6.1

• What are aseptic techniques?

  Microbiological techniques used which minimise or prevent contamination by microorganisms, used to produce uncontaminated cultures so results are reliable and repeatable.
• Basic aseptic techniques

  1. Wipe surfaces with antibacterial cleaner

  2. Set up Bunsen burner nearby to create convection currents that prevent microbes from entering culture

  3. Flame inoculating loop and neck of bottles before use

  4. Minimise time that vessels containing bacteria are open

  5. Sterilise all equipment e.g. using an autoclave

  6. Wear protective clothing
• How to culture microorganisms

  1. Transfer bacteria to agar plate using sterile inoculating loop or pipette

  2. Tape lid on at 2 ends then invert the dish and incubate. In school lab conditions, ensure dish is not airtight and do not incubate above 25°C to avoid growth of pathogens
• Spread plate

  Distribute microorganisms evenly with a sterile spreader
• Streak plate

  Aim to obtain single colonies by rotating the plate to build layers of the culture on at least 3 separate streaks
• Types of nutrient medium

  • Contain nitrogen, carbon and minerals, often enriched with protein from extract of yeast, blood or meat, may be liquid broth or solid agar
  • Selective mediums contain highly specific nutrient balance, only certain microorganisms grow
• Advantages of using a broth medium

  • Can provide anoxic and oxic conditions depending on the depth, which helps to identify microbes/determine their optimum conditions
  • Can grow a very large volume of bacteria
• Advantage of using agar as the medium

  • Can obtain a single, discrete pure colony for study
• Name the 4 phases of a bacterial growth curve

  • Lag phase
  • Log phase
  • Stationary phase
  • Death phase
• What happens during the lag phase?

  Microorganisms need to adjust to the environment before reproducing so population size only increases slowly
• What happens during the log phase?

  After every round of division, population size doubles (exponential growth)
• What happens during the stationary phase?

  Reproduction rate = Death rate, so population size stabilises at its maximum
• What happens during the death phase?

  Microorganisms die due to buildup of toxic waste products and lack of nutrients
• Name 3 methods to estimate the growth of a bacterial culture

  • Cell count
  • Turbidity measurement (type of colorimetry to measure opacity)
  • Dilution plating
• How to conduct a cell count

  1. Dilute broth sample with equal volume of trypan blue to stain dead cells blue

  2. Use a calibrated haemocytometer with volume 0.1mm3, count the cells in each of the sets of squares and calculate mean

  3. Number of bacterial cells = number counted x 104 per cm3
• How to conduct a turbidity measurement

  1. Use colorimeter, measure absorbance or % transmission of samples with known microorganism count

  2. Plot calibration curve: absorbance/% transmission (y-axis), number of microorganisms (x-axis)

  3. Record absorbance/% transmission of unknown sample, interpolate graph
• How to conduct dilution plating

  1. Grow a colony from a single microorganism

  2. Perform serial dilution with distilled water to see single colonies

  3. Prepare a lawn plate and count colonies

  4. Number of cells = number of colonies x dilution factor
• Suggest advantages and disadvantages of using a cell count

  + only counts living cells

  – slow

  – expensive equipment

  – large margin for human error
• Suggest advantages and disadvantages of using a turbidity measurement 


  + quick

  + can be conducted in the field

  – expensive equipment

  – counts both living and dead cells

  – assumes equal density of cells across culture
• Suggest advantages and disadvantages of dilution plating

  + no complex or expensive equipment needed

  + only counts living cells

  – incubation period needed (slow)
• What is Agar cultures?

  Culturing organisms using a solid agar jelly medium
• What are broth cultures?

  The culturing of microorganisms using a liquid medium
• What is dilution plating?

  The process of diluting a sample of microorganisms by a known amount so that the individual cells can be counted
• What is a growth medium?

  A sterile liquid or gel which is used to support the growth of microorganisms by providing required nutrients in the correct quantities