FINAL EXAM- Microbio 251

• why do microbes need staining?

  Most microbes are translucent and hard to see under the microscope

  
  

  Staining provides contrast to the cell and cell structures which aids in identification. It also provides information about bacteria
• simple staining

  Basic dyes (+ charge) stain the cell

  
  

  Acidic dyes (- charge) stain the background
• gram stain

  Determines whether a bacteria is gram-positive or gram-negative depending on the color
• acid-fast stain

  Determines if the bacteria has a mycolic-acid coat
• what instruments are used to visualize microbes?

  Microscopes are used to visualize microorganisms (light microscopes and electron microscopes are used)

  
  

  Also oil immersion is used to prevent light refraction

  
  

  Limit for resolution for light microscope is 0.2 microns
• gram stain(again)

  Separates bacteria into 2 major groups: gram positive and gram negative; reflects differences of the chemical structure of the cell wall
• gram stain reagents and steps

  Crystal violet: Primary stain added to specimen

  -Stains cell purple or blue

  
  

  Iodine: Makes dye less soluble so it adheres to cell walls

  -Cell remains purple or blue

  
  

  Alcohol: Decolorizer washes away stain from gram-negative cell walls

  -Gram-positive cells remain purple/blue

  -Gram-negative cells are now colorless

  
  

  Safranin: Allows dye adherence to gram-negative cells

  -Gram-positive cells remain purple/blue

  -Gram-negative cells appear pink/red
• gram-variable species

  Gram-variable species don't gram stain +/-. Aged cultures stain variably because they lose peptidoglycan integrity.
• Peptidoglycan (part of the cell wall)

  Unique to bacteria; major component of cell wall

  
  

  Protects cell from harsh conditions in the outside environment

  
  

  Layer of meshwork or fabric

  
  

  Composed of long chains of alternating molecules of NAG and NAM

  
  

  Lay down alternating molecules of NAG and NAM, then cross link them (tetra/pentapeptide chains), and stack sheets; gram positive, peptide interbridge; gram-negative - direct links
• gram positive cell walls

  contain a thick cell wall (many layers, 30 to 100 nm) of peptidoglycan

  
  

  Teichoic acid molecules enhance rigidity and binding (exclusive to gram-positive)

  
  

  Porous, fully permeable to many small molecules (ex. Sugars, amino acids, ions)
• gram negative cell walls

  more complex

  
  

  Thin layer of peptidoglycan (~4 nm)

  
  

  May be a barrier to some antibiotics

  
  

  Contains outer layer; bilayer composed of phospholipid and lipopolysaccharide (LPS); endotoxin
• microorganism distribution

  Pretty abundant

  
  

  Recycles Nitrogen, Phosphorus, Carbon and Sulfur (these things are pretty essential for Earth)

  
  

  Microbes are used in food production (fermentation - wine; it helps make bread rise too)

  
  

  Degrades pesticides

  
  

  Microbes are used in the production of medical products, agricultural, and industrial products

  -Ex: Antibiotics such as penicillin, steroids, organic solvents and acids, enzymes

  
  

  Microbes are used in recombinant protein production

  
  

  Gene therapy
• taxonomy

  Classification/description, identification and nomenclature

  
  

  Based on shared characteristics

  
  

  Establishes phylogenetic or evolutionary relationships

  
  

  Provides identification schemes

  
  

  Continually evolving

  
  

  Provides consistent naming terminology for related organisms
• taxonomic hierarchies

  Domain, Kingdom, Phylum, Class, Order, Family, Genus, Species

  
  

  Dear King Phillip Came Over For Good Spaghetti
• naming system

  developed by Linnaeus in the 18th century

  Binomial system of Genus species

  Naming is often shortened to First letter of genus name and species
• species

  basic unit of classification

  
  

  Includes a group of related strains and isolates

  
  

  Individual strains within a species vary in minor characteristics
• strain

  genetic variant; subtype or subculture of a biological species
• cell wall

  Cell shape in prokaryotes in maintained by the cell wall

  
  

  Cell wall is a rigid structure that surrounds the cell
• plasmids

  Smaller, circular, double stranded DNA molecule(s) in many Bacteria and Archaea; Copy number (# per cell) varies

  
  

  Self-replicating

  
  

  Code for resistance to antibiotics, disinfectants, ability to metabolize unusual nutrients, or ability to produce certain toxins

  
  

  Contains non-essential accessory genes (bonus DNA, can give prokaryotes extra advantages)

  
  

  Extrachromosomal piece of DNA, is transferable (via conjugation and transformation), can also be gained or lost

  
  

  Common in microbial world, codes for useful phenotypes (such as antibiotic resistance) but are not absolutely necessary
• genes on plasmids

  Pilus-synthesis genes (transfers via conjugation), can still transfer via transformation without pilus

  
  

  Origin of transfer gene

  -Plasmid needs pilus-synthesis and origin of transfer genes in order to be transferable

  
  

  Origin of replication

  
  

  Some antibiotic resistant genes are found on R plasmids
• plasmid replication

  One strand of F plasmid DNA transfers from donor cell to recipient cell

  
  

  Donor synthesizes complementary strand to restore plasmid. Recipient synthesizes complementary strand to become F⁺ cell with pilus
• endospores

  Resting cells

  
  

  Created in an inhospitable environment

  
  

  Protects genome until environment is favorable

  
  

  Formed in cytoplasm

  
  

  Contains DNA

  
  

  Highly resistant to heat and harsh chemicals

  -Not indestructible

  
  

  Organism can remain dormant for over 100s of years
• bacteria that are spore formers

  Bacillus subtilis

  Bacillus anthracis

  Clostridium botulinum

  Clostridium tetani

  Clostridium difficile
• how are endospores killed?

  Endospores are killed/eradicated with sterilants (chemicals that achieve sterilization) and heat (autoclaving)
• prokaryotes

  Have no membrane-bound nucleus (nucleoid)

  
  

  Capsule, cell wall

  
  

  Single, circular chromosome
• eukaryotes

  Have a membrane-bound nucleus

  
  

  More complex; contains an abundance of membrane-bound organelles (endoplasmic reticulum, golgi apparatus, mitochondria, lysosomes, chloroplasts in plants)

  
  

  Multiple, rod-shaped chromosomes
• similarities between prokaryotes and eukaryotes

  Chemically similar (nucleic acids, proteins, lipids, carbohydrates)

  
  

  They both have ribosomes

  
  

  Both have DNA

  
  

  Both have a cell membrane
• virus structure

  Genetic information (DNA or RNA, not both) contained in protein coat called a capsid, subunits called capsomeres

  
  

  Requires host to replicate
• Steps of viral replication

  attachment(adsorption), virus enters target cell, virus uncoated, assembly and maturation, release
• attachment(adsorption)

  Virus binds to one or more receptor sites on host cell (lock and key interaction)

  
  

  Limited host range
• virus enters target cell

  Entire virus enters (bacteriophages just inject DNA)

  
  

  Enveloped virus enters via fusion with host cell membrane or endocytosis (nucleocapsid released directly into cytoplasm)

  
  

  Non enveloped virus enters only via endocytosis
• virus uncoated

  genome released from nucleocapsid
• assembly and maturation

  Spontaneous self assembly when viral nucleic acid and capsid proteins accumulate in host cell

  
  

  Site of assembly varies with virus types and affects release

  
  

  Synthesis of viral nucleic acids & proteins
• release

  Most enveloped viruses leave via budding (Viral protein spikes insert into host cell membrane; matrix proteins accumulate; nucleocapsid extruded; covered with matrix protein and liquid envelope; some obtain envelope from organelles)

  
  

  Non enveloped viruses released when host cell dies, often by apoptosis initiated by virus or host - viral particles spill out
• gene transfer

  Process by which new donor genes are introduced into a recipient cell
• the 3 mechanisms of gene transfer

  transformation, transduction, conjugation
• transformation

  Uptake of free cell ("naked") DNA from environment (the DNA may be fragments of chromosomal DNA or plasmids)

  
  

  Competent cells; able to take up DNA (most bacterial cells are competent)

  
  

  May remain separate as a plasmid or be incorporated into host genome

  
  

  If DNA is integrated into genome, the bacterium gains new phenotypic properties

  
  

  Important mechanisms for acquiring virulence factors and antibiotic resistance (ex: penicillin-resistance gonorrhea is a result of transformation)

  
  

  Does not require donor to be alive
• transduction

  Transfer of DNA from one bacterium to another via bacteriophage

  
  

  Bacteriophage infects bacterial cell, normally packages its own phage DNA during assembly

  
  

  Occasionally packages fragment of host chromosomal DNA

  
  

  Transfers host chromosomal DNA to other bacteria

  
  

  Recombination integrates new DNA into chromosome

  
  

  Now have recombinant strain (transducing particle)

  -Generalized: Phage picks up random piece of host DNA during assembly and transfers it to a new host cell

  -Specialized: A specific piece of host chromosome is excised as part of the phage genome when phage enters lytic cycle

  
  

  Does not require donor to be alive
• conjugation

  Pilus of donor cell attaches to the recipient cell. Pilus contracts, draws cells together to make contact with one another

  One strand of F plasmid DNA transfers from donor cell to recipient cell

  Donor synthesizes complementary strand to restore plasmid. Recipient synthesizes complementary strand to become F⁺ cell with pilus

  Conjugation pilusfrom donor brings both cells into contact

  Only a single strand of the plasmid is transferred, which is then made a double strand

  Both bacterial cells need to be alive for this process to work
• the significance of gene transfer

  Can allow for antibiotic resistant bacteria to transfer plasmids to other bacteria so they can become resistant