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Microbiology 1&2
| Question | Answer |
|---|---|
| Microbiology | a specialized area of biology that studies living things ordinarily too small to be seen without magnification |
| Microorganisms include | Bacteria Archaea Protozoa Fungi Viruses Prions Helminths Algae |
| Acellular microorganisms | Viruses and Prions |
| Cellular Microorganisms | Bacteria, Archaea, and Eukaryote |
| differences between Prokaryotes and Eukaryotes | Prokaryotes lack a membrane enclosed nucleus and do not have organelles within cell. |
| What is a cell? | A cell is an individual membrane-bound living entity capable independent existence. A cell is the basic fundamental unit of life. |
| Common features of all cells | overall shape, protoplasm/cytoplasm enclosed in membrane, chromosomal contents (DNA, RNA), ribosomes, and proteins |
| Cellular microbes | Fungi, Protists, Bacteria, and Archaea |
| Acellular microbes | Viruses, Viroids, Satellites, and Prions |
| How long have microbes been on earth? | 3.5 billion years old |
| Microbes are found: | Deep in the earth’s crust, In geothermal vents (Ex. Deferribacter desulfuricans), In polar ice caps (Ex. Polaribacter) and oceans, Inside/outside plants and animals, In/on the earth’s landscape, and In the clouds |
| Phylogeny | the taxonomic scheme that represents the natural relatedness between groups of living beings |
| Taxonomy | the science of classifying of living things |
| Areas that affect classification | Physiology/ Biochemistry, Electron microscopy, Molecular Biology |
| Woese’s comparison | small subunit ribosomal RNA sequences (ssu 16S rRNA), divides microorganisms into: Bacteria (true bacteria), Archaea, and Eukarya (eukaryotes) |
| Domain Bacteria | Usually single-celled, Cell wall with peptidoglycan, Most lack membrane-bound nucleus, Ubiquitous, Microbiota (human microbiome/nature). |
| Domain Archaea | Distinguished from Bacteria by unique rRNA sequences, Lack peptidoglycan in cell walls, Some have unusual metabolic characteristics, and Many live in extreme environments (thermophiles, halophiles). |
| Domain Eukarya | Mostly plants, animals—but also microbes such as protists, fungi, look at power point for more details. |
| Nomenclature | the assignment of scientific names to the various taxonomic categories and to individual organisms |
| Classification | the orderly arrangement of organisms into a hierarchy |
| Who created taxonomy? | System developed by Carolus Linnaeus |
| ranks of taxonomy | • Domain • Kingdom • Phylum or division • Class • Order • Family • Genus • Species |
| Binomial system of nomenclature: | Scientific name is a combination of the genus and species names with the Genus capitalized and species name lower case. If typed it is italicized and if in print is underlined. |
| Spontaneous Generation | Since the time of Aristotle (4th century B.C.) people believed that living organisms were generated from non-living matter. |
| what did Louis Pasteu do? | Swan-necked flask experiments used to disprove spontaneous generation. |
| earliest microscopic observation | it was of bees/weevils by Stelluti (using scope from Galileo) |
| Robert Hook | published drawings of fungus, Mucor, in the scientific book Micrographia –the first drawings of microbes published |
| Leeuwenhoek | a clothier by trade, became the first microscopist, building simple scopes, could magnify 50-300X, saw bacteria & protists in a drop of water & from tooth scrapings (‘animalcules’) |
| Germ Theory | microscopic organisms—pathogens like bacteria, viruses, fungi, and protists—are the root cause of many diseases, invading hosts from the outside |
| pasteurization | invented by Louis Pasteur, heats liquids to kill pathogens, not all microbes, preserving taste and nutrition. |
| Koch’s postulates | series of logical steps that establish whether or not an organism is pathogenic and which disease it caused. Showed that anthrax was caused by Bacillus anthracis in 1875, then proved tuberculosis caused by M. tuberculosis |
| Macromolecules | Very large • Four main types: • Carbohydrates • Lipids • Proteins • Nucleic acids |
| Monomers | subunits of macromolecules |
| Polymers | chains of various lengths of monomers |
| Cell Membrane Components | Cell membranes contain fatty acids, cholesterol, DNA, and RNA |
| Carbohydrates: CHO Structure | Carbohydrates have general formula C_x(H_2O)_y with glucose as an example |
| Disaccharides and Polysaccharides | Lactose, sucrose and polysaccharides like starch (C_6H_10 O_5)_n and chitin are key carbohydrates |
| Cellulose and Agar Polysaccharides | Agar (C_12H_18O_9n) from seaweed is vital for microbial culturing |
| Peptidoglycan and Microbial Walls | Peptidoglycan amount determines gram {Gram}+ lacks lipopolysaccharides, {Gram}- contains. component of bacterial cell wall. |
| Chitin | cell wall found in fungi |
| Lipopolysaccharide | component of gram-negative cell wall |
| Glycocalyx | protective outer layer; role in attachment of cells to other cells/ surfaces |
| Lipids and Triglycerides | Lipids are nonpolar, energy-storing molecules, insoluble in water but soluble in nonpolar solvents |
| Phospholipid Bilayer Structure | Phospholipid bilayer arranges tail-to-tail as $2$ layers with polar heads outward |
| Cell Membrane Structure | Lipid bilayer forms with hydrophilic heads out, hydrophobic tails in: phospholipid bilayer |
| Amino Acids in Proteins | Proteins consist of sequences of up to 20 amino acids; polypeptides typically greater than equal to (<_)50 amino acids |
| Protein Structure Levels | Protein structure evolves: primary (amino acid sequence), secondary (alpha-helix, beta-sheet), tertiary (3D fold), quaternary (polypeptides) |
| Protein Structural Hierarchy | Amino acid sequence forms primary, secondary, tertiary, and quaternary structures: {primary}-> {secondary}-> {tertiary} ->{quaternary} |
| Protein Structure and Function | A protein's function depends on its unique amino acid sequence and three-dimensional folding |
| Denaturation and Enzyme Activity | Denaturing disrupts protein structure so enzyme function ceases above 80 degrees Celsius |
| Enzyme and Antibody pH Sensitivity | Enzyme and antibody function depends on optimal pH, usually around pH = 7 |
| Nucleic Acids Structure | Nucleic acids are polymers of nucleotides: phosphate, sugar, and base (A, G, C, T, U) |
| DNA Structure and Base Pairing | DNA strands form base pairs: A with T,C with G |
| DNA Helix and Supercoiling | DNA forms a helix, then supercoils for compact storage: Helix->Supercoiling |
| Microscopes | use magnification, resolution, contrast to get a clear image of the specimen. They are a collection of lenses. |
| objective lens | found closest to specimen |
| ocular lens | those with which specimen viewed |
| Parfocal microscopes | remain focused when changing from one objective lens to another. |
| Microscopy in Microbiology | Studying microorganisms requires magnification, typically with microscopes(e.g., 10x, 100x) |
| Resolution | ability of lens to separate/distinguish between small objects close together |
| Magnification | of an image increases its size, but it may be unclear |
| Resolving power of the human eye | 0.2 mm |
| Resolving power of a light microscope using the oil immersion lens | 0.2 μm |
| what do Lenses in a microscope do? | create images by bending light |
| Refractive Index | measures deflection oflight ray from a straight path as it travels from one medium (ie air) to another (ie glass). |
| Refractive index gives a high degree of contrast, which is? | the ability to distinguish magnified image from its surroundings. |
| Bright-field Microscopy | Illumination by visible light transmitted through specimen, Background light, object dark, Sample can be live, preserved, stained, unstained |
| Dark-field Microscopy | by visible light manipulated with stop disc placed on condenser, Light not sent directly through specimen, but is blocked except that which is reflected off the sides of specimen, Dark background with a light specimen, Useful in live, unstained specimen. |
| Phase Contrast Microscopy | illumination with visible light, Deviation is converted into changes in density by use of a condenser annulus & phase plate that transform subtle changes in light waves into differences in light intensity. Observed as differences in image contrast. |
| Fluorescence Microscopy Basics | UV-excited fluorochromes reveal cellular details in dark backgrounds: emission --> fluorescence |
| Fluorescent Stains in Microscopy | Different fluorochromes stain DNA or antibodies, visualized under specific wavelengths lambda. |
| Confocal and Electron Microscopy | Electron microscopes use electrons for imaging; resolution exceeds light microscopes by 1000x |
| Electron Microscopy Principles | Electron dense areas appear darker, scattering more electrons; vacuum needed for electron travel |
| Microscopy Specimen Preparation | Embedding and thin sectioning specimens is essential for electron microscopy imaging |
| Electron vs Atomic Force Microscopy | Atomic force microscopy allows imaging of non-conductive samples; electron microscopy requires conductivity |
| Scanning Tunneling Microscopy | STM achieves up to 100,000,000x magnification; enables imaging DNA and surfaces underwater |
| Atomic Force Microscopy | Atomic force microscope enables surface imaging without electrical conduction using a fixed probe |
| Microscope Specimen Preparation | Staining and mounting enhance visualization of microscopic samples |
| Sample Fixation Techniques | Fixation secures samples to slides via heat or chemicals, preserving morphologies and structures |
| Heat Fixation Technique | Passing slide over flame fixes sample; overexposure risks damage |
| Chemical Fixation Methods | Fixation uses chemicals like glutaraldehyde or ethanol for gentle preservation of structures |
| Ionic and Covalent Staining | Acidic dyes (- charge)bind positives, basic dyes (+ charge)bind negatives |
| Positive vs Negative Staining | Positive stains color cells, negative stains color background; charge interactions determine adherence |
| Negative Staining Technique | Negative stains like nigrosin highlight cell capsules for identification of pathogenic cells |
| Simple Stain Technique | A simple stain uses one dye for fast, single-step cell visualization |
| Identifying Cell Morphology | Shape, arrangement, and Gram stain help identify microorganisms; Gram stain distinguishes cell walls |
| Gram Stain Procedure | Crystal violet binds gram positive cells; decolorizer differentiates {Gram}+ vs {Gram}- cells |
| Counterstain in Gram Staining | After alcohol, counterstain colors all cells; those not violet become red-pink (safranin) |
| Gram Stain Mechanism | Cell wall peptidoglycan thickness affects Gram stain: {thick} ->{purple},{thin/ outer membrane} ->{pink} |
| Endospores and Pathogenicity | Bacterial endospores form under stress; some like Clostridium and Bacillus are pathogenic |
| Endospore Staining Techniques | Endospore stains require heat to force dye into spores: heat enables dye penetration |
| Spore Formation and Survival | Spores resist destruction more than vegetative cells; require stronger measures to kill {spores} |
| Capsule Staining Technique | Negative staining reveals capsules as halos; important in identifying pathogenic organisms. |
| Flagella Staining Techniques | Flagella stains help visualize $\text{flagella}$ for microbial identification |
| Microorganism Culturing Methods | To identify bacteria, we must culture and isolate them from samples |
| Identifying Anaerobic Infections | Anaerobic organisms grow without oxygen: O_2 exposure kills them |
| Five I's of Microbiology | Inoculation, incubation, isolation, inspection, and identification help identify organisms |
| Microbial Inoculation Process | Inoculation introduces an inoculum into sterile growth media for microbial culturing |
| Inoculation and Incubation Steps | After inoculation, incubate cultures at controlled conditions for microbial growth {growth} = f(temperature}, {oxygen}, {pressure}) |
| Bacterial Growth and Culture | Incubation time varies; E. coli replicates every 20 min, Mycobacterium takes weeks |
| Types of Culture Media | Media supports growth and transport of microorganisms in labs |
| Transport Media Function | Transport media preserves sample; prevents culture overgrowth before clinical analysis |
| Broth and Agar Media | Liquid media is called broth; solidified using agar as a gelling agent |
| Agar as Growth Medium | Agar resists degradation and supports diverse microbial cultures at various temperatures |
| Microbial Culture Media | Microbes are cultured using liquid broths or solid $\text{agar}$ media |
| Defined vs Complex Media | Defined media have known compositions; complex media have unknown exact amounts of components |
| Complex Versus Defined Media | Complex media like nutrient agar have undefined compositions, unlike defined media where ingredients are known |
| Types of Growth Media | Enriched, selective, and nutrient agar differ by added components and growth support |
| Selective Media Overview | MacConkey agar detects gram-negative bacteria using selective biochemical reactions (e.g., E. coli) |
| Selective and Differential Media | Media like MacConkey and blood agar differentiate organisms using agents and metabolic products MacConkey agar: bile, crystal violet; blood agar: hemolysis |
| Hemolysis on Blood Agar | Alpha, beta, and gamma hemolysis distinguish organisms by red blood cell lysis patterns |
| Differential and Selective Media | Media type distinguishes organisms based on metabolic traits, e.g., sugar use, hemolysis, motility |
| Transport Media and Isolation | Transport media preserve samples; isolation is needed to identify organisms accurately |
| Bacterial Colony Formation | A colony =visible cluster from one microorganism; formed after binary fission growth |
| Isolation Streak Plate | Quadrant streaking isolates colonies for pure culture identification |
| Spread and Pour Plate Methods | Spread plate evenly distributes inoculum; pour plate mixes inoculum with 45°C agar |
| Serial Dilutions in Plating | Serial dilution required to quantify high bacterial concentration N |
| Colony Counting Range | Countable colonies per plate: 30 greater than equal to N greater than equal to 200 for accurate counts |
| Serial Dilution Process | Each dilution step reduces concentration by 10^-1, resulting in 10^-4 after four steps |
| Calculating Original Cell Count | Original count is 127 times 10^3 cells from 10^-3 dilution plate |
| Microbial Colony Analysis | Dilution and plating yield countable colony numbers: N = DF times C |
| Microbe Identification Methods | Biochemical and genetic tests identify microbes via DNA sequence: {DNA sequence} = {microbial identity} |
| Fish Species Identification Project | Genetic analysis can reveal mislabeling and contamination in fish samples, e.g., pseudomonas |
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