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.