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Microbiology ✔✔The study of microbes and their biological processes at the micro (microscopic) level. Microbes ✔✔1. A general term that includes microorganisms and viruses; an organism or virus too small to be seen without a microscope
- Beneficial when aiding in food digestion to protecting us when we are exposed to potentially harmful foreign invaders to helping crops grow
- Detrimental when harmful strains of bacteria, fungi, protozoa, and viruses kill millions of people each year and sicken even more Microorganisms ✔✔1. Usually consist of a single cell
- Examples: bacteria, archaeons, fungi, protozoa, and algae Prokaryotic or eukaryotic ✔✔1. The most common distinction between living organisms
- Prokaryotic cells (before kernel) lack a nucleus, whereas eukaryotic cells (true kernel) have a defined nuclear region
- Difference: size, complexity, unicellular or multicellular, abundance, examples, nucleus & DNA, and membrane-bound organelles
BIOD 171 Microbiology Module 1 – 6
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- Similarity: macromolecules, plasma membrane, cytosol, chromosomes, and ribosomes 3 classifications of life ✔✔1. Bacteria, Archaea, and Eukarya
- The first two categories are prokaryotic microorganisms that are different in cellular composition Bacteria (Prokaryotes) ✔✔1. Morphologies: coccus (round/spherical), bacillus (rod), vibrio (curved rod), or spirillum (spiral/corkscrew).
- Cellular organization: isolated (individual) cells, in chains, or in clusters
- Examples: Streptococcus (round chains); E. coli (rod); Mycobacterium tuberculosis (a unicellular bacterium) Archaea (Prokaryotes) ✔✔1. Also referred to as extremophiles
- Survive in extremely harsh environmental conditions, such as high salt levels, acid conditions, high temperatures, and oxygen-poor conditions Eukarya ✔✔Animalia, Plantae, Fungi, and Protista Animalia ✔✔1. Multicellular eukaryotic organisms includes animals and humans
- Heterotrophic and the general characteristic of motility Heterotrophic ✔✔Incapable of producing one's own energy, must absorb (consume) nutrients from the environment
Cell membrane/plasma membrane/cytoplasmic membrane ✔✔1. A semi-permeable barrier surrounding the cytoplasmic space of a cell acts as a barrier to the outside environment while also retaining and preventing the intra (within) cellular components from being lost to the surrounding environment
- A bilayer composed primarily of amphipathic phospholipids that contain a polar hydrophilic (water loving) head group and a non-polar hydrophobic (water fearing) tail region
- A relatively high degree of fluidity dependent on the types of lipids, the temperature, and additional molecules (proteins)
- Sterol lipids (such as cholesterol), which play a major role in eukaryotic membrane composition Cell walls/outer membrane ✔✔1. Found in bacteria, plants, fungi, and algae and help to form the shape of the cell, providing support, structure, and protection
- Peptidoglycan for bacterial wall: a peptide-polysaccharide matrix; vary in thickness to protect the cell from potentially damaging environmental stresses
- Surface-layer proteins (S-layer) for archaea cell: physical and chemical protective barrier to the harsh surrounding environments
- Cellulose and chitin for fungi
- Mannoproteins (highly glycosylated polypeptides called mannan and mannose) and chitin for yeasts
- Silica (main component in glass) for Diatoms (algae) : very rigid
- Some eukaryotic cell walls may contain polysaccharides, like cellulose and pectin
Cell envelope ✔✔The combination of the cell membrane and the outer membrane (cell wall) Cytoplasm ✔✔1. Constitutes the open volume within a cell;
- Comprised mostly of water containing dissolved substances important to the functioning of the cell. Organelles ✔✔Membrane-enclosed structures within the cytoplasm that perform specific functions. Nucleus ✔✔1. A porous double lipid bilayer that contains the genomic content of the cell (DNA)
- The command center of the cell
- The defining organelle of all eukaryotic cells Nucleolus ✔✔Within the nucleus, the site of ribosome synthesis Ribosomes ✔✔1. Responsible for protein synthesis.
- Located in the cytoplasm of prokaryotic cells and attached to the endoplasmic reticulum in eukaryotic cells Endoplasmic Reticulum (ER) ✔✔1. A series of membrane-enclosed sacs and interconnected tubes (lumen)
Cell ✔✔1. A cell is the smallest, most basic biological unit of life
- At the most generalized level, all cells are comprised of macromolecules
- Retains each characteristics of life: responsiveness, growth and reproduction, metabolism, homeostasis, and organization Macromolecules ✔✔1. complex molecules that are composed of smaller subunits
- Four main types: (1) proteins, (2) nucleic acids, (3) lipids, and (4) polysaccharides
- Building blocks: (1) amino acids, (2) nucleotides, (3) fatty acids, and (4) sugars Proteins ✔✔1. Polymers (a large molecule comprised of many smaller subunits) of amino acids, and the most abundant form of macromolecule
- Functions: facilitate the movement of materials in or out of a cell; act as enzymes that catalyze/speed up biochemical processes; play a structural role, while other proteins, such as filaments, enable movement.
- There are 20 different amino acids and 9 are considered essential amino acids
- The primary structure of the protein: its own unique sequence of amino acids Titin ✔✔The largest known protein made up of about 33,000 amino acids Nucleic acids ✔✔1. Chemical molecules that carry genetic information within the cell
- Two major types: DNA and RNA
DNA (deoxyribonucleic acid) ✔✔1. 3 parts of nucleotides: a nitrogenous (nitrogen-containing) base, a sugar called deoxyribose, and a phosphate group.
- 4 kinds of nitrogenous bases: adenine or guanine (purine bases) and cytosine or thymine (pyrimidine bases), abbreviated, A, G, C, and T.
- Structure: the sugar and phosphate portions form the backbone of a DNA strand, while the nitrogenous base protrudes outward from the deoxyribose sugar. Strands of DNA form a double helix (2 strands held together by hydrogen bonding)
- Complementary base pairs: [adenine and thymine] or [guanine and cytosine]. Base pairings between A-T = 2 hydrogen bonds, while G-C pairings = 3 hydrogen bonds
- Contains a vast amount of hereditary information, responsible for the inheritable characteristics of living organisms
- Location: nucleus, mitochondria, and chloroplasts RNA (ribonucleic acid) ✔✔1. Composed of nucleotides
- Contains the sugar ribose rather than deoxyribose.
- Always single-stranded
- Contains uracil (U) pairing with adenine (A), and guanine (G) pairing with cytosine (C)
- Responsible for deciphering the hereditary information in DNA and using it to synthesize proteins
- Location: nucleus, cytoplasm, and ribosomes Lipids ✔✔1. Mainly composed of non-polar hydrophobic (water fearing) hydrocarbons (hydrogen + carbon atoms)
Cellular metabolism ✔✔A controlled set of biochemical reactions that occur in living organisms to maintain life Enzyme ✔✔A protein, or group of proteins, that catalyze (speed up) chemical reactions Cofactor ✔✔A small chemical component, usually metal ions, that assist enzymes during the catalysis reactions Catabolism ✔✔The process of breaking down larger molecules into useful energy sources Anabolism ✔✔The building up or biosynthesis of macromolecules from smaller molecular units into larger complexes ATP and ADP ✔✔ATP has energy to donate, while ADP can accept energy (phosphate group) to become ATP. By donating available phosphate groups, ATP transfers energy from catabolic (breaking down) reactions to be used for anabolism (building up). This process of donating and/or accepting energy is what fuels a cell to carry out the necessary biochemical reactions for survival. Phototrophs ✔✔Acquire energy from photons of light to generate ATP from ADP
Chemotrophs ✔✔Acquire energy from preformed (already existing) chemicals found in the environment Organotrophs ✔✔Remove electrons from organic molecules (such as glucose) Lithotrophs ✔✔Remove electrons from inorganic molecules (such as elemental sulfur) Heterotroph ✔✔A microorganism that derives its carbon from organic molecules, such as sugars Autotrophs ✔✔Organisms that derive their carbon from inorganic molecules, most often from carbon dioxide (CO2) Phosphorylation ✔✔1) Photophosphorylation: Light energy is used to power the formation of ATP from ADP.
- Substrate-level phosphorylation: The phosphoryl (PO3) group of a chemical compound is transferred and donated (added) directly to ADP. The chemical compound losing the phosphate group is referred to as the phosphorylated reactive intermediate.
- Oxidative phosphorylation: used by chemotrophs. The energy released by the chemical oxidation of nutrients is used to reform ATP. This process occurs in the mitochondria of eukaryotic cells and is highly efficient.
- The transfer of these electrons will fuel the generation of ATP via the electron transport system. Electron transport system/chain ✔✔1. Either aerobical or anaerobical
- Result in the production of up to 34 molecules of ATP
- Electrons transferred from NADH/FADH2 to terminal electron acceptors (O2) - Energy is released and captured by electron acceptor proteins - Electrons are passed down a chain of electron acceptors - protons (H+) to be pumped out of the membrane - a strong differential across the membrane - forms the proton motive force that drives H+ back through the ATP synthase complex - production of up to 34 molecules of ATP Non-Glucose Alternatives ✔✔1. Bacteria may use alternative sugars, such as fructose (in fruits) and lactose (in milk)
- These microorganisms simply insert additional enzymatic steps at the beginning of its catabolism to convert these complex sugars into the usable forms of either glucose or a glucose intermediate (i.e. glucose- 6 - phosphate)
- Polysaccharides, proteins (amino acids), and lipids can also be used as sources of energy Proteases/proteinase ✔✔Enzymes that break down proteins into smaller subunits or individual amino acids Lipases ✔✔1. Catalyzes the breakdown of lipids (fats)
- Separate the fatty acid chains from the glycerol backbone. The backbone is processed for carbon and energy, whereas the fatty acid chain is degraded via the β-oxidation pathway into acetyl-CoA, which feeds directly into the Krebs cycle Chloroplasts ✔✔Double membrane-enclosed organelles specific to algae and plants and house the photosynthetic pigment chlorophyll Photophosphorylation/light reaction ✔✔1. Converts light energy into chemical energy to be used by the cell in the forms of ATP and NADPH.
- Light is absolutely required.
- Always occur in the membrane to generate a proton concentration gradient to generate ATP The Calvin cycle/dark reaction ✔✔1. The ability of a microorganism to use the ATP and NADPH generated in the light reaction to convert CO2 and H2O into organic carbon compounds and useful carbohydrates (carbon fixation)
- Light is no longer required. Nor does darkness.
- It requires 6 turns/repetitions to generate one molecule of glucose
- Formula: 6 CO2 + 18 ATP + 12 NADPH + 12 H20 → C6H12O6 + 18 ADP + 12 NADP+ Catalase ✔✔Accelerates the decomposition of hydrogen peroxide (H2O2) into water and oxygen Hydrolases ✔✔Catalyze hydrolysis—the cleavage or breaking apart of chemical bonds by the addition of water. (i.e. A-B + H2O → A-OH + B-H)
Units of measurement for microbial world ✔✔1. The micrometer (μm) is one-millionth of a meter and is commonly designated at 10^- 6 m.
- The nanometer (nm) equals 10^-9 m or one-billionth of a meter.
- For perspective, the unaided eye can resolve (see clearly) objects typically > 100 μm. Resolution and contrast ✔✔1. The resolution refers to the distance between two objects at which the objects still can be seen as separate.
- The contrast is the difference in light absorbance between two areas (objects). Brightfield microscopes (halogen bulb) (0.2 μM) ✔✔The light first passes through a condenser converging the light beams into a focused area on the sample. The iris diaphragm controls the amount of light that passes through the sample and into the objective lens. To image samples clearly at higher magnifications, more light is required. Then the light is directed through the ocular lens/eyepiece to your eye. The total magnification ✔✔For a microscope using two lenses (objective and ocular) the total magnification of a specimen is multiplicative. Thus, a 40x objective and a 10x ocular result in a total magnification of 400x. Phase contrast microscopes (0.2 μM) ✔✔1. By using specialized condensers and objectives, a phase contrast microscope amplifies the slight differences between cells and the surrounding medium (background) to make the cells highly distinguishable.
- Can provide detailed images of live cells without staining and visualize cell movements (motility), such as swimming or gliding. Dark field microscopes ✔✔1. Greatly increase the contrast between a specimen and background, resulting in a dark background with bright objects in it.
- Reflects light off of the specimen at an angle. This reflective approach does not permit the visualization of intracellular structures. Fluorescence microscopes (UV light) (0.2 μM) ✔✔1. Take advantage of fluorescent molecules called fluorophores to visualize cells on a dark background.
- The energy of the incoming light is in the form of the ultraviolet (UV) spectrum.
- UV light excites different fluorophores at varying wavelengths, enabling scientists to use a wide array of colors during imaging like the green, yellow, and red fluorescent proteins (GFP, YFP, and RFP). Confocal (laser scanning) microscopes (0.2 μM) ✔✔1. Combine the usefulness of fluorescence microscopy with the ability to visualize cells in 3 - D (or 2 - D).
- Use lasers to focus on a single plane within an object and with a higher degree of accuracy, rendering a 3 - D reconstruction of the sample. Electron microscope (TEM and SEM) (<1 nm) ✔✔1. Use beams of electrons, which have significantly shorter wavelengths than light, to increases its resolution capacity to less than 1nm—that's 200x better!
Gram staining ✔✔1. Different types of bacteria react differently to various dyes.
- This differentiation, based on color, divided bacteria into two categories: Gram-positive or Gram-negative. Gram-positive bacteria ✔✔1. Have a thick cell wall with many overlapping strands of peptidoglycan.
- Appear purple by using the dye combinations of crystal violet and iodine. Gram-negative bacteria ✔✔1. Have a relatively thin peptidoglycan layer followed by an outer membrane composed of lipopolysaccharides (LPS).
- Retain a pink color after decolorization wash and a secondary (counterstain) dye called safranin is added. Differential staining ✔✔1. A generalized term used for any staining technique that separates specimens into further subgroups. This process most often utilizes at least two dyes.
- Includes gram stain, acid-fast staining, and giemsa. Heat fixation ✔✔Samples are added to a glass slide and then passed through a flame until all liquid in the sample has been removed. Chemical fixation ✔✔Include the use of paraformaldehyde, ethanol, or methanol.
Wet mount ✔✔1. A small liquid culture (usually just a drop) containing a microorganism of interest is prepared, added to a slide, and then covered with a glass coverslip.
- To observe the motility and behavior of an organism Simple staining ✔✔1. Uses a solution of a positively charged dye, such as methylene blue, crystal violet, safranin, or fuchsin, to bind to and stain the negatively charged membrane of the microorganism.
- To quickly observe the size, shape, and arrangement of cells Negative staining ✔✔1. By applying the dark stain nigrosin (or India ink) to a sample, its negative charge is repelled by the negatively charged membrane, resulting in a sharp contrast between the unstained specimen and the now dark background.
- Only mildly invasive and may not kill the microorganism and is contraindicated for pathogenic samples. Acid-fast staining/Ziehl-Neelsen stain ✔✔1. To identify bacterial stains showing a high degree of resistance to decolorization
- Mycobacterium tuberculosis is the most common use for an acid-fast stain as it is the causative agent of tuberculosis
- Red dye carbolfuchsin - decolorization wash step (acid-fast cells remain red) - counterstained with methylene blue (stain non-acid-fast bacterium)
- Vital in the diagnosis of tuberculosis and is most often performed on sputum samples
