Bacteriology AB2

• Cocco-bacillus


  Short, round, rod
• Gram (+)


  Gram-stain is retained by the thicker cell wall
• Gram (-)


  Thin cell wall doesn't retain stain, is then stained pink using counter stain
• Gram + and Gram - cell walls contain peptidoglycan which provides the primary structural strength to the cell wall
• Gram + and Gram - bacteria

  Possess an inner cytoplasmic membrane
• Gram (-) bacteria


  Also possess an outer lipid membrane containing lipopolysaccharide (LPS), making the cell wall more impermeable to factors such as antibiotics
• Endotoxins


  Lipopolysaccharides found in the cell wall of Gram-negative bacteria, which can induce inflammation and fever as an immune response in higher organisms
• Acid-fast


  There is another group of organisms (eg. Mycobacterium spp) that have a cell wall similar to Gram-positive organisms but with additional components including increased lipid content that alters the staining properties of these bacteria
• Gram staining


  1. Before staining → neither is visible

  2. Crystal violet → both purple

  3. Iodine as dye fixative → both purple

  4. Alcohol destain → +ve is purple and -ve lost dye

  5. Fuchsin or safranin stain → +ve purple and -ve pink
• Ziehl-Neelsen stain


  1. Acid-fast → PINK

  2. Non-acid fast → BLUE (if methylene blue is used)
• Capsule


  Protective coating secreted outside of the normal bacterial cell wall, normally composed of polysaccharides but some are made of peptides/amino acids
• Capsule


  • Highly hydrated
  • May loosely bind bacterial cells together in microcolonies or biofilms
• Flagella


  Filamentous protein organelles which are primarily responsible for bacterial motility
• Flagella


  • Anchored into the bacterial cell wall; ~1-15 µm long
  • Made up of flagellin monomers that assemble as polymers to form a helical structure attached to a flagellar motor embedded in the cell membrane and cell wall
• Flagellin


  Structural protein of the flagellum, a surface filament, recognised by TLR5 to induce inflammation, often known as H- antigens and can be used for identification of some bacteria, such as Salmonella and Listeria
• Fimbriae or pili


  Hair-like adhesins which project from the surface of the bacterial cell
• Fimbriae or pili


  • Confer binding to specific host cell surface receptors, extracellular matrix components or extracellular ligands
  • Some specialised pili (e.g. F pili) are involved in bacterial conjugation, a process by which genetic information is transferred between donor and recipient bacteria
• Microbial-associated molecular patterns (MAMP)


  Recognised by specific host pattern recognition receptors (PRRs) in the host, including Toll-like receptor
• Toll-like receptor recognition of bacterial components

  • TLR1 – lipopeptides
  • TLR2 – lipoproteins and lipoteichoic acids
  • TLR4 – Lipid A of LPS
  • TLR5 – Flagellin monomers
  • TLR6 – lipopeptides
  • TLR9 – unmethylated repeated 'CG' motifs in bacterial DNA
  • TLR13 – may recognise specific bacterial ribosomal RNA?
• Cytoplasmic membrane


  Phospholipid bilayer that can exist in many different chemical forms as a result of variation in the groups attached to the glycerol backbone
• Cytoplasmic membrane


  • Permeability barrier: prevents leakage and functions as a gateway for transport of nutrients into, and wastes out of the cell
  • Protein anchor: site of many proteins that participate in transport, bioenergetics, and chemotaxis
  • Energy conservation: site of generation and use of the proton motive force
• Peptidoglycan


  Provides the primary structural strength to the cell wall, consists of chains of alternating N-acetyl glucosamine (NAG) and N-acetyl muramic acid (NAM) residues that are regularly cross-linked through peptide bridges to form a rigid permeable mesh
• LPS (lipopolysaccharide)


  Hydrophobic barrier that resist phagocytosis, Lipid A acts as a PAMP that is recognised by PRRs, including Toll-like receptor 4 (TLR4) leading to inflammation
• Bacterial Sporulation


  Occurs during the stationary phase and is a response of some bacteria to nutrient limitation or other stress, Spores are dormant structures which resist adverse conditions of heat, dehydration or freezing and survive many chemicals toxic to actively growing bacteria
• During sporulation


  1. Structural and biochemical alterations occur within the bacterium

  2. Many components of vegetative cells disappear and a thick wall is formed to protect the genetic material along with transcription and translation machinery

  3. Germination of spores occurs under appropriate 'positive' environmental conditions, e.g. moisture and nutrients
• Binary fission


  Bacteria multiply by forming a new cell membrane and cell wall, the chromosome duplicates and segregates, and a septum (new cell wall) splits the bacterial cell into two compartments, each containing a complete bacterial chromosome
• Peptidoglycan Synthesis and Bacterial Cell Division

  Preexisting peptidoglycan needs to be severed to allow newly synthesised peptidoglycan to form, Small openings in the wall are created by autolysins, New cell wall material is added across the openings, Transpeptidation: final step in cell wall synthesis, Forms the peptide cross links/cross-bridges between Naceytylmuramic acid residues in adjacent glycan chains, Inhibited by β-lactam antibiotics eg. penicillin
• Extracellular and Intracellular lifestyles

  Most bacteria are extracellular, However, a subset has evolved to also infect and multiply inside eukaryotic cells (even within phagocytes that normally engulf and destroy bacteria), Facultative intracellular bacteria within phagocytes (eg. Salmonella and Mycobacterium), Obligatory intracellular (they have to replicate in eukaryotic cells) (eg. Chlamydia and Rickettsia)
• Bacterial growth conditions


  • Nutrients
  • Temperature
  • Atmosphere/Oxygen
  • pH
  • ion concentrations
  • Hydration
• Cardinal temperatures


  The minimum, optimum, and maximum temperatures at which an organism grows
• Oxygen requirements


  • Obligate aerobes
  • Microaerophiles
  • Facultative (an)aerobes
  • Aerotolerant anaerobes
  • Obligate anaerobes
• Acidophiles


  Organisms that grow best at low pH (<6), some are obligate acidophiles with membranes destroyed at neutral pH
• Alkaliphiles


  Organisms that grow best at high pH (>9), some have sodium motive force rather than proton motive force
• Typical bacterial growth curve

  1. Lag phase

  2. Exponential or log phase

  3. Stationary phase

  4. Death phase
• Exponential growth


  Growth of a microbial population in which cell numbers double at a constant time interval
• Most bacteria have shorter generation times than eukaryotic microbes
• Direct microscopic counting


  Limitations: Cannot distinguish between live and dead cells without special stains, Small cells can be overlooked, Phase-contrast microscope required if a stain is not used, Cell suspensions of low density (<106 cells/ml) hard to count, Motile cells need to immobilised, Debris in sample can be mistaken for cells, Time consuming
• Flow cytometer


  Microscopic counting where cells are counted in liquid samples, Uses laser beams, fluorescent dyes, and electronics, Requires expensive machine
• Viable Counts


  Measurement of living, reproducing population, Two main ways to perform plate counts: To obtain an appropriate colony number, the sample to be counted should always be diluted to have a manageable number to count on the plate, When too many colonies are present you can't see colony morphology properly, Not all cells may growth because of competition for space and nutrients on plates, Mixed cultures might be overlooked eg. small and large colonies
• Turbidimetric Methods


  Indirect, rapid, and useful method of measuring microbial growth, Turbidity proportional to cell number (within limits), Most often measured with a spectrophotometer and measurement referred to as optical density (O.D.), Quick and easy to perform, Typically do not require destruction or significant disturbance of sample, Sometimes problematic (e.g., microbes that form clumps or grow as biofilms), Not accurate at high densities, Doesn't show if culture mixed, Doesn't differentiate between live and dead cells