Lecture Notes for Exam 1, Biology 250

  1. Introduction
    1. Bacteria
      1. Prokaryotic cells (simple structure, no internal membranes)
      2. Some are pathogens (cause disease)
      3. Many are not pathogenic (found in soil, water, on body as normal flora)
      4. Categorized in Kingdom Monera
      5. Binomial naming conventions: Genus species, Salmonella typhi, or S. typhi
      6. Criteria for classification (characteristics of bacteria used for identification)
        1. Morphology (microscopic)
        2. Biochemical differences (use of nutrients, production of waste materials, growth requirements)
        3. DNA characteristics
    2. Viruses
      1. Noncellular particles
      2. Obligate intracellular parasites: need host cell in order to reproduce (replicate)
      3. Host- and/or tissue specific: usually limited to a specific type of host cell
        1. Chicken virus infects chicken cells
        2. Bacterial viruses infect bacteria cells (often only a single genus or species)
        3. Human liver viruses infect human liver cells (hepatitis)
    3. Protists and fungi (eukaryotic, classified in Kingdoms Protista and Fungi)
  2. Study of bacteria
    1. Microscopy
      1. Three principal factors: magnification, resolution, illumination
      2. Visible light microscopes use visible light for illumination, glass lenses
      3. Oil immersion lens
        1. 100x objective lens x 10x ocular gives total magnification of 1000x
        2. Increases illumination, which increases resolving power, which makes higher magnifications useful)
        3. Allows observation of most bacteria to determine cell size, shape, grouping of stained cells
      4. Electron microscope
        1. Uses electron beam for illumination (shorter wavelength allows greater resolution and magnification)
        2. Uses magnetic lenses to focus beam
        3. Scanning, transmission electron microscopes
        4. Makes viruses and smaller particles visible, as well as bacteria
      5. Staining to improve contrast in visible light microscopes
        1. Simple stain: stains all cells the same
        2. Differential stain: stains some cells or cell parts differently from others
        3. Examples of differential staining procedures: Gram stain, acid fast stain, endospore stain, flagella stain
    2. Bacterial morphology (shape of cell and grouping determined by microscopy)
      1. Size (can be measured if microscope has micrometer)
      2. Cell morphology (shape of cells, grouping determined by separation after cell division)
        1. Bacillus, bacilli (rod-shaped cells, longer than they are wide)
          1. Examples of genera: Escherichia (food poisoning), (anthrax, food poisoning), Clostridium (botulism, tetanus), (typhoid fever), Corynebacterium (diphtheria)
          2. Common groupings: chains (Bacillus), palisading (Corynebacterium)
        2. Coccus, cocci (spheres, flattened spheres)
          1. Examples: Staphylococcus (wound infections, etc.), Streptococcus (strep throat, pneumococcal pneumonia), Neisseria (gonorrhea, meningococcal meningitis)
          2. Common groupings: chains (many Streptococcus species), pairs or diplococci (Streptococcus pneumoniae), tetrads or 8-cell packets (Micrococcus), clusters (Staphylococcus
        3. Spirochete (curved or spiral cells, usually found as single cells)
          1. Spiral cells: Borrelia (Lyme disease), Treponema (syphilis)
          2. Curved cells: Vibrio (cholera)
    3. Observation of growth characteristics on artificial media
      1. Colony morphology on solid media (appearance of group of cells growing together on solid medium)
      2. Characteristics of colony: color, texture (shiny, matte, fuzzy), edge (smooth, jagged), light transmission (opaque, translucent, transparent),
      3. Growth of isolated colonies on streak plate
        1. Mixture of cells from source such as throat or mouth
        2. Aseptic inoculation of sterile solid media with cells, well separated by streaking technique
        3. Incubation at appropriate temperature for source (37oC)
        4. One cell divides by binary fission to make many identical daughter cells (colony)
        5. Samples of each type of colony transferred to new solid media to prepare pure cultures
        6. Incubation
        7. Further study of pure cultures to identify organisms (staining, biochemical tests)
      4. Colonies of same organism have different characteristics grown on different media or under different conditions
      5. Utilization of food sources (different organisms can use different nutrients, such as sugars, proteins)
      6. Production of waste products (different organisms produce different waste products, such as acidic, alkaline, neutral products)
      7. Preferred environmental characteristics (high or low in oxygen, high or low temperature, acidic/alkaline)
  3. Bacterial Cell Structure
    1. Capsule (part of cell envelope)
      1. Slippery, protein or polysaccharide, protects from phagocytosis, not essential for survival of cell
      2. Example: Streptococcus pneumoniae
        1. Encapsulated/smooth colony/pathogenic/not easily phagocytized by macrophages in lungs
        2. Nonencapsulated/rough colony/nonpathogenic/easily phagocytized by macrophages in lungs
    2. Cell wall (part of cell envelope)
      1. Unique structural element: peptidoglycan (crosslinked amino acids and amino sugars in rigid 3-dimensional grid)
      2. Gram positive cell wall (purple in Gram staining procedure): thick peptidoglycan layer with teichoic acids attached
      3. Gram negative cell wall (pink/red in Gram staining procedure): outer membrane with phospholipids, lipopolysaccharide (endotoxin), lipoproteins, thin layer of peptidoglycan
      4. Contributes to shape, rigidity of cell
      5. Acts as target of action of some antibiotics (penicillin prevents peptidoglycan synthesis, most effective on rapidly growing cell population))
      6. Protects cell membrane from rupture due to osmotic pressure
      7. Associated with certain symptoms of disease
      8. Determines gram reaction (how cell stains in differential Gram staining procedure)
      9. Essential for survival of most bacteria unless protected by high osmotic pressure environment
      10. Acid fast bacteria (Mycobacterium, cause tuberculosis and leprosy): unique cell wall rich in lipids makes cells hard to stain/destain, important in pathogenesis)
    3. Cell membrane (part of cell envelope)
      1. Phospholipids (40%) and proteins (60%), semipermeable, controls entrance and exit of molecules from cell, contains enzymes responsible for transport, nutrient breakdown, and energy production, essential for survival of cell
      2. Lipid bilayer membranes similar in all cells
    4. Flagella
      1. Thin, fiber-like extensions anchored in membrane
      2. Simple repeating structure made of protein (flagellin) responsible for motility
      3. Not necessary for survival
      4. Movement toward stimulus requires coordinated rotatory movement of flagella, like corkscrew, not whip (no reverse movement)
      5. Must be stained to be visible in light microscope (too thin to see otherwise)
      6. Polar (monotrichous, lophotrichous) arrangements of flagella (at one or both ends of cell)
      7. Peritrichous arrangements (flagella all around cell, Proteus mirabilis
      8. Other types of bacterial motility exist that are not dependent on flagella: gliding
      9. Vibration of nonmotile cells due to bombardment by fluid molecules (Brownian movement)
    5. Pili
      1. Fine, hairlike extensions from cell surface, made of protein (pilin), involved in attachment, not essential for survival
      2. Attachment to other bacteria in conjugation (DNA transfer) (F pili of Escherichia coli)
      3. Attachment to other bacteria to hold oxygen-preferring bacteria at surface of liquid (Bacillus)
      4. Attachment to other cells: Neisseria gonorrhoeae to epithelial cells of genital tract, Bordetella pertussis to lining of respiratory tract
    6. Nuclear material (no true nucleus)
      1. Genome: DNA in one circular chromosome, not enclosed in nuclear membrane
      2. Plasmids: small circles of extrachromosomal DNA, contain genes for virus and antibiotic resistance, used in preparing recombinant DNA
    7. Ribosomes
      1. Ribosomal protein and RNA in two subunits involved in protein synthesis in cytoplasm
      2. Polyribosomes: ribosomes linked by mRNA, actively engaged in protein synthesis
    8. Cytoplasmic storage granules
    9. Endospores
      1. Protected form of cell, not a means of reproduction, but of preservation of DNA (a survival mechanism)
      2. Resistant to heat, cold, drying, radiation, many chemicals including stains
      3. Metabolically inert (no chemical reactions)
      4. Examples of sporulating genera: Bacillus (anthrax, food poisoning), Clostridium (tetanus, botulism, gas gangrene)
      5. Cycle of sporogenesis and germination
      6. Endospores usually observed in aging cultures (incubated more than 48 hours) with decreasing amounts of nutrients and water, and increasing amounts of toxic waste products
    10. Contrasts between prokaryotic (P) and eukaryotic (E) cells
      1. P cell walls complex, E have none or have simple cellulose CW
      2. P lipid bilayer membranes protected by cell wall, E lipid bilayer membranes have sterols to strengthen them
      3. P have no internal membranes in cells, E do
      4. P have no nuclear membrane and only one genome, E do and have more than one chromosome
      5. P have no mitochondria or other specialized organelles, E do
      6. P have pili, E don't
      7. Some P have simple flagella structures that rotate, E may have complex flagella structures that wave
      8. Some E have cilia, P don't
      9. Both have ribosomes, although structure is different
      10. Some P have capsule, few E do
      11. E spores are reproductive, P endospores are not
  4. Microbial growth
    1. What would you think if after you inoculated and incubated a plate, nothing grew?
      1. Maybe your loop was too hot, and the organisms were all dead. (but you were very careful and cooled your loop....)
      2. The medium didn't have the proper nutrients.
      3. The medium was the wrong pH, since different organisms grow best at different pH's.
      4. The medium was incubated at the wrong temperature, since different organisms grow best at different temperatures.
      5. The culture was incubated for too short of time, since different organisms have different rates of growth.
      6. The organism needed more or less oxygen or carbon dioxide than was available to it--different organisms have different oxygen and carbon dioxide requirements.
      7. The shortest time between cell divisions is the shortest generation time, which is also the most rapid growth of the organism
      8. Microorganisms grow with the shortest generation time when the conditions are most appropriate for enzyme function.
      9. For each growth condition, an organism will tolerate a range of values, but will exhibit the most rapid growth only at its optimum level for that condition.
    2. Growth requirements
      1. Trace elements (Na, K, etc.)
      2. Essential elements (CHONPS)
      3. Carbon source (CO2, organic C)
      4. Nitrogen source (NO2, organic N)
      5. Oxygen
        1. aerobic--prefer an environment with abundant oxygen (Bacillus, Pseudomonas)
        2. facultatively anaerobic--prefer an oxygen-rich environment, but can survive in an oxygen-poor environment (Escherichia, Staphylococcus, Streptococcus)
        3. anaerobic--require an oxygen-poor environment, oxygen is often toxic (Clostridium)
        4. extremely oxygen sensitive--grow only in a reduced environment (no oxidized compounds)
      6. Carbon dioxide (capnophiles--carbon dioxide loving, prefer an environment enriched in carbon dioxide, such as a candle jar (Neisseria))
      7. Halophiles (salt-loving)
      8. pH preference
        1. Some prefer or tolerate acidic conditions (acidophiles)
        2. Some prefer or tolerate alkaline conditions--Vibrio cholerae)
        3. Most prefer conditions close to neutral pH 7.0
      9. Temperature preference
        1. Mesophile--most human pathogens
        2. Psychrophile--some Pseudomonas
        3. Thermophile--organisms found in hot springs, etc.
      10. Water
    3. Type of metabolism based on carbon source and energy production
      1. Autotrophs
        1. Can use all inorganic nutrients, including carbon dioxide for C-source (strict autotrophs), except glucose for carbon)
        2. Some autotroph use inorganic forms for all nutrient except carbon, for which they require glucose
        3. Capable of synthesizing their own organic compounds
        4. Photoautotrophs--use light to produce energy, some release oxygen (cyanobacteria, etc.)
        5. Chemoautotrophs--obtain energy from chemical reactions (various soil and water bacteria)
      2. Heterotrophs
        1. Require preformed organic molecules
        2. Lack certain enzymes necessary for synthesis of some organic compounds
        3. Photoheterotrophs--use light for energy, need organic materials
        4. Chemoheterotrophs--obtain energy from chemical reactions (humans and other animals, most human pathogens)
      3. Facultative autotroph
        1. Can grow as autotroph if they must; grow better as heterotrophs
        2. Escherichia coli growing as a heterotroph grows more rapidly, with a generation time of approx. 20 minutes, in an enriched medium with organic nutrients and abundant oxygen (allows use of aerobic respiration to produce more energy).
        3. Escherichia coli growing as an autotroph grows more slowly, with a generation time of approx. 35 minutes, in a glucose plus salts medium, without oxygen (forces organism to use fermentation which produces less energy.
    4. Measurement of bacterial growth (skipped in spring, 2001)
      1. Wet cell mass (centrifuged pellet) (measures live and dead cells)
      2. Dry cell mass (centrifuged pellet, dried) (measures live and dead cells)
      3. Turbidity (cloudiness, measured by light transmission through culture) (measures live and dead cells)
      4. Colony count (transfer small amt. culture to plate, grow, count colonies, live cells only)
    5. Asynchronous growth curve (cells in all stages of synthesis and division)
      1. Initial stationary phase (# cells dividing = # cells dying, rich in nutrients, low in wastes)
      2. Exponential growth phase (# cells dividng greatly exceeds # dying, nutrients being used rapidly, wastes begin to accumulate, cells very sensitive to antibiotics)
      3. Maximum stationary phase (# cells dividing = # cells dying, population stabilized at higher level, low in nutrients, high in waste products)
      4. Exponential death phase (# cells dying greatly exceeds # dividing, waste products at toxic levels, few nutrients remain, spore formers may form endospores toward end of max. stationary phase or in exp. death phase)
    6. Synchronous cultures can be established and then maintained by adding fresh media and removing old media to keep cells in exponential growth for study.
  5. Microbial metabolism (energy production and storage mechanisms)
    1. Catabolic reactions (breakdown of larger molecules into smaller molecules, usually releasing energy which is trapped in chemical bonds of ATP)
    2. Anabolic reactions (biosynthesis of larger molecules from smaller molecules, usually requiring consumption of energy in the form of ATP)
      1. Amino acids used to make proteins
      2. Nucleotides used to make nucleic acids
      3. Glycerol and fatty acids used to make lipids
      4. Simple sugars used to make complex sugars and starches (carbohydrates)
      5. Macromolecules used to make new cell components