Measuring the Growth of Microorganisms (Edexcel IAL Biology)

Spec point — 6.2

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

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

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.