Culturing and counting microorganisms

Aseptic technique

Aseptic technique prevents contamination of the culture and of the person:

• Disinfect the bench before and after; wash hands.
• Work near a Bunsen flame, whose updraught carries airborne microbes away.
• Sterilise inoculating loops by heating until red hot, and let them cool before use.
• Flame the neck of culture bottles after removing and before replacing lids.
• Lift the Petri dish lid only slightly and for as short a time as possible.
• Secure lids with two strips of tape (not sealed all round, so aerobic conditions are kept and anaerobic pathogens are not encouraged).
• Incubate at about 25 °C in schools, which reduces the chance of growing human pathogens.
• Sterilise media and equipment in an autoclave (121 °C under pressure) before use, and autoclave cultures before disposal.

Measuring growth

• Direct cell count: count cells in a known volume using a haemocytometer (counting chamber) under a microscope. Counts living and dead cells.
• Dilution plating (viable count): make a serial dilution, spread a known volume of a dilution on agar, incubate and count colonies. Each colony is assumed to come from one living cell. Choose a plate with 30 to 300 separate colonies.
• Mass: filter and dry cells and weigh them (dry mass).
• Turbidity: measure cloudiness with a colorimeter (absorbance or optical density). Quick, but counts dead cells too, so it needs a calibration curve.

Calculating from dilution plating

Cells per cm³ in the original culture = number of colonies / volume plated (cm³) x dilution factor

A tenfold serial dilution repeated 5 times gives a dilution factor of 10⁵.

Worked example

0.1 cm³ of a 10^-5 dilution is spread on a plate and 45 colonies grow. Estimate the number of bacteria per cm³ in the original culture.

1. Colonies per cm³ of the dilution = 45 / 0.1 = 450
2. Multiply by the dilution factor: 450 x 10⁵

Answer: 4.5 x 10⁷ bacteria per cm³.

Check yourself

1. 0.2 cm³ of a 10^-4 dilution gives 60 colonies. Estimate the number of bacteria per cm³ in the original culture.
   Show answer

   3000000 per cm³. 60 / 0.2 = 300; 300 x 10⁴ = 3 x 10⁶ per cm³.

2. 1 cm³ of culture is added to 9 cm³ of sterile water, and this is repeated three times in series. What is the total dilution factor?
   Show answer

   1000. Each step is tenfold: 10 x 10 x 10 = 1000.

3. Why is an inoculating loop heated until red hot before use?
   • To kill any microorganisms on it
   • To make the agar melt
   • To help bacteria grow faster
   • To sterilise the bench
   Show answer

   To kill any microorganisms on it. Heating sterilises the loop.

4. Why are school cultures incubated at about 25 °C rather than 37 °C?
   • Because bacteria cannot grow at 37 °C
   • To kill the bacteria
   • To make colonies merge
   • To reduce the chance of growing human pathogens
   Show answer

   To reduce the chance of growing human pathogens. 37 °C is human body temperature, ideal for pathogens.

5. Which method counts only living bacteria?
   • Measuring turbidity with a colorimeter
   • Dilution plating and counting colonies
   • Using a haemocytometer
   • Measuring dry mass
   Show answer

   Dilution plating and counting colonies. Only living cells divide to form colonies.

6. What is a limitation of measuring turbidity to estimate bacterial growth?
   • It takes several days
   • It only works on agar plates
   • It needs colonies to be counted
   • It includes dead cells as well as living ones
   Show answer

   It includes dead cells as well as living ones. Dead cells still make the broth cloudy.

7. Why is a Petri dish lid taped with two strips rather than sealed all round?
   • So oxygen can enter and anaerobic pathogens are not encouraged
   • So the agar dries out quickly
   • So the lid can fall off
   • So bacteria can escape
   Show answer

   So oxygen can enter and anaerobic pathogens are not encouraged. Keeping aerobic conditions discourages harmful anaerobes.

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Sources

• Pearson Edexcel International Advanced Level in Biology specification (XBI11/YBI11, first teaching 2018)

Last updated 7 November 2026. Spotted a mistake? Tell us.