of chemicals or drugs to treat disease. Antimicrobial drugs can be produced by bacteria or fungi (natural antibiotics) or developed from man-made chemicals (synthetic antimicrobials). Semi-synthetic drugs are derivatives of natural antibiotics with increased bacterial range and stability but reduced activity.
Comparing Microorganisms
Microorganisms can be classified as acellular (not composed of cells), prokaryotic, or eukaryotic. Acellular microbes include viruses, viroids, and prions. Prokaryotic microbes include bacteria and archaea, which lack a true nucleus and membrane-bound organelles. Eukaryotic microbes include algae, fungi, protozoa, and helminths, which have a true nucleus and membrane-bound organelles.
Binomial Nomenclature
Binomial nomenclature is a system that names each species with a two- word name, the first identifying the genus and the second the species. The purpose of this system is to provide a consistent and unique way to name species, avoiding confusion from common names.
The Three Domains of Life
The three domains of life are Archaea, Bacteria, and Eukarya. Archaea are prokaryotes that resemble bacteria but have significant differences in genetics, metabolism, and cell structure. Bacteria are prokaryotes with peptidoglycan/lipopolysaccharide cell walls and the ability to form spores. Eukarya contains all eukaryotic organisms, including unicellular and multicellular forms.
Interpreting Microscopy Techniques
Magnification is the ability of a lens to enlarge an object compared to its real size. Total magnification is the product of the ocular and objective magnifications. Other important microscopy concepts include resolution, contrast, and illumination.
Microbial Staining
Staining is the coloring of microorganisms with dyes to make structures visible. Differential stains can differentiate bacteria based on their reactions to the stains. Smears are thin films of material used for microscopic examination.
Recognizing the Importance of Microbiology
Chemotherapy involves the use of chemical substances to treat disease. Antibiotics are chemicals produced by bacteria or fungi that act against other microorganisms. Synthetic drugs are chemotherapeutic agents prepared from chemicals in a laboratory.
source and then emitting this energy as visible light, creating contrast. This is used to identify pathogens and molecules in cells.
Electron Microscope : Electron microscopes use short-wavelength electron beams rather than light to increase magnification and resolution. The two basic types are the transmission electron microscope (TEM) and the scanning electron microscope (SEM). This allows for the viewing of subcellular structures.
Microbial Staining Techniques
Stains : Stains are salts composed of a positive or a negative ion, one of which is colored. Basic dyes have a positive ion carrying the color, while acidic dyes have a negative ion carrying the color.
Positive Stain : A positive stain will be used to color the cell or organism to be observed, making it stand out from the clear or opaque background. For example, chive pollens stained by aceto carmine dye for a viability test under a light microscope.
Negative Stain : A negative stain produces an outline or silhouette of the organism against a dark background. Negative staining requires an acidic dye such as India Ink or Nigrosin, which is an acidic stain that readily gives up a hydrogen ion (proton), and the chromophore of the dye becomes negatively charged. Since the surface of most bacterial cells is negatively charged, the cell surface repels the stain, and the bacteria will show up as clear spots against a dark background.
Mordant : A mordant is a chemical added to the stain to intensify the depth of the stain.
Gram Staining
Gram staining provides valuable information for the treatment of disease and distinguishes cells as Gram-positive or Gram-negative based on the composition of their cell wall.
Gram Staining Results: Purple = Gram-positive Pink = Gram-negative
The steps of Gram staining are: 1. Crystal violet - Primary dye, stains all cells initially. 2. Iodine - Mordant, intensifies the stain. 3. Alcohol - Decolorizes cells with lipopolysaccharides in the cell wall. 4. Safranin - Counter stain, visualize decolorized cells.
Acid-Fast Staining
The acid-fast stain is an important diagnostic tool. It is able to differentiate two types of Gram-positive cells: those with a waxy mycolic acid in their walls and those that do not. Mycolic acid is found in Mycobacterium leprae
and Mycobacterium tuberculosis, the causative agents of leprosy and tuberculosis, respectively.
The steps for Acid-Fast Staining are: 1. Carbolfuchsin: Primary Stain - Carbolfuchsin is applied to a fixed smear, and the slide is gently heated to enhance penetration and retention of the dye. The slide is then cooled and washed with water to remove the excess dye. 2. Decolorization - The slide is then treated with alcohol to decolorize or remove the red stain from the bacteria that are not acid-fast. The acid-fast microorganisms retain the red, as they have mycolic acid in their cell walls. 3. Methylene Blue: Counterstain
- The slide is then stained with methylene blue counterstain. Non-acid-fast cells appear blue after the counterstain is applied.
Capsule Staining
The capsule is a gelatinous covering found on some bacteria. The presence of a capsule is directly related to an organism's virulence, which is the degree to which a pathogen can cause disease. Capsule staining is more difficult than other differential stains because capsular materials are water- soluble and may be dislodged or removed during the rigorous washing. Capsules do not accept most biological dyes, such as safranin, and therefore a halo appears around each stained bacterium.
Endospore Staining
Endospores are a resistant, dormant structure formed within a cell that protects a bacterium from adverse environmental conditions. They are uncommon in most bacterial cells and cannot be stained by ordinary methods such as simple staining or Gram staining because the dyes don't penetrate the endospore's wall.
Flagella Staining
Flagella are structures of locomotion too small to be seen with a light microscope without staining. Flagella, while not always clinically important, are used to identify bacteria in a mixed sample. It is a tedious process of using a mordant and carbolfuchsin to build the diameter of the flagellum until it is visible.
Prokaryotic and Eukaryotic Cell Structures
Prokaryotic Cell Structures
Cytoplasm - Gel-like substance within the plasma membrane. Ribosome - Internal structure where protein synthesis occurs. Prokaryotic cells have 70S ribosomes. Nucleoid - Internal structure where the chromosome is located. Plasmid - Internal structure, small, circular, double-stranded DNA molecules.
Bacterial cells of the same species can assume special arrangements: - Diplococcus - pair of cocci - Tetrad - a group of 4 cells arranged in a square - Streptococcus - chain of cocci - Staphlyococcus - cluster of cocci - Streptobacillus - chain of rods
Similarities and Differences between Prokaryotic and
Eukaryotic Cells
Similarities: - 70S Ribosome - Pili - Fimbriae - Endospore - Capsule - DNA - Flagella - Cell Wall - Cytoplasm - Plasma Membrane
Differences: Prokaryotic: - 70S Ribosome
Eukaryotic: - 80S Ribosome - Nucleus - Mitochondria - Membrane-bound organelles
Gram-Positive and Gram-Negative Cell Walls
Vocabulary Terms
Peptidoglycan - Only found in bacteria, a unique meshwork found in the cell wall of Gram-positive and Gram-negative cells. Cell Wall - Protects the cell from harsh conditions. Gram-Positive - Contain many layers of peptidoglycan. Outer Membrane - External to the peptidoglycan layer, contains the LPS. Gram-Negative - More complicated cell wall with a thin layer of peptidoglycan between the plasma membrane and the outer membrane. Lipopolysaccharide (LPS) - Found in Gram-negative cells only, functions as an endotoxin.
Gram-Positive Cell Walls
Gram-positive cells have a cell wall that contains many layers of peptidoglycan. Some well-known Gram-positive bacteria include Staphylo- and Streptococcus species. The thick peptidoglycan layer in Gram-positive cell walls is the target of many antibiotics.
Gram-Negative Cell Walls
Gram-negative cells have a more complicated cell wall compared to Gram- positive cells. The peptidoglycan layer is thinner than in Gram-positive cell walls. Gram-negative cells have the following additional cell wall components: 1. Outer Membrane 2. Periplasmic Space 3. Lipopolysaccharide (LPS) a. Lipid A = endotoxin
Atypical Cell Walls
Mycobacterium species contain high levels of a waxy substance in their cell wall called mycolic acid. This helps protect the bacterium from phagocytosis and resists dyes used for staining. Mycoplasma are the smallest known bacteria that can replicate outside a host cell and do not contain a cell wall. Because they lack a cell wall, they are referred to as pleomorphic, meaning they can have a variety of different shapes and resemble small animal cells. The most notable species is M. pneumoniae, which causes 'walking pneumonia.'
Relating Selective Toxicity to Cellular
Differences
Vocabulary Terms
Selective Toxicity - Selectively kills or inhibits certain microbial targets with minimal harm to the host. Antimicrobial Drugs - Drugs that target and inhibit microbial cells. Ribosome - Many antimicrobial drugs target the 70S ribosome of prokaryotic cells. Peptidoglycan - Many drugs target the peptidoglycan in prokaryotic cells.
Selective Toxicity
Selective toxicity refers to the ability of a drug to target or inhibit specific microbial cells and cause little to no harm to host cells. There are many different ways to target differences between prokaryotic and eukaryotic cells, such as: - Ribosomes - Cell Wall - Metabolic Pathways
Membrane Transport Mechanisms
Vocabulary Terms
Facilitated Diffusion - The diffusion of solutes through a protein channel; a passive transport. Passive Transport - The passive movement of molecules down their concentration gradient. Active Transport - The transport of molecules across a membrane that requires energy. Osmosis - The movement of water down its concentration gradient, does not require energy. Diffusion - The movement of molecules across a membrane down their concentration gradient. Hypertonic Solution - Solution containing a higher concentration of solutes compared to the inside of the cell, resulting in water rushing out of the cell (plasmolysis).
Active Transport Mechanisms
Active transport mechanisms require energy in the form of ATP to move large molecules or to move molecules against their concentration gradient from areas of low concentration to a high concentration. These include:
Endocytosis : The process of bringing large particles into the cell, such as phagocytosis.
Exocytosis : The process of exporting particles, such as waste or signaling molecules, outside of the cell.
Pumps : The most commonly discussed pump is the Sodium-Potassium Pump, which requires energy to move sodium and potassium ions against their concentration gradients.
Metabolic Diversity of Microorganisms and
Metabolism
Organic Molecules, Structure, and Function
Carbohydrates : Monosaccharides: Simple sugars or monomers, such as glucose, ribose, and deoxyribose. Disaccharides: Formed through a dehydration synthesis reaction between two monosaccharides, such as maltose, lactose, and sucrose. Polysaccharides: Hundreds of monosaccharides strung together, forming polymers like starch, glycogen, cellulose, peptidoglycan, chitin, and beta-glucans.
Functions: Energy source, structural components, and genetic information.
Lipids :
Fatty acids: Long-chain hydrocarbons that can be saturated or unsaturated. Triglycerides: Made of a glycerol molecule plus three fatty acids. Phospholipids: Made of two fatty acids and a phosphate head, forming a bilayer in cell membranes. Sterols: Molecules made of hydrophobic rings, including cholesterol and ergosterol.
Functions: Energy storage and structural components of cell membranes.
Proteins :
Amino acids: The building blocks of proteins, with a central carbon, hydrogen, amino group, carboxyl group, and a variable R-group side chain.
Protein structure: Primary (amino acid sequence), secondary (alpha and beta-pleated sheets), tertiary (polypeptide chain), and quaternary (assembled subunits). Glycoproteins and lipoproteins: Proteins conjugated with carbohydrates or lipids. Functions: Enzymes, membrane structures, cell signaling, receptors, virulence factors, self-identity, and transport.
Structure of Nucleic Acids
Nucleic Acids
Nucleic acids include DNA, RNA, and ATP. The monomer is a nucleotide, which is composed of one or more phosphate groups, a pentose sugar, and a nitrogen base.
DNA
DNA is a double-stranded molecule made up of the following components: - Deoxyribose sugars - Phosphate groups - Nitrogenous bases
The nitrogenous bases in DNA undergo complementary base pairing, where adenine (a purine with two rings) pairs with thymine (a pyrimidine with one ring), and guanine (a purine) pairs with cytosine (a pyrimidine).
RNA
RNA is the working form of DNA. It is single-stranded, with the sugar ribose replacing deoxyribose. RNA is shorter than DNA and only copies the gene of interest. The nitrogenous bases in RNA are adenine, cytosine, guanine, and uracil (which replaces thymine). When RNA is being made, the DNA strand acts as the template, and the same complementary base pairing applies, except that uracil replaces thymine.
There are three forms of RNA: messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA).
ATP
Adenosine triphosphate (ATP), while not technically a nucleic acid, is a nucleotide. ATP contains the base adenine, the sugar ribose, and three phosphate groups. It is the energy currency of the cell.
Functions of Nucleic Acids
DNA
DNA encodes genetic information, holding the genes (genome) and instructions for proteins.
Anabolic Reactions
Anabolic reactions are those that combine smaller molecules into larger ones, storing energy in the bonds. These reactions are endergonic, meaning they require an input of energy.
Energy Currency
Adenosine triphosphate (ATP) is the energy currency of all cells. Cells use ATP to store energy generated through catabolic reactions and to build larger molecules through anabolic reactions.
ATP Production
Cells can produce ATP through two main mechanisms: substrate-level phosphorylation and oxidative phosphorylation. Oxidative phosphorylation, which occurs during cellular respiration, generates most of the ATP inside a cell.
