Lecture 4 - Microbiology

Preparing Specimens The method of specimen preparation depends on: – The condition of the specimen: living or preserved  Fresh, Living Preparations Wet mounts or hanging drop mounts: – Cells are suspended in a suitable fluid: water, broth, or saline – Fluid maintains viabilit...

Preparing Specimens The method of specimen preparation depends on: – The condition of the specimen: living or preserved  Fresh, Living Preparations Wet mounts or hanging drop mounts: – Cells are suspended in a suitable fluid: water, broth, or saline – Fluid maintains viability and provides a medium for movement – Provide a true assessment of size, shape, arrangement, color and motility of cells  Fresh, Living Preparations Wet Mounts (cont’d): – Consist of a drop or two of culture placed on a slide overlaid with a coverslip – Advantages: quick and easy to prepare – Disadvantages: Can damage larger cells Susceptible to drying Can contaminate the handler’s fingers  Fresh, Living Preparations Hanging Drop preparation: – Prepared with concave (depression) slide, Vaseline adhesive or sealant, and a coverslip – Overcomes the disadvantages of wet mounts Fresh, Living Preparations  Fixed, Stained Smears Smear technique: – Spread a thin film made from a liquid suspension of cells on a slide – Allow the slide to air dry – Heat fixing Heat the slide gently after it has been air dried  Fixed, Stained Smears Important functions of heat fixing: – Kills the cells – Secures the specimen to the slide – Preserves cellular components in a natural state with minimal distortion Alcohol and formalin can also be used for heat fixation.  Fixed, Stained Smears Staining: – Provides contrast – Makes inconspicuous features stand out – Dyes impart colors to cells by becoming affixed to them through a chemical reaction  Fixed, Stained Smears Dyes used in microbial staining: – Basic (cationic): have a positive charge, attracted to acidic, negatively charged components on bacterial cell walls – Acidic (anionic): have a negative charge, repelled by acidic, negatively charged components on bacterial cell walls  Negative vs. Positive Staining Positive stain: – Positively charged stain is attracted to negatively charged cell walls – Stick to the cell and give it color Negative stain: – Negatively charged dye is repelled by negatively charged bacterial cell walls – Produces a black background around the cells Negative vs. Positive Staining  Simple vs. Differential Stains Simple stains: – Require a single dye – Uncomplicated procedure Differential stains: – Use two differently colored stains to clearly contrast cell types or cell parts – Complex staining technique  Gram Stain Gram stain is a laboratory test that checks for bacteria at the site of a suspected infection or in certain bodily fluids. A medical laboratory scientist processes the Gram stain, which gives relatively quick results – Developed in 1884 by Hans Christian Gram – Delineates two major groups of bacteria Gram-positive Gram-negative – Differences lie in the structure of the cell envelope Gram-positive vs. Gram-negative Cells Gram-positive – Thick cell wall composed of peptidoglycan – Inner cytoplasmic membrane Gram-negative – Outer membrane – Thin cell wall – Inner cytoplasmic membrane Gram-positive vs. Gram-negative Cells The Gram staining process includes four basic steps, including: 1.Applying a primary stain (crystal violet). 2.Adding Gram’s iodine. 3.Rapid decolorization with ethanol, acetone or a mixture of both. 4.Counterstaining with safranin. Gram – Vs + N. gonorrhoeae Streptococcus pyogenes  What conditions do Gram stains help diagnose? urinary tract infections (UTIs) and bacterial pneumonia.  Gram-positive organisms include Staphylococcus species. Streptococcus species. Corynebacterium species. Clostridium species. Listeria species.  Gram-negative organisms include: Neisseria gonorrheae and Neisseria meningitides. Moraxella species. Escherichia coli (E. coli). Pseudomonas species. Proteus species. Klebsiella species.  Nontypical Cell Walls Mycolic acid – Found in the cell walls of Mycobacterium Very- long-chain fatty acid – Contributes to pathogenicity of these organisms – Acid-fast stain used to diagnose tuberculosis and leprosy Bacteria and Archaea vs. Eukaryotes Packaging of DNA – Bacteria and archaea have nuclear material that is free in the cytoplasm – Eukaryotes have a nucleus Cell wall makeup: – Bacteria: cell wall made of peptidoglycan – Archaea: cell wall distinct from bacteria and eukaryotes Internal Structures – Bacteria and archaea: no membrane-bound organelles Bacterial Cell Structure Bacterial Arrangements and Sizes Bacterial cells are capable of carrying out all necessary life activities – Reproduction – Metabolism – Nutrient processing Bacteria can also act as a group – Colonies – Biofilms Bacterial Arrangements and Size Considerable variety in size, shape, and colonial arrangement. – Average size 1 μm – Mycoplasma: 0.15 – 0.30 μm – Nanobacteria: 0.05 – 0.2 μm  Nanobacteria: 0.05 – 0.2 μm Nanobacteria produce a rock-hard calcium phosphate substance that is chemically identical to the substance found in hardening arteries, prostate disease, kidney disease, periodontal disease and breast cancer Bacterial Arrangements and Size Bacterial Arrangements and Size Three general shapes of bacteria: – Coccus: spheres, oval, bean-shaped, pointed – Bacillus: cylindrical – Spirillum: rigid helix Pleomorphism: – Variations in size and shape among cells of a single species Bacterial Arrangements and Size Bacterial Arrangements and Size Bacterial Arrangements and Size Arrangements and groupings of bacilli: – Diplobacilli: pairs of cells with their ends attached – Streptobacilli: chains of cells – Palisades: cells of a chain remain partially attached and fold back, creating a side-by-side row of cells Spirilla occasionally found in short chains. Spirochetes rarely remain attached after cell division.  External Structures Two major groups of appendages: – Flagella and axial filaments: provide motility – Fimbriae and pili: provide attachment points or channels Flagella Flagellar Arrangement  Flagellar Arrangement Polar: flagella attached at one or both ends of the cell. – Monotrichous: single flagellum – Lophotrichous: small bunches or tufts – Amphitrichous: flagella at both poles of the cell Peritrichous: flagella are dispersed randomly over the surface of the cell.  Flagellar Function Chemotaxis: movement in response to chemical signals – Positive chemotaxis: movement of a cell in the direction of a favorable chemical stimulus – Negative chemotaxis: movement of a cell away from a repellant or potentially harmful compound  Periplasmic Flagella Axial filament: – Two or more long coiled threads found in spirochetes – Internal flagellum enclosed between the cell wall and cell membrane – Impart a twisting or flexing motion to the cell Periplasmic Flagella  Appendages for Attachment and Mating Attachment can enhance pathogenicity in some bacteria. – Pilus (plural: pili) – Fimbria (plural: fimbriae) Both provide adhesion but not locomotion – Flagella can also be used for attachment in some species  Fimbriae Small, bristle-like fibers sprouting off the surface of certain species of bacteria – Composition varies, but most contain protein – Have the inherent tendency to stick to each other and to surfaces – May be responsible for the formation of biofilms – E. coli and the gonococcus use fimbriae to adhere to epithelial cells Fimbriae  Pili Also known as a sex pilus – Long, rigid tubular structure made of pilin protein – Only found in gram-negative bacteria – Used in conjugation, the partial transfer of DNA from one cell to another – Production of pili is controlled genetically  Surface Coatings S layer: – Thousands of copies of a single protein linked together – Provides protection from environmental conditions – Only produced in hostile environments  Glycocalyx Slime layer: – Forms loosely around the cell – Protects the cell from loss of water and nutrients Capsule: – More tightly bound to a cell than a slime layer – Denser and thicker than a slime layer Glycocalyx Glycocalyx Biofilms  The Cell Envelope Composed of two or three basic layers: – Cell wall – Cell membrane – Outer membrane (in some bacteria) Act as a single protective unit.  Structure of the Cell Wall Characteristics of the cell wall – Helps determine the shape of a bacterium – Provides strong structural support to keep the cell from bursting or collapsing due to osmotic pressure  Structure of the Cell Wall Peptidoglycan: – Found in the cell walls of most bacteria – Unique macromolecule composed of glycan chains cross linked with short peptide fragments – Provides a strong but flexible support framework Structure of the Cell Wall  The Gram-Positive Cell Wall Thick, homogenous sheath of peptidoglycan, 20 – 80 nm thick. – Cell wall maintenance – Enlargement during cell division – Acidic charge on cell surface  The Gram-Negative Cell Wall Single, thin sheet of peptidoglycan, 1 – 3 nm thick. Somewhat rigid structure Thinness gives gram-negative bacteria greater flexibility and sensitivity to lysis  Nontypical Cell Walls Mycolic acid – Found in the cell walls of Mycobacterium Very- long-chain fatty acid – Contributes to pathogenicity of these organisms – Acid-fast stain used to diagnose tuberculosis and leprosy  Mycoplasmas and Cell-Wall Deficient Bacteria Mycoplasmas – Naturally lack a cell wall – Membrane is stabilized by sterols and is resistant to lysis – Pleomorphic shape, range from 0.1 – 0.5 μm, ranging from filamentous to coccus Mycoplasmas The Gram-Negative Outer Membrane Contains specialized polysaccharides and proteins. – Lipopolysaccharide Polysaccharide chains function as antigens and receptors Endotoxin: stimulates fever and shock reactions – Lipoproteins: anchor the outer membrane to peptidoglycan  The Gram-Negative Outer Membrane (cont’d) Porin proteins: – Completely span the outer membrane – Only allow relatively small molecules to penetrate – Size can be altered to block the entrance of harmful chemicals – Act as a defense against certain antibiotics  Cell Membrane Structure 5 – 10 nm sheet molded completely around the cytoplasm: Lipid bilayer embedded with proteins: – 30 – 40% phospholipids – 60 – 70% proteins Mycoplasmas contain high amounts of sterols. Archaea contain unique branched hydrocarbons rather than fatty acids. Functions of the Cell Membrane Energy reactions Nutrient processing Synthesis Transport: – Passage of nutrients into the cell – Discharge of wastes Functions of the Cell Membrane Selective permeability: – Water and small uncharged molecules diffuse freely – Special carrier mechanisms exist for passage of most molecules Secretion: discharge of metabolic products into the extracellular environment Differences in Cell Envelope Structure Outer membrane in gram-negative bacteria: – Makes them impervious to antimicrobial chemicals – More difficult to kill than gram-positive bacteria – Infections with gram-positive bacteria are treated differently than infections with gram- negative bacteria. Differences in Cell Envelope Structure The cell envelope can interact with human tissues and cause disease. – Proteins in the outer cell wall of gram-positive bacteria can be toxic. – Lipids in the cell wall of Mycobacterium can be harmful to human cells. – Macromolecules in the cell wall are seen as foreign and can stimulate antibody production.  Bacterial Internal Structure Cytoplasm: gelatinous solution contained by the cell membrane – Prominent site for the cell’s biochemical and enzymatic activities – 70 – 80% water – Complex mixture of sugars, amino acids, and salts – Also contains chromatin, ribosomes, granules, and fibers that act as the cytoskeleton Bacterial Chromosomes and Plasmids Bacterial chromosome: – Single circular strand of DNA – Aggregated in a dense area called the nucleoid – DNA is tightly coiled around basic protein molecules to fit into the cell compartment. Bacterial Chromosomes and Plasmids Plasmids: – Non-essential pieces of DNA – Separate, double stranded circles of DNA – Duplicated and passed onto offspring during replication – Confer protective traits – Important in genetic engineering  Ribosomes Made of RNA and protein. Dispersed throughout the cytoplasm, often found in chains. Svedberg (S) units: – Measurement of the relative size of cell parts through sedimentation during centrifugation – Bacterial ribosomes: 70S – Eukaryotic ribosomes: 80S Ribosomes  Inclusion Bodies and Microcompartments Inclusion bodies: – Storage sites for nutrients during periods of abundance – Single-layered membranes – Vary in size, number, and content Inclusion Bodies  The Cytoskeleton Long polymers of proteins similar to eukaryotic actin. – Arranged in helical ribbons around the cell just under the cell membrane – Contribute to cell shape The Cytoskeleton  Bacterial Endospores Endospores: – Withstand hostile conditions and facilitate survival Two phase life cycle: – Vegetative cell: metabolically active – Endospore: inert, resting condition – Sporulation: spore formation induced by environmental conditions Bacterial Endospores  Bacterial Endospores Endospores can resist: – Heating – Drying – Freezing – Radiation – Chemicals Endospore Formation and Resistance Stimulus for endospore formation: – Depletion of nutrients, especially carbon and nitrogen sources Sporangium: – Sporulating cell – Transformation takes 6 – 8 hours in most species  Germination of Endospores Germination begins when favorable conditions arise. – Exposure to water and a germination agent – Germination agent stimulates the formation of hydrolytic enzymes that break down the cortex – Core rehydrates and takes up nutrients and bacterium grows out of the endospore coats Medical Significance of Endospores Some diseases are related to the persistence and resistance of their spores – Bacillus anthracis: Anthrax – Clostridium tetani: Tetanus – C. difficile: pseudomembranous colitis – C. perfringens: gas gangrene – C. botulinum: botulism Medical Significance of Endospores Endospores are constant intruders where sterility and cleanliness are important. – Resist ordinary cleaning methods: boiling water, soaps and disinfectants – Frequently contaminate cultures and media – Hospitals must protect against endospores in wounds – Destruction of endospores important in the food- canning industry  The Archaea More closely related to Eukarya than Domain Eubacteria – Share ribosomal RNA sequences not found in bacteria – Protein synthesis and ribosomal subunit structures are similar The Archaea  The Archaea Differences from other cell types: – Certain genetic sequences are only found in their rRNA – Unique method of DNA compaction – Unique and chemically distinct cell walls  The Archaea The most primitive of all life forms. – Most closely related to cells that originated 4 billion years ago – Live in habitats that are similar to the extremes found anciently – heat, salt, acid, pH, pressure, atmosphere – Methane producers, hyperthermophiles, extreme halophiles, and sulfur reducers  The Archaea Methanogens: – Convert CO2 and H2 into methane gas (CH4) – Common inhabitants of anaerobic swamp mud, bottom sediments of lakes and oceans, and the digestive systems of animals – Gas produced in swamps may become a source of fuel – May contribute to greenhouse gases and global warming  The Archaea Extreme halophiles: – Require salt to grow – Can multiply in 36% NaCl that would destroy most cells – Exist in inland seas, salt lakes, salt mines, and in salted fish – Use a red pigment to synthesize ATP in the presence of light The Archaea  The Archaea Psychrophiles: adapted to grow at very low temperatures. Hyperthermophiles: – Flourish at temperatures between 80° and 113°C and cannot grow below 50°C – Live in volcanic waters and soils and submarine vents – Often salt and acid tolerant as well as heat tolerant  The Archaea rRNA sequencing is used in identification of archaea. – This technique has also advanced the general understanding of transcription, translation, and cellular evolution  Bergey’s Manual The definitive published source for bacterial and archaea classification. – Phenotypic traits were the basis for early classification: shape, cultural behavior, and biochemical reaction – Still used by clinical microbiologists and researchers who need to quickly identify unknown bacteria  Bergey’s Manual of Systematic Bacteriology Comprehensive view of bacterial and archaeal relatedness. – Combines phenotypic information with rRNA sequencing for classification – 5-volume set  Diagnostic Scheme Divisions in the diagnostic scheme: – Gram-positive – Gram-negative – Bacteria without cell walls – Cell shape – Arrangements – Oxygen usage: Aerobic: use oxygen in metabolism Anaerobic: do not use oxygen in metabolism Facultative: may or may not use oxygen