advanced science topic a

• Branches of microbiology


  • Bacteriology - the study of bacteria
  • Mycology - the study of fungi
  • Virology - the study of viruses
• Prokaryotes


  • Single celled organisms
  • Do not have a nucleus or membrane-bound organelles
  • Genes found on a single loop of DNA
  • Some have plasmids - rings of DNA
  • DNA lacks histone proteins and lies free in the cytoplasm
  • Do not have the "9+2" arrangement of microtubules
• Structures found in bacteria


  • Cytoplasm
  • Ribosomes (size 70s)
  • Nuclear Zone (or Nucleoid)
  • DNA (circular)
  • Plasmid
  • Flagellum
  • Cell Surface Membrane
  • Mesosome
  • Capsule (or Slime Layer)
  • Cell Wall

  
• Gram positive bacteria


  Stain purple, have a thick murein (peptidoglycan) cell wall outside their cell membrane
• Gram negative bacteria


  Stain pink, have a thin layer of periplasm, a thin murein (peptidoglycan) cell wall and an outer membrane containing lipopolysaccharides
• Bacterial nutrition


  • Phototrophs - obtain energy from sunlight
  • Organotrophs - obtain energy by oxidising organic compounds
  • Lithotrophs - obtain energy by oxidising inorganic compounds
• Binary fission


  1. The single chromosome replicates itself forming two identical loops of DNA

  2. The two identical chromosomes move apart from each other

  3. The cell membrane tucks in down the mid line between the two chromosomes

  4. The cytoplasm splits into two new cells
• Growth curve for bacteria

  • Lag phase
  • Log (or exponential) phase
  • Stationary phase
  • Death (or decay) phase
• Generation time (G)


  The time taken for a population of microbes such as bacteria to double in number

  
  

  G = t (time per generation) / n (number of generation)
• Factors affecting the growth of micro-organisms include temperature, pH, oxygen, carbon dioxide, water potential, light, chemicals such as antibiotics, and nutritional factors
• Bacterial groups based on temperature

  • Mesophiles - 20-45°C
  • Thermophiles - >45°C
  • Psychrophiles (cryophiles) - <20°C
• Many microbes need molecular oxygen for respiration and are termed obligate aerobes
• Some microbes are facultative anaerobes and can grow with or without oxygen
• Thermophiles


  • Useful in biotechnology, as their enzymes do not denature easily (e.g. in PCR or biological detergents)
• Psychrophiles (cryophiles)


  Soil bacteria or polar ocean dwellers, can cause food spoilage in refrigerators
• pH


  • Most microbes grow best in neutral pH and die in extreme acid or alkali conditions as their enzymes slow down
  • Optimum pH for different microbes varies from 5 to 7.5
  • A few acid-tolerant species can grow at pH 2, but almost all microbes are killed below pH 4, which is why human stomach acid is so effective
• The pH of a growth medium can change over time due to production of waste acids or alkalis, so it is important to use a pH buffer when growing microbes
• Obligate aerobes


  Many microbes need molecular oxygen for respiration, just as animals do, and will only grow on the surface of a solid agar medium or require a well-aerated liquid medium
• Facultative anaerobes


  Will use aerobic respiration if oxygen is available, but can switch to anaerobic respiration if oxygen is absent, include yeasts and lactic acid bacteria
• Obligate anaerobes


  Die in the presence of oxygen, useful industrially as they will grow throughout a medium, whereas aerobes will only grow on surfaces, or in well-stirred liquid media
• Any of these factors (temperature, pH, oxygen, carbon dioxide, water potential, light, chemicals) can be controlled to select particular microbes, or particular metabolic products, or to control the growth rate
• Carbon dioxide


  Some organisms, including many human pathogens, grow best in increased carbon dioxide levels
• Water potential


  Microbes must have water available for growth to occur. If the water potential of the medium is higher (less negative) than that of the microbial cell, the microbe will take up water by osmosis. If the water potential of the medium is much lower (more negative) than that of the microbial cell, water leaves the cell by osmosis and the microbe may die. This concept has been applied in the preservation of food, where high levels of either salt or sugar have been used to preserve food. Some microbes, known as halophiles, are adapted to grow at very low osmotic potentials.
• Light


  Photoautotrophs require visible light as an energy source. Some other wavelengths can be used to kill microbes, such as short wavelength ultraviolet light to kill pathogens in food shops and hospitals, and X-rays to sterilise medical equipment
• Chemicals, such as antibiotics


  Many chemicals, such as antiseptics and disinfectants, inhibit the growth of microbes. Antibiotics also affect growth – they are either bacteriostatic (prevent bacterial replication) or bactericidal (kill bacteria)
• Nutritional factors required by microbes

  • Carbon, hydrogen, oxygen, nitrogen, sulphur, phosphorus
  • organotrophs need Simple carbohydrates like glucose or sucrose for respiration (and some may be able to digest starch)
  • Minerals, especially a source of nitrogen such as ammonia or amino acids to make proteins
  • Growth factors like amino acids, purines, pyrimidines, vitamins
• Organotrophs


  Require simple carbohydrates such as glucose or sucrose for respiration (and some may be able to digest starch), together with minerals, especially a source of nitrogen such as ammonia or amino acids to make proteins
• Lithotrophs


  Require much simpler inorganic nutrients, such as methane, carbon dioxide, water and minerals
• Bacteriological investigations usually involve growing pure cultures in a nutrient medium under controlled conditions of temperature (usually 37°C) and pH (usually around pH 7.4)
• Components of nutrient media

  • Carbon (e.g. glucose, another simple sugar, or a salt of an organic acid, e.g. sodium ethanoate)
  • Nitrogen (usually amino acids, peptides, or ammonium salts)
  • Sulphur
  • Phosphorus
  • Mineral salts (calcium, potassium, sodium, magnesium, iron)
  • Water
• Heterotrophs gain energy by the oxidation of sugars, photoautotrophs have the power of photosynthesis and require light, and chemoautotrophs gain energy by oxidising inorganic chemicals such as ammonia and nitrite
• Bacteria are usually cultured in a solid medium or a liquid medium, with media solidified with agar, which acts as a gelling agent into which the nutrients are mixed
• Culture containers


  • Wire loops and wires
  • Sterile (autoclaved) Pasteur pipettes and 1.0, 5.0 and 10.0 cm3 pipettes
  • Autoclave (or a good pressure cooker)
  • Constant temperature incubator
  • Inoculating cabinet
• Sterilisation methods


  1. Red heat

  2. Moist heat (autoclave)

  3. Filtration

  4. Ionising radiation
• Preparing liquid media (nutrient broth)

  1. Dissolve meat extract, peptone and sodium chloride in water

  2. Heat to dissolve ingredients

  3. Adjust pH to 7.4

  4. Dispense into containers

  5. Autoclave
• Preparing solid media (nutrient agar)

  1. Prepare in the same way as nutrient broth but add agar

  2. Autoclave

  3. Dispense into plates, slopes or deeps as required
• Plating out


  1. Melt down stored media

  2. Dispense into sterile petri dishes
• Inoculation and culture techniques

  1. Streak plates

  2. Pour plates

  3. Confluent plates

  4. Agar slopes

  5. Agar deeps

  6. Broth cultures
• Agar deeps


  Used to grow anaerobic organisms or to separate aerobic and anaerobic organisms
• Methods of measuring bacterial growth

  • Total counts ( include dead and living cells, all cells included)
  • Viable counts ( only living cells included )