Microbiology

• Bacteria


  • Prokaryotic
  • Smallest organisms with a cellular structure
  • Average diameter about 1µm
  • Range in length from 0.1µm to 10µm
  • Occupy many environments such as soil, water, air, and on animals and plants
• Importance of bacteria to man

  • Cause diseases
  • Fundamental to the recycling of nutrients through the decay of organic materials
  • Used to manufacture food and drinks (bread, cheese, yoghurt, wine, and beer)
  • Used to make antibiotics, enzymes, and insulin
• Bacterial cell structures


  • Peptidoglycan cell wall
  • Plasmisa
  • Nuclear material
  • Ribosomes
  • Plasma membrane
  • Outer membrane
  • Flagella
  • Capsule
  • Pili
  • Mesosome
• Peptidoglycan cell wall


  • Provides support, mechanical strength, and rigidity
  • Protects cell from bursting
• Plasmisa


  Exchange of DNA between bacterial cells
• Nuclear material


  Replication and expression
• Ribosomes


  Site of protein synthesis
• Plasma membrane


  • Removal of waste products from the body
  • Acts as a semipermeable membrane
• Outer membrane


  Protection against harsh environments
• Flagella


  Helps the cell move and spin
• Capsule


  Protection against phagocytic engulfment, osmotic stress, attacks by anti bacterial agents, harsh environments
• Pili


  Fixes bacteria to surfaces
• Mesosome


  DNA replication, separation of nucleotides, oxidative phosphorylation
• Many bacteria have a thick layer of jelly-like material surrounding them called a capsule or slime layer
• The capsule is made of polysaccharides which absorb water to form a slimy material
• The capsule protects the bacterium from attack by viruses and from antibodies
• Endospores


  • Thick walls and long lived
  • Resistant to heat and shortwave radiation
  • Produced during unfavourable conditions to aid survival
  • Reason bacteria responsible for botulism persists in improperly sterilised cans bottles and then is able to multiply within them
• Bacterial cell wall types

  • Gram positive
  • Gram negative
• Gram positive bacterial walls


  • Thicker peptidoglycan layer on top
  • Membrane on bottom layer
  • No outer membrane
• Gram negative bacterial walls

  • Thinner peptidoglycan layer in the middle
  • Membrane on bottom layer
  • Outer membrane on top
• Gram staining


  • Gram positive bacteria stain purple as their cells wall retain crystal violet
  • Gram negative bacteria stain red by the counterstain as the cell walls don't retains crystal violet
• Gram negative bacteria have a more complex wall structure so when treated with acetone the gram negative bacteria lose their outer lipopolysaccharide membrane, washing the crystal violet stain/iodine complexes from the cell exposing the inner peptidoglycan layer which stains red with the counterstain safranin
• Criteria for classifying bacteria

  • Shape
  • Staining characteristics
  • Size
  • Metabolic features
  • Antigenic features
  • Genetic features
• Bacterial shapes


  • Cocci - spherical
  • Spirilla - spiral
  • Bacilli - rod
  • Vibrio - comma
• Only bacteria and fungi can be cultured on agar plates in the laboratory
• Reasoning behind aseptic technique


  • Flame loop until red hot
  • Disinfect bench
  • Flame neck of tube
  • Work next to a roaring flame
  • Don't remove the lid of the dish totally
  • Hold open bottles at an angle
  • Don't mouth pipette
  • Sterilise agar plates in an autoclave
• Aseptic technique


  • Prevent contamination of the environment by the microbes being handled
  • Prevent contamination of the microbes being cultured by unwanted microbes from the environment
• Equipment sterilisation methods


  • Autoclave at 121oC for 15 minutes
  • Irradiated for the heat stable plastics
• Factors affecting growth of microbes

  • Nutrients
  • pH
  • Oxygen
  • Temperature
• Nutrients required for microbial growth

  • Water
  • Vitamins and minerals and micronutrients
  • Respiratory substrate
  • Source of carbon, nitrogen, and phosphorus
• pH range for microbial growth

  Between 5 and 7.5
• Oxygen requirements for microbes

  • Obligate aerobes
  • Facultative anaerobes
  • Obligate anaerobes
• Temperature ranges for microbial growth

  • Thermophiles (above 45C)
  • Mesophiles (20C to 45C)
  • Psychrophiles (below 20C)
• Isolating individual species from a mixed population

  1. Spread a mixed culture so thinly on a plate of solid media that individual cells are separated and will grow into separate, distinct colonies

  2. Use a sterile wire loop to make three streaks of mixed culture near the edge of an agar plate

  3. Resterilise loop in a bunsen burner flame, then streak three more times at an angle across the previous streaks

  4. Repeat several times

  5. Incubate plate for several days

  6. Pick a sample of a clearly separate colony off the plate with a sterile loop and streak onto a fresh sterile plate to obtain a pure culture
• Counting microorganisms


  1. Dilute sample as there are usually far too many microbes in the original suspension to count

  2. Make total counts using a microscope sample of the culture

  3. Make viable counts by transferring a small volume of diluted sample to a sterile petri dish and spreading over the surface of the agar, then incubating and counting the colonies
• Serial dilution


  • Take a known volume of stock and place it into a known volume of distilled water to produce a 10-fold dilution
  • Repeat this process sequentially using more and more dilute solutions to produce further 10-fold dilutions
• Plate count


  Transfer a small volume from specific dilutions to sterile petri dishes, spread over the agar surface, incubate, and count the colonies to estimate the number of viable cells in the original sample
• Dilutions which contain too many bacteria will give carpets of growth or uncountable numbers of colonies, while some dilutions will contain too few bacteria to give an accurate count
• It is usually impractical to count more than about a hundred colonies on a petri dish
• As each colony arises from one cell, the number of colonies can be used to calculate the number of bacteria in the dilution and therefore the number in the original sample