Chapter 3 Microscopy PDF

2024-09-11 Chapter 3 Microscopy 1 Light microscope – Any kind of microscope that uses visible light to observe a specimen Compound light microscope – uses two lenses to magnify the image 1) Ocular le...

2024-09-11 Chapter 3 Microscopy 1 Light microscope – Any kind of microscope that uses visible light to observe a specimen Compound light microscope – uses two lenses to magnify the image 1) Ocular lens (the eyepiece): Magnifies by 10x. 2) Objective lens: Lens closest to the specimen Magnifies between 10x - 100x 2 Calculating magnification For a compound microscope: Total magnification = Ocular lens x Objective lens Resolution Ability to distinguish fine detail and structure Ability to distinguish 2 points a certain distance apart ex. Resolving power of 4 nm Two points can be distinguished if they are at least 4 nm apart. https://www.youtube.com/watch?v=Y6e_m9iq-4Q https://www.nei.nih.gov/learn-about-eye-health/eye-conditions-and-diseases/floaters https://microscopewiki.com/microscope-slides/ 3 1  2024-09-11 Light must pass between 2 objects for them to be seen as 2 separate things Need light of a short-enough wavelength to fit between them, otherwise will appear as 1 object Resolution general principle: the shorter the wavelength, the better the resolution. https://www.quora.com/Why-is-a-shorter-wavelength-of-light-needed-to-observe-small-particles-or-organisms-that-are-not- visible-to-the-naked-eye 4 Electron Microscope Uses electrons instead of light Electrons travel in much shorter waves than light Resolving power is greater Allows greater magnification (up to 500 000 x) Allows us to view viruses and internal cell structures. https://www.nagwa.com/en/videos/725154569076/ https://www.globalsino.com/EM/page4787.html https://www.nanoscience.com/techniques/scanning-electron- microscopy/ 5 Two types of Electron microscope Transmission – (TEM) to see internal structures - Very thin slices can be cut from sample – thin sections - Samples generally stained with a metal (ex. Osmium, uranium) to make structures opaque to electrons Scanning – (SEM) to see surfaces; less powerful Atomic force – (AFM) to see molecules Uses thin metal probe to scan a specimen revealing bumps and depressions. https://www.differencebetween.com/difference-between-sem-and-tem/ 6 2  2024-09-11 Human eye can see an object about 0.2 mm Compound light microscope can view an object about 0.2 µ m Electron microscope can view an object about 2 nm. https://www.npr.org/2022/06/23/1107012619/largest-bacteria-ever-discovered-thiomargarita-magnifica 7 Clinical use of the light microscope Since microorganisms are colorless when seen through a microscope, we must prepare them for viewing using stains Stains are composed of positively and negatively charged ions, one of which is colored – chromophore Simple Stain - Only one dye used to highlight the entire microorganism Following these four simple steps: – Smear sample on slide – Fix with heat – Add stain – Wash, dry and view. 8 How stains work Bacteria have a net negative charge on their outer surface This charge attracts stains with positively charged chromophores, and repels stains with negatively charged chromophores Positive stains Stain will bind to the bacterium Bacterium appears colored Background appears clear Ex. Crystal violet Negative stains Will not bind to the bacterium Bacterium appears clear Background is colored Ex. Nigrosin, India ink 9 3  2024-09-11 Differential Stains React differently with different bacteria, thus can be used to distinguish between them Ex. Gram stain Differentiates bacteria based on the structure of the cell wall. https://www.thoughtco.com/gram-stain-procedure-4147683 10 The Gram stain Bacteria with a thick cell wall retain the primary stain crystal violet and appear purple – Gram positive Ex. Streptococcus pyogenes Bacteria with a thin cell wall lose crystal violet during destaining, take on the color of the counterstain safranin and appear pink – Gram negative Ex. E. coli. 11 Spore stain Stains an internal structure of some bacteria Primary stain colors endospores green Counterstain (safranin) colors the rest of cell red (pink) ex. Bacillus anthracis Flagella stain Stains an external structure Mordant thickens the flagella so they can be observed under light microscope. https://pubmed.ncbi.nlm.nih.gov/19885934/ 12 4  2024-09-11 Other differential stains Acid-fast stain* Detects the presence of a waxy compound in cell wall Used to identify the genus Mycobacterium ex. Mycobacterium tuberculosis Mycobacterium leprae Mycobacterium cell wall retains the dye carbol fuchsin Counterstain with methylene blue stains non acid-fast bacteria and tissues blue. * https://www.sciencedirect.com/topics/medicine-and-dentistry/acid-fast 13 Capsule stain Detects a thick layer of polysaccharide outside the cell – capsule a negative stain colors the background a positive stain colors the cell the capsule does not take up most dyes and remains colorless Ex. Streptococcus pneumoniae 14 Prokaryote – Pro (before), Karyon (nucleus) DNA is not enclosed within a nucleus Usually DNA is arranged as one circular chromosome They lack membrane-enclosed organelles Single celled organisms: Bacteria, Archaea* Eukaryotes – Eu (true), Karyon (nucleus) DNA is found in the nucleus: surrounded by a nuclear membrane DNA arranged as multiple chromosomes They have membrane-enclosed organelles Single celled or multicellular organisms ex. Algae, Protozoa, Fungi, Plants, Animals. * https://microbiologysociety.org/why-microbiology-matters/what-is-microbiology/archaea.html https://www.youtube.com/watch?v=73c1RIqi0uw 15 5  2024-09-11 16 The Bacteria Morphology (shape) Coccus (pl. Cocci) – Spherical Bacillus (pl. Bacilli) – Rod shaped Vibrio – curved Spirillum (pl. Spirilla) – Spiral shaped Spirochete – Corkscrew shaped. 17 Bacterial cell structure: 18 6  2024-09-11 External structures Capsules and slime layers Sticky, gelatinous layer external to the cell Composed of polysaccharide, protein or both If the layer is organized and firmly attached to the cell wall it is known as a capsule If the layer is unorganized and loosely attached to the cell wall it is known as a slime layer. https://www.sciencedirect.com/topics/immunology-and-microbiology/slime-layer 19 In some bacteria capsules a play a role in virulence Protection against phagocytosis ex. Streptococcus pneumoniae* With a capsule: causes disease Without capsule no disease * https://academic.oup.com/cid/article/46/7/1038/290579 Slime layers often allow bacteria to attach to surfaces Medical implants, water pipes, teeth ex. Streptococcus mutans Makes polysaccharide slime from sucrose Attaches to teeth, which can lead to cavities. 20 Flagella (sing. flagellum, “whip”) Long protein appendages Used in motility Semi-rigid, helical, turns like a propeller Bacterial cells have four typical arrangements of flagella: Monotrichous = a single polar flagellum Lophotrichous = two or more flagella originating from one pole Amphitrichous = tufts of flagella originating from opposite poles Peritrichous = flagella distributed all over the cell. 21 7  2024-09-11 Flagellar motility Flagella turn causing cell to move in one direction – “run” Periodically flagella reverse direction causing a random change in direction – “tumble” Flagella allow chemotaxis movement toward or away from a stimulant toward a nutrient (attractant) away from a toxin (repellent)* ex. E. coli will move toward glucose Flagellar protein can be used to distinguish among strains of a species ex. E. coli O157:H7 ** * https://www.ncbi.nlm.nih.gov/pmc/articles/PMC134409/ ** https://academic.oup.com/af/article/6/2/37/4638727 22 Fimbriae and Pili Short, hair-like appendages Hollow Fimbriae allow the cell to adhere to surfaces contribute to pathogenicity Ex. some strains of E. coli have fimbriae that allow them to attach to the intestinal wall Pili allows attachment of two bacteria to each other involved in transfer of genetic material between bacteria Ex. E. coli’s sex pilus. 23 https://en.wikipedia.org/wiki/Pilus 24 8  2024-09-11 Bacterial Cell Wall Semi-rigid structure giving shape to the cell Major function is to prevent rupture of the cell – protects against environmental changes Useful in the identification of bacteria – ie. The Gram stain Composed of the complex macromolecule: PeptidoGlycan Mesh-like structure composed of polysaccharide and amino acids Polysaccharide portion is composed of two alternating monosaccharides: N-acetyl glucosamine (NAG) N-acetyl muramic acid (NAM) Peptide portion composed of short chains of amino acids. 25 A generalized view of peptidoglycan Polysaccharide chains run parallel Peptide chains link polysaccharides together Forms a mesh-like net surrounding the cell. 26 The Gram positive cell wall Made of thick layers of peptidoglycan outside of plasma membrane Also contains teichoic acids Wall teichoic acids: attached to the peptidoglycan Lipoteichoic acids: attached to plasma membrane and extend through the peptidoglycan Gram positive bacteria have only one membrane = cytoplasmic membrane. 27 9  2024-09-11 The Gram negative cell wall Thin peptidoglycan layer that is sandwiched between two membranes Outer membrane made of lipids (phospholipids), proteins and lipopolysaccharides (LPS) Polysaccharide portion of LPS is composed of O-sugars Useful for distinguishing Gram negative bacteria ex. E. coli O157:H7 Lipid portion of LPS is toxic Referred to as endotoxin. https://www.mdpi.com/1422-0067/21/2/379/htm 28 How the Gram stain works Gram positive cells Thick peptidoglycan traps the crystal violet – stain purple Gram negative cells Thin peptidoglycan does not trap crystal violet, and the outer membrane gets disrupted by alcohol Crystal violet is washed away Safranin counterstain stains the cells pink. 29 What’s so special about peptidoglycan? Completely different from anything found in animal cells Many antibiotics have been discovered that act against peptidoglycan ex. Penicillin – inhibits production of peptidoglycan Also degraded by one of our own natural defenses: lysozyme found in tears, saliva, mucous. 30 10  2024-09-11 The cytoplasmic membrane (Plasma or cell membrane) Composed of a phospholipid bilayer Separates the interior (cytoplasm) from the outside environment Serves as a semi- permeable barrier Selectively allows inflow and outflow of materials Exists in a semi-fluid state Antimicrobial agents Alcohols disrupt the membrane can be used as a disinfectant. https://origin.bg/2020/04/16/why-is-70-the-most-effective- concentration-of-denatured-ethanol-for-disinfection/ 31 Internal components Cytoplasm The substance inside the plasma membrane About 80% water Contains most of the ‘stuff’ needed for life sugars, amino acids, nucleotides etc enzymes some functional structures. 32 The Nucleoid Contains the bacterial chromosome (DNA) All genetic information required for cell’s structures and functions Not surrounded by a nuclear membrane May also contain plasmids: Smaller double stranded DNA molecules Contain non-essential genes ex. Genes for antibiotic resistance. 33 11  2024-09-11 Ribosomes Site of protein synthesis (Translation) Made of protein and ribosomal RNA (rRNA) Two parts: 30S subunit 50S subunit Together form the complete 70S ribosome Ribosomes of bacteria differ from Eukaryotic ribosomes Eukaryotes have 80S ribosomes Several antibiotics target bacterial ribosomes ex. Streptomycin, Erythromycin Prevent the bacteria from making new proteins. 34 Storage granules (inclusion bodies) Usually deposits or granules of nutrients, stored for later use Examples: Sulfur granules Polysaccharides (glycogen) Lipid inclusions Enzymes Magnetite Variety of inclusion bodies occur in different bacterial species - can serve as a basis for identification. 35 Endospores Formed only by some Gram positive bacteria Special resting structure – allows bacteria to enter dormant state Extremely durable Resists heat, desiccation, chemicals, radiation, etc Some endospores can survive in boiling water for hours Remains dormant until good growth conditions occur Can form a new population Examples: Bacillus anthracis – causes anthrax Clostridium botulinum – causes the food borne illness botulism. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3772150/ 36 12  2024-09-11 Sporulation 1. Cell replicates its DNA 2. Septum forms, dividing the cell into unequal compartments 3. Larger compartment engulfs the smaller. 4. Peptidoglycan and other protective material forms around the forespore Spore coat 5. Finished spore is freed from the mother cell as the mother cell dies. 37 Eukaryotic cell structure Includes microorganisms: Algae, fungi, protozoa Higher organisms: Plants and animals Larger and more complex than prokaryotes Genetic material is housed in a nucleus Membrane bound organelles. 38 Cytoplasmic membrane Same basic structure as in prokaryotic cells Contains phospholipids, proteins, and sterols Sterols make membrane relatively rigid compared to bacteria Cell wall Not all eukaryotes have a cell wall Allows endocytosis* Simple structure compared to bacteria Made of: Cellulose (algae, plants) Chitin (fungi). https://www.cell.com/trends/plant-science/fulltext/S1360-1385(15)00081-3 https://doi.org/10.1016/j.cub.2007.01.045 39 13  2024-09-11 Cytoplasm Substance inside the plasma membrane, but outside the nuclear membrane Cytoplasm has complex internal structure – cytoskeleton Protein filaments on the inside of the plasma membrane Provides support and shape Transports substances through the cell Ribosomes Larger and heavier than bacterial ribosomes 80S. 40 41 Membrane bound organelles Structures with specialized functions Not present in bacteria Examples: 1. Nucleus – compartment holding genetic material 2. Mitochondria – site of energy production 3. Chloroplasts – site of photosynthesis in algae and plant cells. 42 14  2024-09-11 Appendages external to the cell Flagellum and cilia Long flexible projections that contain protein and cytoplasm Move in a whip-like fashion Can be used for: Motility Sweeping material past stationary cells. 43 44 15