6-4-13-factors-affecting-the-growth-of-microorganisms

Practical: Factors Affecting the Growth of Microorganisms

• To investigate the effect of different factors e.g. temperature or pH, on the growth of microorganisms

• The agar plates are prepared, using the same procedure as discussed in 6.4.10, to avoid contamination

• After the bacteria have been cultured in the desired conditions, it may become apparent that they are too numerous to count as there are so many colonies or the colonies overlap, forming a lawn

• A solution to this problem is to use a serial dilution method to dilute the bacteria in the broth before plating them onto the agar

Serial dilutions

• Serial dilutions are created by taking a series of dilutions of a stock solution (sample of the microorganism culture broth). The concentration decreases by the same quantity between each test tube

  • They can be created to any chosen dilution factor, but most commonly, either by ‘doubling dilutions’ (where the concentration is halved between each test tube) or a desired range (e.g. 0, 2, 4, 6, 8, 10 mmol dm^-3)

• Serial dilutions are completed to create a less dense culture of cells.

  • This means more countable colonies when studying bacteria or yeast populations

In order to study cultured microorganisms, it might be necessary to produce a serial dilution so that colonies are easier to see.

Investigating the effect of temperature on the growth of microorganisms

Apparatus

• Sterile agar plates

  • The agar can be made sterile by boiling

• Diluted bacterial broth with a concentration of 1 x 10⁸ CFU mm^-3

  • Colony-forming unit (CFU): a live bacterial cell that is able to reproduce and form a colony on the agar plate

• Pipettes

• Spreaders

• Bunsen burner

• Gloves

• Goggles

• Incubator

• Fridge

Method

• Spread a sample of the diluted bacterial broth onto the surface of each of the sterile agar plate

• Tape the lid shut

• Keep three of the agar plates in the fridge overnight 5°C

• Keep three of the agar plates in the incubator overnight at 25°C

• Remove the agar plates the next morning and count the number of colonies, keeping the lid on as you count

• Calculate the average number of colonies that have formed and compare the results at each temperature

Results

• Overnight, the bacterial colonies will grow large enough that they can be easily counted

• The bacteria that were cultured at 25°C are expected to have developed at a much faster rate with many more colonies visible

• The bacteria cultured at 5°C are expected to have formed fewer colonies which are much smaller in size, or even no colonies at all

• This difference is due to the fact that 25°C provides a temperature close to the optimum for enzyme activity in the bacteria

• As a result, the rate of growth is much faster than those cultured at 5°C as enzymes’ cellular reactions will be very slow

These results show the difference in bacterial colony growth at 5°C compared to 25°C

Variations on this investigation

• We can investigate the effect of pH and nutrient availability using a very similar method to the one detailed above.

  • pH can be altered using different buffer solutions with different pH levels to the broth

  • Nutrient availability can be altered by using different agar plates with different nutrient contents

• It is also possible to use turbidity as a measure of growth instead of counting the colonies