Bacterial Growth Tutorial Notes

Bacterial Growth Measurement

Bacterial Growth by Binary Fission

• Bacterial growth occurs via binary fission, where one cell divides into two identical daughter cells (clones).
• Binary Fission: The primary mode of reproduction in bacteria, resulting in exponential population increase.
• Generation Time (Doubling Time):
  • The time required for one bacterial cell to divide into two, or for the entire population to double.
  • Can be as short as 20 minutes for Staphylococcus aureus and Escherichia coli under optimal conditions.
• Due to the speed of reproduction and constraints regarding the amount of bacteria cells being grown, bacterial cultures usually involve millions of cells rather than a single cell.
  • Example: A starting population of 200 E. coli cells with a doubling time of 20 minutes.

Bacterial Growth Curve and Calculation of Bacterial Counts

• It's challenging to plot bacterial counts against time directly due to the exponential increase in numbers.
• Using a logarithmic scale simplifies the representation and analysis of bacterial growth.
• Logarithmic Scale (Log):
  • A mathematical function to handle large numbers.
  • The base-10 logarithm indicates how many times a number can be divided by 10 until reaching 1.
    • Example: The log of 10,000 is 4 because 10000 = 10 \times 10 \times 10 \times 10.
  • Scientific calculators are essential for calculating logarithms of complex numbers.

Classical Bacterial Growth Curve Phases

• The bacterial growth curve consists of four phases:
  • Lag Phase
  • Log (Exponential) Phase
  • Stationary Phase
  • Death Phase
• These phases are determined by:
  1. Measuring the turbidity of the bacterial cell population in broth culture using a spectrophotometer.
  2. Measuring actual number of bacterial cells using viable counts

Experimental Method

1. Inoculation:
   • A four-hour starter culture of E. coli in nutrient broth is inoculated into a 500 mL flask containing 300 mL of fresh nutrient broth.
2. Initial Absorbance Measurement:
   • A sample is immediately transferred to a cuvette, and absorbance is measured using a spectrophotometer at 550 nm.
3. Viable Count Preparation:
   • 100 μL is transferred from the flask into 9.9 mL of 0.1% peptone water (1 in 100 dilution) for viable counts.
4. Incubation and Sampling:
   • The flask is placed in a water bath at 37 ºC, and samples are taken at regular 20-minute intervals.
   • At each time point, absorbance readings are taken, and viable counts are performed from 10^{-4}, 10^{-5}, and 10^{-6} dilutions by spreading 100 μL over the surface of an agar plate.
5. Incubation of Viable Count Plates:
   • All viable count spread plates from different time points are incubated for 24 hours at 37 ºC.
6. Data Analysis:
   • Determine the mean (average) number of colonies for each dilution.
   • Calculate the bacterial count (CFU/mL) for that dilution.
   • Spread plates have 0.1 mL spread over the surface of the agar.

Results Table

Bacterial Growth Curves

1. Absorbance against Time:
   • Plot a graph of absorbance (optical density) values against time.
2. Log Number of Bacteria against Time:
   • Plot a graph of the logarithm of the number of bacteria (from viable counts) against time.

Questions and Considerations

1. Describe the Shape of the Graph:
   • How does the shape of each graph reflect the different phases of bacterial growth (lag, log, stationary, death)?
2. Difference Between the Two Graphs:
   • What are the key differences in the information conveyed by the absorbance graph versus the viable count graph?
3. Advantages and Disadvantages of Measurements:
   • Viable Counts:
     • Advantages: Provides a direct count of living bacteria.
     • Disadvantages: Time-consuming, labor-intensive, and may not detect all viable cells.
   • Optical Density (Spectrophotometry):
     • Advantages: Rapid, easy to perform, and provides a quick estimate of bacterial population size.
     • Disadvantages: Measures both living and dead cells, and may be affected by cell clumping or media turbidity.
4. Calculate Generation Time:
   • Determine the generation or doubling time, which is the time it takes for the bacterial population to double in size during the exponential phase.