Cells and Methods to Observe: Chapter 3 - PDF

Cells and Methods to observe them Chapter 3 In this chapter we will study Prokaryotic cell structures Eukaryotic cell structures Similarities between prokaryotic and eukaryotic cells Differences between prokaryotic and eukaryotic cells Microscopy principles Preparation of sp...

Cells and Methods to observe them Chapter 3 In this chapter we will study Prokaryotic cell structures Eukaryotic cell structures Similarities between prokaryotic and eukaryotic cells Differences between prokaryotic and eukaryotic cells Microscopy principles Preparation of specimens Common simple and differential staining methods Prokaryotic and Eukaryotic cells- similarities Made up of cells Cell membrane, Cytoplasm, DNA, RNA Organization Population, community, ecosystem, biosphere Life processes Reproduction, growth & development, metabolism, communication, response to stimuli, adaptation Some pathogens Prokaryotic vs. Eukaryotic cells Prokaryotic cells Eukaryotic cells Members of Kingdoms- Members of Kingdoms- Bacteria, Archaea Protista, Fungi, Plantae, Animalia Size- smaller Size- larger No nucleus or membrane Nucleus and membrane bound organelles bound organelles present Unicellular Unicellular & multicellular Single circular Multiple linear Chromosome chromosomes Ribosome- 70S Ribosome- 80S Prokaryotic Cell structures – Figure 3.1 Imagine Outer components of bacterial cells Cell membrane Outside cell membrane Cell wall Glycocalyx Slime layer Capsule Adhesion- pili, fimbriae Movement- flagella, pili Archaea- Cannulae, hami (singular- hamus) 3.1- Cytoplasmic membrane of prokaryotes Describe the structure and chemistry of the cytoplasmic membrane, focusing on how it relates to membrane permeability. Describe how the cytoplasmic membrane is involved with proton motive force. Describe the systems prokaryotic cells use to move small molecules across the cytoplasmic membrane. Explain why prokaryotic cells must secrete certain proteins. Cytoplasmic membrane Cytoplasmic membrane defines boundary of cell Phospholipid bilayer embedded with proteins Hydrophobic tails face in; hydrophilic tails face out Proteins serve numerous functions Selective gates Sensors of environmental conditions Enzymes Fluid mosaic model: proteins drift about in lipid bilayer Plasma membra ne  The Cytoplasmic Membrane of Bacteria and Archaea Have same general structure Distinctly different phospholipid compositions Lipid tails of Archaea are not fatty acids (are isoprenoids) Connected differently to glycerol Ether linkage to glycerol instead of ester linkageNot important structure and compositional diversity ensures the required stability at extreme environmental conditions including extreme temperatures, high salinity and extreme pH values Permeability of Cytoplasmic Membrane – Figure 3.3 Cytoplasmic membrane is selectively permeable O2, CO2, N2, small hydrophobic molecules, and water pass freely Some cells facilitate water passage with aquaporins Other molecules must be moved across membrane via transport systems Transport across the membrane Passive transport Active transport Down the concentration Against the concentration gradient gradient Energy provided by the Energy provided by ATP, concentration gradient not the concentration Transport protein not gradient required of most Transport protein required Examples- simple diffusion, osmosis, facilitated diffusion (transport protein required)  Permeability of Cytoplasmic Membrane- Diffusion Simple Diffusion Movement of molecules from high to low concentration until equilibrium is reached The rate of diffusion is affected by Concentration gradient The greater the difference in concentration on either side of a membrane, the higher the rate of diffusion Temperature Lower temperature, slower movement making the diffusion slower Mass of the molecules Heavier molecules move slower reducing the rate of diffusion  Permeability of Cytoplasmic Membrane- Osmosis Diffusion of water across a selectively permeable membrane due to unequal solute concentrations Water diffuses from high water concentration (low solute concentration) to low water concentration (high solute concentration) Water flows from hypotonic to hypertonic solution No net water flow between isotonic solutions  Permeability of Cytoplasmic Membrane- Osmosis- Tonicity Environment of prokaryotes are typically dilute (hypotonic) relative to cytoplasm Water flows into the cell where cytoplasm is a concentrated solution (hypertonic) Cell wall prevents cell from bursting Tonicity High salt/sugar is used in food to prevent growth of bacteria Transport of Small Molecules Across Cytoplasmic Membrane – Figure 3.6 Cells use transport systems to move nutrients and other small molecules across the cytoplasmic membrane Transporters, permeases or carriers Membrane-spanning Highly specific: a single transporter generally moves only one molecule type Facilitated Diffusion - Figure 3.7 A form of passive transport Movement down gradient; no energy required Not typically useful in low-nutrient environments Channel proteins Active Transport - Figure 3.7 Requires energy Moves material against concentration gradient Sometimes driven by proton motive force Example is efflux pump Sometimes driven by ATP (ABC transporter) Protein Secretion – Figure 3.8 Active movement of proteins out of cell Examples: exoenzymes (extracellular enzymes), external structures Polypeptides tagged for secretion via signal sequence of amino acids 3.2- The Cell Wall of Prokaryotic Cells Describe the chemistry and structure of peptidoglycan. Compare and contrast the structure and chemistry of the Gram-positive and Gram-negative cell walls. Explain the significance of lipid A and the O antigen of LPS. Explain how the cell wall affects susceptibility to penicillin and lysozyme. Explain how the cell wall affects Gram staining characteristics. Describe the cell walls of archaea. The Cell Wall of Prokaryotic Cells Cell wall is strong, somewhat rigid structure that prevents cell from bursting Distinguishes two main types of bacteria: Gram-positive with a thick peptidoglycan layer Gram-negative with a lipopolysaccharide layer Some bacteria do not have cell wall- Mycoplasma Peptidoglyc an Figure 3.9 Peptidoglycan A layer of peptidoglycan is found in the cell walls of both gram negative and gram-positive bacteria The Gram-Positive Cell Wall Relatively thick peptidoglycan layer Teichoic acids extend above peptidoglycan layer Important for cell wall integrity, pathogenesis, sensitivity of temperature and salt concentration Gel-like material called periplasm lies below peptidoglycan layer Alternating series of subunits form glycan chains N-acetylmuramic acid (NAM) N-acetylglucosamine (NAG) Tetrapeptide chain attach to the NAM and link glycan chains together Direct link in Gram-negative cells Peptide inter-bridge in Gram-positive cells Peptidoglycan of gram- positive bacteria The Gram-Negative Cell Wall – Figure 3.11 Thin peptidoglycan layer Outside is unique outer membrane Lipopolysacchari de cell wall– Figure 3.11 The Gram-Negative Cell Wall 1 Outer membrane: outside layer is lipopolysaccharide (LPS) Signals immune system of invasion by Gram-negative bacteria Small levels elicit response to eliminate invader LPS is also called endotoxin Has role in infection and survival in the host Large amounts accumulating in bloodstream can be deadly- endotoxemia Includes Lipid A Hydrophobic, anchors LPS to the membrane Recognized by immune system Highly conserved among gram negative bacteria Includes O antigen Can be used to identify species or strains The Gram-Negative Cell Wall 2 Outer membrane blocks passage of many molecules including certain antimicrobial medications Small molecules and ions can cross via porins Secretion systems important in pathogenesis Periplasm Between cytoplasmic membrane and outer membrane Filled with gel-like substance Exported proteins accumulate unless specifically moved across outer membrane Binding proteins of ABC transport systems Antibacterial Substances That Target Peptidoglycan Interference with peptidoglycan can weaken cell wall and allow cell to burst Antibiotics inhibit different steps in peptidoglycan synthesis Penicillin interferes with peptidoglycan synthesis Prevents cross-linking of adjacent glycan chains Usually more effective against Gram-positive bacteria than Gram-negative bacteria Outer membrane of Gram-negative cells blocks access Derivatives have been developed that can cross Lysozyme breaks bonds linking glycan chains Enzyme found in tears, saliva, other body fluids Destroys structural integrity of peptidoglycan molecule Usually more effective against Gram-positive bacteria  Bacteria That Lack Cell Wall Some bacteria naturally lack a cell wall Mycoplasma species one of which causes a mild form of pneumonia (walking pneumonia) flexible because they lack a rigid cell wall. Neither penicillin nor lysozyme affects these organisms. Mycoplasma and related bacteria can survive without a cell wall because their cytoplasmic membrane contains sterols which make it stronger than that of other bacteria. Non typical cell walls Some bacterial groups lack typical cell wall structure Examples-Mycobacterium and Nocardia Gram-positive cell wall structure with lipid mycolic acid (cord factor) Pathogenicity and high degree of resistance to certain chemicals and dyes Basis for acid-fast stain used for diagnosis of infections caused by these microorganisms  Cell Walls of Archaea Members of Archaea have variety of cell walls Probably due to wide range of environments including extreme environments Archaea less well studied than Bacteria No peptidoglycan, but some have similar molecule pseudopeptidoglycan (or pseudomerin) Many have S-layers that self-assemble Built from sheets of flat protein or glycoprotein subunits 3.3-Structures Outside the Cell Wall of Prokaryotic Cells Compare and contrast the structure and function of capsules and slime layers. Describe the structure and arrangements of flagella and explain how they are involved in chemotaxis. Compare and contrast the structure and function of fimbriae and sex pili.  Capsules and Slime Layers – Figure 3.12 Gel-like layer outside cell wall protects or allows attachment Capsule: distinct, gelatinous Slime layer: diffuse, irregular Most composed of glycocalyx (sugar shell), but some are polypeptides Once attached to a surface, cells can grow as biofilm Polymer-encased community Example: dental plaque Some capsules allow bacteria to evade host immune system  Flagella – Figure 3.13 Flagella-involved in motility Spin like propellers to move cell Some important in disease Helicobacter pylori Numbers and arrangements help with characterization of bacteria Peritrichous – flagella distributed around surface of the cell Polar (monotrichous) - a single flagellum at one end  Flagella – Figure 3.14 Three parts of bacterial flagellum: Basal body: anchors to cell wall and cytoplasmic membrane Hook Filament: made up of flagellin subunits Use energy from ion motive force instead of ATP Archaella (flagella of Archaea): Chemically distinct from those of Bacteria About half the diameter of bacterial flagella Use energy from ATP instead of proton motive force Chemotaxis – Figure 3.15 Chemotaxis: Bacteria sense a chemical and move toward it (nutrient) or away from it (toxin) Movement is series of runs (straight line) and tumbles (changes in direction) due to coordinated rotation of flagella Chemotaxis – Figure 3.16 Other responses observed: Aerotaxis response to O2 Magnetotaxis response to earth’s magnetic field Thermotaxis response to temperature Phototaxis response to light  Pili – Figure 3.17 Pili (singular: pilus) are shorter and thinner than flagella and the function is different Common pili, or fimbriae, allow the bacterial cells to attach to specific surfaces Some pili help bacterial cells move with a twitching or gliding motility Sex pili are used to join one bacterium to another for DNA transfer Outer Structures of Archaea Cannulae unique to archaea have been discovered in some marine archaeal strains hollow tube-like structures appear to connect cells after division could serve as a means of anchoring a community of cells to a surface Hamus (hami- pleural) unique to archaea have been discovered in some marine archaeal strains Long helical tube with 3 hooks at the end appear to allow cells to attach one another and to surface Helps forming a community of cells Outer structures of bacteria and archaea  3.4-Internal Components of Prokaryotic Cells Describe the structure and function of the chromosome, plasmids, ribosomes, storage granules, gas vesicles, and endospores. Describe the significance and processes of sporulation and germination. Inside prokaryotes Internal Components of Prokaryotic Cells – Figure 3.18 DNA Chromosome Plasmids Ribosomes Cytoskeleton Storage granules, inclusion bodies Protein based compartments Endospores- in some DNA- Chromosome, plasmids Chromosome Plasmids forms gel-like region: the Similar structure to nucleoid chromosome, but much Single circular double- smaller and multiple copies stranded DNA molecule May be shared with other Packed tightly via binding bacteria; antibiotic resistance proteins and supercoiling can spread this way No histones attached as in Do not encode genetic eukaryotes information essential for Holds genes responsible for species but adds characters species characteristics with advantages Used in genetic engineering to transfer genes into bacteria Ribosomes Involved in protein synthesis Facilitate joining of amino acids Relative size and density expressed as S (Svedberg unit) that reflects how fast they settle when centrifuged Prokaryotic ribosomes are 70S Composed of 30S and 50S subunits Eukaryotic ribosomes are 80S Important medically: antibiotics impacting 70S ribosome do not affect 80S ribosome Cytoskeleton and Storage granules Cytoskeleton Storage granules Interior protein framework Synthesized from nutrients available in excess Bacterial proteins similar to Carbon, energy storage: eukaryotic cytoskeletal Glycogen proteins Poly-β-hydroxybutyrate (PHB) Metachromatic granules stain red with Likely involved in cell division methylene blue and controlling cell shape Protein-based compartments: separate reactions or functions Small rigid structures Identical protein subunits forming the semi-permeable container Functions may be more diverse than previously recognized  Protein Based Compartments Small, rigid structures that physically separate certain reactions or functions from the cell's cytosol Gas vesicles - provide aquatic prokaryotes with a mechanism of adjustable buoyancy Allowing cells to move up or down in the water column. Allow only gases to flow in freely, thereby decreasing the density of the cell Bacterial microcompartments (BMCs) - contain enzymes required for certain metabolic reactions. By confining these in a compartment, the cell prevents unwanted side reactions and protects the cytosol from toxic metabolites. Encapsulin nanocompartments - the most recently discovered type of compartment. hold certain proteins in isolation (example an iron binding protein)  Endospores – Figure 3.20 Endospores: unique type of dormant cell Produced by members of Bacillus, Clostridium May remain dormant for 100 years or longer Extremely resistant to heat, desiccation, chemicals, ultraviolet light, boiling water Not eliminated by pasteurization Can germinate to become vegetative cells that can multiply Found virtually everywhere Endospores Sporulation triggered by limited carbon or nitrogen Starvation begins 8-hour process Endospore layers prevent damage Exclude molecules (for example, lysozyme) Cortex maintains core in dehydrated state, protects from heat Core has small proteins that bind to and protect DNA Calcium dipicolinate seems to play important protective role Germination triggered by heat, chemical exposure Not a means of reproduction Endospores – Figure 3.21 Prokaryotic Cells – Figure 3.1- for comparison Eukaryotic Cell Structure and Their Functions- Figure 3.22a and b Eukaryotic cells are highly variable Protozoa are self-contained units with no cell wall Animal cells also lack a cell wall Fungal cells have a cell wall containing polysaccharides such as chitin Plant cells have a cell wall composed of cellulose Eukaryotic Cell Structures and Their Functions – Figure 3.22c Eukaryotic cells are larger and more complex than prokaryotic cells Membrane-enclosed compartments called organelles Nucleus contains cell’s DNA Cells within an organism vary Similar cells form tissues Various tissues form organs  Eukaryotic cell Common to all “cells” Plasma membrane Cytoskeleton/cytoplasm DNA- chromosomes RNA- ribosomes Unique to eukaryotes Organelles common to animal and plant cells Nucleus Endomembrane system- rough and smooth ER, Golgi complex Other Membrane bound organelles- mitochondria, peroxisomes Organelles unique to animal and plant cells Plant cells- cell wall, plasmodesmata, chloroplast, central vacuole Animal cells- lysosomes, centrioles 3.5-Eukaryotic cytoplasmic membrane Describe the structure and function of the eukaryotic cytoplasmic membrane- compare and contrast it with the prokaryotic counterpart. Describe the mechanisms eukaryotic cells use to transfer molecules across the cytoplasmic membrane. Cytoplasmic Membrane of Eukaryotic Cells Similar to that of prokaryotic cells Phospholipid bilayer embedded with proteins Proteins in outer layer serve as receptors Bind specific molecule called a ligand Important in cell communication May contain sterols to increase strength Cholesterol in mammals, ergosterol in fungi Lipid rafts: allow cell to detect, respond to signals Many viruses use to enter, exit cells Electrochemical gradient maintained by sodium or proton pumps Transfer of Molecules Across Cytoplasmic Membrane Similar to prokaryotes Passive transport Active transport Down the concentration Against the concentration gradient gradient Energy provided by the Energy provided by ATP, concentration gradient not the concentration Transport protein not gradient required of most Transport protein required Examples- simple diffusion, osmosis, facilitated diffusion (transport protein required) Transfer of Molecules Across Cytoplasmic Membrane – Figure 3.24 and 3.25- different from prokaryotes Endocytosis: cell takes up material from surrounding environment by forming invaginations in cytoplasmic membrane Exocytosis: internal vesicles fuse with the cytoplasmic membrane and release their contents Membrane bound budding– Figure 3.23- different from propkaryotes Membrane-bound vesicles bud off from one organelle and fuse with another, delivering material to lumen of organelle Secretion- different from prokaryotes Proteins destined for secretion carry a signal sequence that acts as a tag Ribosomes synthesizing protein with a signal sequence attach to endoplasmic reticulum (ER) Protein enters lumen of ER Membrane of ER buds off and binds to cytoplasmic membrane, releasing protein outside the cell Proteins going to other organelles have specific tags 3.6- Protein Structures Within Eukaryotic Cells Describe the structure and function of eukaryotic ribosomes, the cytoskeleton, flagella, and cilia. Eukaryotic Ribosomes Eukaryotic ribosome is 80S, made up of 60S and 40S subunits Prokaryotic ribosomes are 70S Antibacterial medications typically do not interfere with 80S ribosome Cytoskeleton– Figure 3.26 Cytoskeleton: cell framework Actin filaments (microfilaments) allow movement Polymers of actin polymerize and depolymerize Microtubules are thickest component Long, hollow structures made of tubulin Found in mitotic spindles, cilia, flagella Framework for organelle and vesicle movement Intermediate filaments Perspective 3.1 Pathogens Hijacking Actin  Flagella and cilia Flagella and cilia function in motility Covered by extensions of cytoplasmic membrane Composed of microtubules in 9 + 2 arrangement Eukaryotic flagella are very different from prokaryotic flagella Propel cell or pull it forward Cilia are shorter than flagella, move synchronously Can move cell forward or move material past stationary cells 3.7- Membrane-Bound Organelles of Eukaryotic Cells Describe the function of the nucleus, mitochondria, chloroplasts, endoplasmic reticulum, Golgi apparatus, lysosomes, and peroxisomes.  Nucleus and nucleolus Nucleus contains the genetic information Surrounded by two phospholipid bilayer membranes Nuclear pores allow large molecules to pass Nucleolus is region inside the nucleus where ribosomal RNAs synthesized  Mitochondria – Figure 3.29 Generate ATP Bounded by two phospholipid bilayers Inner membrane forms folds (cristae), increasing surface area for ATP generation Mitochondrial matrix contains DNA, 70S ribosomes, divides by binary fission Chloroplasts – Figure 3.30 Site of photosynthesis Found only in plants, algae Harvest sunlight energy to generate ATP ATP used to convert CO2 to sugar and starch Contain DNA and 70S ribosomes, divides by binary fission, two membranes Stroma contains thylakoids containing pigments that capture radiant energy  Endosymbiotic Theory Ancestors of mitochondria and chloroplasts were bacteria residing within other cells Endosymbiont lost key features (cell wall, replication) as partners because indispensable to one another Lines of evidence Carry DNA for some ribosomal proteins, ribosomal RNA Ribosomes similar to bacterial 70s ribosomes Double membrane surrounds both organelles Multiplication is by binary fission controlled by their own DNA Mitochondrial DNA sequences resemble those of obligate intracellular parasites: rickettsias Chloroplast DNA sequences resemble those of cyanobacteria  Endoplasmic Reticulum (ER) – Figure 3.31 System of flattened sheets, sacs, tubes Rough endoplasmic reticulum dotted with ribosomes Synthesize proteins that need to be modified and/or secreted Smooth endoplasmic reticulum lipid synthesis and degradation, calcium storage Golgi Apparatus – Figure 3.32 Membrane-bound flattened compartments Macromolecules synthesized in ER are modified here Addition of carbohydrate, phosphate groups Molecules sorted and delivered in vesicles Other Membrane-Bound Organelles Lysosomes contain degradative enzymes Could destroy cell if not contained Endosomes, phagosomes fuse with lysosomes Material taken up by cell is degraded Old organelles, vesicles fuse with lysosomes: autophagy Peroxisomes use O2 to degrade lipids, detoxify chemicals Peroxisome generates, contains, and ultimately degrades hydrogen peroxide, superoxide Protects cell from toxic effects of these molecules 3.8- Microscopy Discuss the principles and importance of magnification, resolution, and contrast in microscopy. Compare and contrast light microscopes, electron microscopes, and scanning probe microscopes. Microscopy Light microscope can magnify 1,000x Common, important tool in microbiology Electron microscope (1931) can magnify more than 100,000x Scanning probe microscope (1980s) can produce images of individual atoms on a surface Principles of Light Microscopy 1 Light passes through specimen and then series of magnifying lenses Most common type is Bright-field microscope - evenly illuminates the field of view and generates a bright background Three key concepts: Magnification: apparent increase in size Resolution: resolving power, or ability to distinguish two objects that are very close together Contrast: difference in color intensity between an object and the background; determines how easily cells can be seen  Principles of Light Microscopy - 2 Magnification: apparent Resolution: resolving power, or increase in size ability to distinguish two objects Modern compound that are very close together microscope has two lens types: objective and ocular Defined as minimum distance between two points at which those Magnification is product of points can be observed as separate objective (4x, 10x, 40x, or 100x) and ocular lens Depends on quality and type of (10x) lens, wavelength of light, Condenser lens (between magnification, and specimen preparation light source and specimen) focuses light on specimen; Maximum resolving power of light does not magnify microscope is 0.2 micrometer Principles of Light Microscopy – Figure 3.33  Principles of Light Microscopy – Figure 3.34 Immersion oil used to displace air between lens and specimen when using high powered 100x objective Has same refractive index (measure of speed of light passing through medium) as glass Prevents refraction of light; keeps rays from missing opening in Principles of Light Microscopy – Figure 3.35, 3.36 Bright-Field Microscope Dark-Field Microscope Principles of Light Microscopy – Figure 3.37, 3.38 Differential Interference Phase-Contrast Microscope Contrast (DIC) Principles of Light Microscopy – Figure 3.39, 3.40 Scanning Laser Microscopes Fluorescence Microscope (SLM) Super-Resolution Microscope – Figure 3.41 Can improve resolution down to 10 nanometers Light vs Electron Microscope Electron Microscopes Can clearly magnify objects 100,000x Uses electromagnetic lenses, electrons, and a fluorescent screen to replace glass lenses, visible light, and the eye Image photographed as black-and-white electron photomicrograph May be artificially colored Wavelength of electrons approximately 1,000 shorter than light Resolving power approximately 1,000-fold greater: approximately 0.3 nanometer Electron microscopes- Figure 3.43, 3.44 Transmission Electron Scanning Electron Microscopes Microscopes 3.9- Viewing specimens Describe the principles of a wet mount, a simple stain, the Gram stain, and the acid- fast stain. Describe the special stains used to observe capsules, endospores, and flagella. Describe the benefits of staining with fluorescent dyes and tags. Preparing Specimens for Light Microscopy – Figure 3.46 Simple Staining Simple staining uses a single dye to stain the specimen Basic dyes carry positive charge Attracted to negatively charged cellular components Examples are methylene blue and crystal violet Acidic dyes can be used for negative staining Cells repel the negatively charged dye; colorless cells stand out against background Can be done as wet mount  Staining Simple Staining Differential Staining Simple staining uses a single dye to Differential staining used to stain the specimen distinguish different structures or groups of bacteria Basic dyes carry positive charge Gram stain most widely used for Attracted to negatively charged cellular bacteria components Examples are methylene blue and crystal Two groups: Gram-positive bacteria violet and Gram-negative bacteria Acidic dyes can be used for negative Reflects fundamental difference in staining cell wall structure Cells repel the negatively charged dye; colorless cells stand out against background Can be done as wet mount Gram Stain Figure 3.47 Gram stain and results Acid-Fast Stain Acid-fast stain used to detect organisms that do not readily take up dyes Detects Mycobacterium species such as causative agents of tuberculosis and Hansen’s disease (leprosy) Cell wall contains high concentrations of mycolic acids Multi-step procedure Primary stain is concentrated red dye Acid-fast cells retain red dye after being flooded with acid-alcohol Methylene blue used as counterstain Capsule Stain Allows observation of gel- like layer that surrounds some microbes Capsules stain poorly, so background is stained to make capsule visible India ink added to wet mount is common method Endospore Stain Allows visualization of endospores, resistant dormant cells often formed by Bacillus and Clostridium Endospore resists Gram stain; appears as clear object Endospore stain uses heat to facilitate uptake of the primary dye malachite green by endospore Counterstain (usually safranin) colors other cells pink Flagella Stains – Figure 3.52 In this chapter we studied Prokaryotic cell structures Eukaryotic cell structures Similarities between prokaryotic and eukaryotic cells Differences between prokaryotic and eukaryotic cells Microscopy principles Preparation of specimens Common simple and differential staining methods