Edexcel International A Level Biology

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6.2 Measuring the Growth of Microorganisms

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Measuring the Growth of Microorganisms

  • Bacteria and most other microorganisms are too small to count with the naked eye
  • There are a variety of methods that can be used to count microorganisms and it is important to be able to make an appropriate choice when conducting investigations
  • These methods include
    • Cell counts
    • Dilution plating
    • Measuring area and mass
    • Optical methods

Cell counts

  • A microscope and haemocytometer can be used to count single-celled microorganisms
    • A haemocytometer is a microscope slide with a rectangular chamber that is marked with grid lines
    • The chamber can hold a standard volume of 0.1 mm3
    • Haemocytometers were originally used to count blood cells
      • Haemo = blood
  • Counting cells using a haemocytometer involves the following
    • A nutrient broth is diluted with an equal volume of trypan blue
      • This is a dye that will stain dead cells blue
      • It enables the investigator to only count the living cells
    • The chamber of the haemocytometer is filled with the stained nutrient broth
    • The number of living cells in the four corner squares of the grid are counted
      • Each corner square consists of 16 smaller squares
      • Consistency needs to be used when deciding whether to count cells that are on the lines that border the corner squares
        • E.g. Counting cells on the top and right-hand borders and ignoring cells on the bottom and left-hand borders
    • The mean number of cells from the four corner squares can be calculated
  • The haemocytometer is calibrated to allow the calculation of the number of cells in a known volume of broth
    • Because the haemocytometer chamber can hold exactly 0.1 mm3 of liquid, it is possible to estimate of the cell count in 1 ml of nutrient broth using the following calculation

No. of cells per ml nutrient broth = mean cell count x dilution factor x 104

    • Multiplying by the dilution factor enables calculation of the bacterial cell count in the original broth rather than the diluted broth
    • The multiplication by 104 enables calculation of the bacterial cell count in 1 ml rather than in 0.1 mm3
      • 1 ml = 1 cm3
      • 1 cm3 = 0.1 mm3 x 10 000
      • 10 000 = 1 x 104
  • E.g. in a scenario where a nutrient broth is diluted by a factor of 100 and the four corner grid squares contain 20, 14, 19, and 16 cells
    • This gives a mean cell count of 17.25
    • No. cells per ml nutrient broth = 17.25 x 100 x 104 = 17 250 000 


The living cells in the corner squares of a haemocytometer grid can be counted to determine the number of microorganisms in a standard volume of nutrient broth

Dilution plating

  • This method can be used to determine the total viable cell count in a nutrient broth
    • The nutrient broth is transferred to agar where the bacteria use nutrients in the agar gel to reproduce
    • A single cell that lands on agar reproduces by cloning itself, resulting in a mass of identical cells known as a colony
    • Each microbial colony that grows on agar gel originated with one viable microorganism, so can be counted as one viable cell 
  • Individual microbial colonies can be difficult to identify on an agar plate as they tend to form one large mass
  • This problem can be overcome by diluting the original cultures before transferring the samples to agar; this reduces the number of cells in the original sample so that individual colonies are visible on an agar plate
    • This is why the technique is known as dilution plating
  • To calculate the total viable cell count, the number of colonies are multiplied by the dilution factor
  • A mean can be determined if more than one plate is used

Serial dilutions of microorganisms 1Serial dilutions of microorganisms 2

Dilution plating can provide a way to determine the total viable cell count of a microbial culture

Area and mass of fungi

  • Fungi do not always live as single-celled organisms, but can form a mass of elongated cells known as a fungal mycelium (plural mycelia); this means that the methods described above can be unsuitable for measuring the growth of some fungi
  • Measuring the diameter of individual areas of the mycelium can be used to determine the growth of fungi
    • Petri dishes of agar are inoculated with fungal spores and incubated at a suitable temperature
    • The resulting areas of fungal mycelia are then measured
  • This can be used to compare growth rates in different conditions, e.g. at different temperatures
    • The larger the mean diameter, the greater the growth of the fungi
  • Testing the dry mass of fungi is another effective way to measure fungal growth
    • A liquid nutrient broth is inoculated with fungal spores
    • Samples of the nutrient broth are removed at set time intervals
    • The fungal mycelia are removed by filtering or centrifugation
    • The material is dried in an oven overnight and its mass measured
  • The higher the mass, the more fungal growth has occurred

Optical methods

  • Turbidimetry is a specialised form of colorimetry that can be used as an alternative method to measure the number of cells in a sample
    • Turbidity is a measure of how cloudy a solution is 
      • More turbid = more cloudy
      • Less turbid = less cloudy
    • Colorimetry uses a machine called a colorimeter to shine a beam of light at a sample and measure the amount of light that is either transmitted through or absorbed by the sample
  • The higher the number of cells the more turbid the solution becomes
  • More turbid solutions will absorb more light and allow less light through; this can be measured by a colorimeter
  • This provides an indirect measure of the number of microorganisms present
  • A calibration curve can be constructed by measuring the turbidity of a series of control cultures while also counting the cells in each culture using a haemocytometer; the results are plotted in a graph of turbidity against cell count
  • This curve can then be used to estimate the cell count of unknown samples by measuring their turbidity and then reading their cell count from the graph

Colorimetry Set Up

A colorimeter can be used to determine the turbidity of a solution containing microorganisms. The solution containing bacteria is placed into a container called a cuvette and the amount of light that can pass through, or is absorbed by, the solution can be measured.

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Author: Marlene

Marlene graduated from Stellenbosch University, South Africa, in 2002 with a degree in Biodiversity and Ecology. After completing a PGCE (Postgraduate certificate in education) in 2003 she taught high school Biology for over 10 years at various schools across South Africa before returning to Stellenbosch University in 2014 to obtain an Honours degree in Biological Sciences. With over 16 years of teaching experience, of which the past 3 years were spent teaching IGCSE and A level Biology, Marlene is passionate about Biology and making it more approachable to her students.