Microbiology: Measuring Growth of Bacterial Cultures

Aims of Study

  • Understand methods of measuring the growth of microorganisms
    • Illustrated by:
    • Cell counts
    • Dilution plating
    • Mass and area measurements
    • Optical methods (turbidity)

Measuring the Growth of Microorganisms

  • Most microorganisms too small for naked-eye counting.
  • Various methods available to count microorganisms:
    • Cell counts: e.g., using a haemocytometer
    • Dilution plating
    • Measuring area and mass
    • Optical methods (turbidity)

Cell Counts: Haemocytometer

  • A haemocytometer is a microscope slide with a rectangular chamber marked with grid lines.
  • Chamber volume: 0.1 mm³.

Procedure for Cell Counts Using a Haemocytometer

  1. Dilute nutrient broth with an equal volume of Trypan blue (a dye that stains dead cells blue).
  2. Fill the haemocytometer chamber with the stained nutrient medium.
  3. Count living cells in the four corner squares of the grid.
    • Only living cells counted (Trypan blue stains dead cells).

Counting Process Details

  • Each corner square consists of 16 smaller squares.
  • Consistency required when counting cells on the lines separating the corner squares.
  • Calculate the mean number of cells from the four corner squares.

Calibration of Haemocytometer

  • Calibrated such that the number of cells in one set of 16 squares equals the number of cells × 10^4 per cm³ of broth.
  • If the broth is diluted, adjustments to calculations are necessary.

Example Calculation for Cell Count

  • Situation: Nutrient broth diluted by a factor of 100; corner squares contain counts of 20, 14, 19, and 16 cells.
  • Calculation of cells per mL of nutrient broth required.

Optical Methods: Turbidity

Turbidimetry

  • Turbidimetry is a specialized form of colorimetry used to measure the number of cells in a sample.
  • Turbidity measures cloudiness of a solution:
    • More turbid = more cloudy = more cells.
    • Less turbid = less cloudy = fewer cells.

Measuring Turbidity

  • Utilizes a COLORIMETER to measure light transmission through a sample.
  • More turbid solutions absorb more light and transmit less.
    • This provides an indirect measure of the number of microorganisms present.

Calibration Curve Creation

  • Construct a calibration curve by measuring turbidity of control cultures and counting cells using a haemocytometer.
  • Plot results in a graph: turbidity vs. cell count.
    • This curve enables estimation of cell counts for unknown samples by measuring turbidity and referencing the graph.

Dilution Plating

  • This method determines the total viable cell count.
  • Process involves:
    • Transferring nutrient broth to agar, allowing bacteria to reproduce.
    • Each single cell that lands on agar reproduces to form a colony.
    • Each microbial colony counted as one viable organism.

Problem and Solution in Dilution Plating

  • Problem: Individual colonies may merge, forming a large mass.
  • Solution: Dilute original cultures before transferring to agar to allow visibility of colonies.

Total Viable Cell Count Calculation

  • Total viable cell count = number of colonies × dilution factor.
  • A mean can be determined if multiple plates are analyzed.

Area and Mass Measurements for Fungi

  • Fungi can form mycelium, which complicates standard growth measurement methods.
  • Growth can be determined by:
    • Measuring diameter of fungal mycelium areas.

Method to Measure Fungal Growth

  1. Inoculate agar plates with fungal spores and incubate at suitable temperature.
  2. Measure resultant areas of fungal mycelium.
    • Larger diameters indicate greater growth.

Dry Mass Measurement for Fungi

  • An effective way to measure fungal growth is to test dry mass:
  1. Inoculate liquid nutrient broth with fungal spores.
  2. Take samples at intervals, removing mycelia by filtering/centrifugation.
  3. Dry collected mycelia in an oven overnight and measure mass.
    • An increase in mass indicates more fungal growth.

Past Paper Questions and Discussions

  • 1. Bacterial growth observed over 14 hours using optical method (turbidity), with calibration curve translating turbidity to bacteria count.
    • Calibration curve enables conversion of turbidity readings into the number of bacteria per cm³.

Analysis of Results Over Culture Period

  • Analysis table shows parameters such as turbidity and cell number increase over time.
  • Completion queries regarding cell number increases between specified hours.

Explanation on Growth Changes

  • Explanation regarding bacterial growth observed over initial hours as well as subsequent hours indicated in questions.

Comparison of Methods (Optical vs. Dilution Plating)

  • Explain observed differences in bacteria counts between methods after specific time intervals.
    • Discuss variations between optical methods (turbidity) and dilution plating result interpretations.