Microbiology

• Bacterial Reproduction and Growth

  • Bacteria can undergo binary fission and reproduce quickly, given a suitable environment
  • Yeast may reproduce by budding
  • In optimum conditions bacteria divide every twenty minutes
• Conditions required for microbial growth

  • Temperature
  • Nutrients
  • pH
  • Oxygen requirement
• Temperature


  Bacterial metabolism is regulated by enzymes, the range of 25-45°C is suitable for most bacteria. The optimum for mammalian pathogens is around 37°C, the temperature of the human body
• Nutrients


  In the laboratory nutrients are supplied in nutrient media, which may be provided as a nutrient agar or nutrient liquid broth. The carbon source is usually glucose, and the nitrogen needed for amino acid and nucleic acid synthesis is provided as nitrate ions
• Bacterial growth environments


  • Incubator for growing bacterial cultures
  • Bacteria grown in liquid broth
  • Bacteria grown on nutrient agar
• pH


  Most bacteria favour slightly alkaline conditions, whereas most fungi grow better in neutral to slightly acidic conditions
• Glucose metabolism


  1. Enters glycolysis

  2. Fatty acids undergo beta-oxidation to acetyl-CoA

  3. Glycerol phosphorylated and enters glycolysis as triose phosphate

  4. Amino acids (keto acid) enter Krebs cycle
• Oxygen requirement


  • Obligate aerobes can only survive and metabolise in the presence of oxygen
  • Obligate anaerobes can only survive and metabolise in the absence of oxygen
  • Facultative anaerobes prefer to metabolise in the presence of oxygen but can survive and metabolise in the absence of oxygen
• Obligate anaerobes


  • Clostridium perfringens - grow in wounds, producing toxins that cause gas gangrene. Symptoms include blisters under the skin, foul smelling fluid and jaundice
• Treatment for gas gangrene

  Use of a hyperbaric oxygen chamber with air pressure 2.5 x higher than atmospheric pressure
• How hyperbaric oxygen treatment works

  • 1) Oxygen inhibits respiration and growth of obligate anaerobes like Clostridium bacteria (toxic to the bacteria)
  • 2) Patient's immune system can kill any current bacteria
  • 3) Less pathogenic bacteria means less toxins produced by bacteria - patient begins to improve
• Directly Counting Cells


  1. Serial dilution-total viable count technique

  2. Place 9 cm³ of sterile distilled water into five sterile test tubes

  3. Place 1 cm³ of the original bacterial culture sample into the first tube and gently mix

  4. Transfer 1 cm³ of this 10-fold dilution from the first tube into the second tube containing 9 cm³ of sterile distilled water and gently mix

  5. Repeat this procedure for the remaining tubes

  6. Transfer 0.1 cm³ of each diluted sample onto a sterile nutrient agar plate and spread with a sterile spreader

  7. Repeat this twice more to give a total of 3 plates per dilution

  8. Seal each agar plate with tape but not all the way around and incubate each agar plate at 25°C for 24-48 hours

  9. After incubation, look for the dilution that shows distinct colonies without merging

  10. Count the number of distinct colonies on each plate

  11. Multiply the number of colonies by the dilution factor to give the number of bacteria in the original 1 cm³ bacterial culture sample
• Bacterial colony appearance


  • Merged colonies (clumping)
  • Distinct colonies
• Aseptic Techniques


  • Preventing contamination of pure culture by microbes from the environment
  • Preventing contamination of the environment by cultures being grown
• Inoculating an agar dish

  1. Pass the metal inoculating loop through a flame until it is red hot. Allow it to cool

  2. Hold the bottle containing the bacterial culture in one hand; remove the cap with the little finger of the other hand. Do not place the cap down on the work surface

  3. Flame the neck of the culture bottle for about 2-3 seconds. Dip inoculating loop into bacterial culture

  4. Lift lid of the petri dish to 45° to allow entry of inoculating loop and streak the agar with bacterial culture

  5. Secure the petri dish with adhesive tape; Do not seal all the way around the petri dish as this could lead to anaerobic conditions and potentially lead to the growth of pathogenic organisms

  6. Incubate at suitable temperature 25-37°C for 24-48 hours

  7. Sterilise all equipment after use