Edexcel A Level (IAL) Biology -6.4 Core Practical 13: Rate of Growth of Microorganisms- Study Notes- New Syllabus

CORE PRACTICAL 13 

Investigating the Rate of Growth of Microorganisms in a Liquid Culture

🌿 Introduction

This practical explores how microorganisms grow in a liquid nutrient broth and how to measure their growth rate safely. The focus is on observing changes over time, using methods like turbidity, cell counts or viable counts, while following proper ethical and safety guidelines.

🌱 Aims of the Practical

• To culture microorganisms in liquid medium.
• To measure changes in population size over time.
• To apply safe handling and disposal methods.
• To understand factors influencing microbial growth.

🧪 Materials Needed

• Sterile nutrient broth
• Sterile culture of a safe organism (commonly E. coli K-12 or Bacillus subtilis)
• Sterile flasks or universal bottles
• Colorimeter or spectrophotometer
• Pipettes and tips
• Incubator set at suitable temperature (usually 25°C for safety)
• Aseptic equipment: Bunsen burner, inoculating loop
• Labels, marker, timer
• Disinfectant for cleanup

🔬 Aseptic Technique

• Work near a flame to create an updraft.
• Flame mouth of bottles when opening and closing.
• Sterilise loops until red hot.
• Avoid leaving containers open.
• Disinfect benches before and after the experiment.
• Wash hands and wear protective clothing.

🌡️ Method Overview

1. Preparation of Liquid Culture

• Pour sterile nutrient broth into labelled flasks.
• Inoculate each flask with a known and consistent volume of the microorganism culture.
• Swirl gently to mix.
• Set up a control flask containing sterile broth only.

2. Incubation

• Incubate the flasks at 25°C (safe for school labs).
• Ensure lids are loose if required for oxygen exchange.
• Avoid shaking by incorrect handling; gentle swirling only when necessary.

3. Measuring Growth Over Time

Measurements are usually taken every 20–30 minutes or hourly.

a. Turbidity (Optical Density)

• Transfer a small sample into a cuvette.
• Measure absorbance using a colorimeter at a fixed wavelength.
• Increasing turbidity means increasing cell number.

b. Viable Cell Count (Dilution Plating)

• Dilute the sample serially.
• Plate each dilution on nutrient agar.
• Count colonies after incubation (only living cells counted).

c. Direct Cell Count

• Use a haemocytometer to count cells under a microscope.
• Measures total cells, including dead ones.

d. Dry/Wet Mass

• Centrifuge and weigh biomass.
• More laborious; less common for school-level practicals.

📊 Plotting the Growth Curve

• Record growth readings against time.
• Plot time on the x-axis and population estimate (OD, mass, or cell count) on the y-axis.
• Expected curve shows: Lag phase → Exponential → Stationary → Death.

🧬 Factors Affecting Growth in the Culture

• Temperature
• pH
• Nutrient availability
• Oxygen supply
• Waste buildup

Controlling these ensures reliable and valid data.

🧷 Safety and Ethical Considerations

• Use non-pathogenic microorganisms only.
• Keep cultures sealed whenever possible.
• Dispose of all cultures by autoclaving or soaking in disinfectant.
• Never open incubated broth tubes unnecessarily.
• Clean up spills immediately with disinfectant.
• Do not eat or drink in the lab.

Ethically, microorganisms must be:

• Handled with respect for lab safety,
• Prevented from being released into the environment,
• Disposed of responsibly.

📋 Summary Table

🧠 Quick Recap
Inoculate broth safely using aseptic technique.
Measure growth at intervals using turbidity or cell counts.
Plot data to see the four phases of growth.
Microbial growth depends on nutrients, temperature, oxygen and pH.