6.1 Microbial Techniques


Microbial Techniques

-important to take care when culturing microorganisms because:

  • even if the microorganism you are culturing is harmless, a mutation may cause some to become pathogenic
  • there is a risk of contaminating the culture with pathogenic microorganisms from the environment
  • when a pure strain is grown, any entry of other microorganisms from the environment contaminates it

-all equipment must be sterile before the culture is started

-once the culture is grown, it must not leave the lab

-all cultures must be disposed of by sealing in plastic bags and then sterilising at 121 degrees C for 15 mins under high pressure before disposal

-microorganisms must be grown with nutrients, wither using nutrient broth (nutrients in liquid form) or nutrient agar (which is a jelly made from seaweed, sets at 50 degrees C and doesn't melt until 90 degrees C)

-selective medium= specific nutrients so only a certain type of microorganism can be grown in it

-selective medium is useful for identifying bacteria that have been genetically modified

-innoculation= getting microorganism onto the agar or broth is called innoculation and is done using an innoculation loop

-when using nutrient broth, it is stoppered with cotton wool to stop contamination with the air and regularly shaken to keep it aerated , allowing oxygen for the growing microorganism

-to isolate bacteria --> grow under anaerobic conditions, use selective medium or use an indicator medium that causes bacteria to change colour 

Measuring the growth of bacterial cultures

-Cell Counts:

  • counted using a haemocytometer which is a specialised thick microscope slide with rectangular chambers hat can hold 0.1mm3 liquid
  • the sameple of nutrient broth is dilued by half with an equal volume of tryptan blue which stains dead cells blue
  • each corner of the haemocytometer has a square divided into 16 smaller squares
  • the no. of cells in each of the 4 sets of 16 squares is counted and a mean calculated
  • the haemocytometer is calibrated so that the no. of bacterial cells in one 16 square is equal to no of cells x 10^4 per cm^3
  • measurements are taken at regular intervals

-Optical Methods (turbidity)

  • light is passed thorugh a sample
  • the more light that gets through, the les turbid the solution therefore the less bacteria are growing in it
  • the more turbid a solution, the less light that can get through, therefore there is more bacteria in it
  • a calibration curve is produced using a control culture 
  • turbidity is measured and a cell count using a haemocytometer is doen to give a relationship between turbidity and cell growt

-Dilution Plating

  • used to find the total viable cell count
  • an orginal culture is diluted until a point is reached when the colonies can be counted
  • if the no. of colonies is multiplied by the dilution factor then a total viable cell count can be found
  • a mean can then be reached by repeating

-Area and mass of fungi

  • when fungi are grown, the diameter of the mycelium can be measured
  • this can be…


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