culturing microorganisms

• Culturing bacteria

  In order to study bacteria, we often need to grow them in a laboratory setting

  • The cultured bacteria can be used to investigate the action of disinfectants & antibiotics
• Factors affecting the speed of bacterial growth:

  • Temperature - most grow fast in warm environments
  • Nutrient availability - Good supply of nutrients to grow rapidly
  • Moisture - Most Grow fast in moist conditions
  • Oxygen - different types of bacteria either need the presence or absence of oxygen for growth
  • build-up waste
• Bacteria need a mixture of nutrients in order to reproduce, including:

  • carbohydrates (for energy)
  • nitrogen compounds (for protein synthesis)
  • vitamins
  • minerals
• Nutrient broth and agar are two nutrient-rich substances used to grow

  bacteria.

  • Often referred to as 'mediums' or 'media'.

  
• Nutrient broth


  A liquid medium (like water)

  
• Agar:


  A gel medium (like firm jelly)

  
• Agar plate:

  A plastic dish containing a thin layer of agar.

  
• An individual bacterium is invisible to the naked eye, but will multiply rapidly when placed in a growth medium.

  • The resulting group of bacterial cells is called a bacterial colony.
  • Each colony is visible to the naked eye, containing millions of bacteria.

  
• Bacteria grow on agar plates as colonies.

  
• Aseptic techniques:

  A set of procedures used to make sure a culture medium is not contaminated (the presence of unwanted microorganisms), when attempting to culture microorganisms.
• Examples of aseptic techniques:

  • Cleaning surfaces with disinfectant
  • Washing hands with antiseptic
  • Sterilising all instruments, solutions, & mediums.
  • Creating a sterile field using a Bunsen burner
  • Growing bacteria in incubators set to a maximum of 25°C.
• Examples of aseptic techniques:

  Sterilising all instruments, solutions, and mediums before they are used.

  • Sterilisation involves heating objects to a temperature at which all contaminating microorganisms are destroyed.
  • So only desired bacteria are transferred to the loop.
• Examples of aseptic techniques:

  Creating a sterile field using a Bunsen burner.

  • Try to work within the sterile field, and minimise the time that cultures and growth media are open to the environment.
• Sterile field:


  A sterilised area created by the updraft of the flame. 
• Examples of aseptic techniques:

  Growing bacteria in incubators set to a maximum of 25°C.

  • To prevent the growth of harmful pathogens, which prefer body temperature (37°C).
• Inoculation:


  The process of transferring bacteria from a broth to an agar plate.

  
• Agar plates are commonly used to investigate the effect of antiseptics

  or antibiotics on bacterial growth.

  By taking filter paper discs, soaking them in different antiseptic or antibiotic solutions.

  • Then placing them on an agar plate, we can find out how good the different solutions are at killing bacteria.
• Bacteria that are not affected by a substance will grow in the agar plate.

  • bacterial growth will be visible close to the paper discs.
• inhibition zone:


  A clear area surrounding the paper discs, if the Bacteria were affected by the substance & died.

  • A larger zone of inhibition means the substance is more effective at killing the bacteria.

  
• The inhibition zone is calculated using:

  area = π r2
• The effectiveness of each antibiotic at killing the bacteria:

  • Antibiotic A is not effective - there's no zone of inhibition.
  • Antibiotic B is somewhat effective - there's a small zone of inhibition.
  • Antibiotic C is very effective - there's a large zone of inhibition.

  
• Steps to take to culture microorganisms found in different places in the classroom:

  • Swab different areas
  • Sterilise the area & the loop
  • Only remove the lid of the petri dish slightly
  • Spread bacteria onto the agar jelly
  • Place tape on two sides
  • Incubate for at least three days at 25 Celsius.
• Reasons why this test does not prove that toothpaste kills all bacteria on teeth:

  • The test only uses one species of bacteria
  • There're still bacteria on the agar plate.
  • The test is not done on teeth
  • The conditions in the mouth are different
  • Toothpaste is only used on teeth for a short time

  
• • Petri dish & agar sterilised before use to kill unwanted bacteria.
  • Inoculating loop passed through flame to kill other bacteria.
  • Loop used to spread bacterium onto agar.
  • Lid of Petri dish opened as little as possible to prevent microbes from air entering.
  • Sealed with tape to prevent microbes from air entering.
  • Incubate to allow growth of bacteria.

  
• The student concluded that disinfectant D would be the best for using.

  • Give one reason why the student might not be correct:
  • Need more evidence as D may be harmful to people / animals / surfaces.
  • May work differently with different bacteria.
  • Disinfectants may be different concentrations.
  • May not last as long.

  
• • after transferring bacteria to an agar plate, the lid of the petri dish should be lightly taped on
  • the petri dish should be stored upside-down during the incubation period
• the bacterium is not resistance to disinfectant A, bc it has an inhabitant zone around it, if the bacterium was resistant, bacteria would be right up to the edge of the disc