Bacterial Growth Curve Protocol – Result, Uses

What is Bacterial Growth Curve?

A bacterial growth curve is the graphical representation of the growth of a bacterial population with respect to time under a closed culture system such as a culture tube or flask. It is the pattern that shows how the number of bacterial cells increases and decreases during growth. Bacteria reproduce by binary fission, where a single cell divides into two daughter cells. Because of this continuous division the number of cells increases rapidly when the environmental conditions are favorable. When the logarithm of the number of living bacterial cells is plotted against time a typical curve is obtained which shows different phases of growth.

This growth curve is divided into four main phases. These are lag phase, log phase, stationary phase and death phase. In the lag phase the bacteria are adjusting with the new environment and synthesis of enzymes and proteins takes place but the number of cells does not increase. After this the culture enters the log phase (exponential phase) where rapid cell division occurs and the population increases at maximum rate. When nutrients become limited and waste products accumulate the culture enters the stationary phase, where the rate of cell division becomes equal to the rate of cell death. Finally the death phase occurs in which the number of living cells decreases gradually because the conditions become unfavorable for bacterial growth.

Principle of Bacterial Growth Curve

The principle of bacterial growth curve is based on observing the increase in number of bacterial cells with respect to time when bacteria are grown in a closed culture system or batch culture. In microbiology, bacterial growth refers to the increase in number of cells and not the increase in size of a single cell. Bacteria multiply by the process of binary fission, where one bacterial cell divides to form two daughter cells. When the logarithm of the number of living bacterial cells is plotted against time, a characteristic curve is obtained which represents the pattern of bacterial growth under given environmental conditions.

This curve shows the physiological changes of bacterial population during growth and is divided into four phases – lag phase, log phase, stationary phase and death phase. In the lag phase, bacteria adjust to the new environment and synthesis of enzymes and proteins occurs but the number of cells does not increase. After adaptation the cells enter the log phase, where rapid cell division occurs and the population increases at maximum rate. As nutrients become limited and waste products accumulate the culture enters the stationary phase, where the rate of cell division becomes equal to the rate of cell death. Finally the death phase occurs where the number of living bacterial cells decreases because the environmental conditions become unfavorable for growth.

Materials Required for Bacterial Growth Curve

• Bacterial culture (e.g., Escherichia coli).
• Liquid culture medium such as Nutrient broth or Luria-Bertani (LB) broth.
• Agar plates (LB agar or nutrient agar) for maintaining bacterial culture.
• Sterile distilled water for preparation and dilution.
• Formaldehyde (optional) – It is used to stop the bacterial growth when readings are to be taken later.
• Bleach solution or 70% ethanol– It is used for sterilization and proper disposal of materials.
• Conical (Erlenmeyer) flasks – It is used for growing bacterial cultures in liquid medium.
• Measuring cylinders – It is used for measuring culture media and other reagents.
• Sterile test tubes and microfuge tubes – These are used for culture handling and sampling.
• Sterile Petri plates– It is used for plating bacterial culture.
• Cuvettes (glass or disposable plastic)– These are used for measuring optical density in spectrophotometer.
• Spectrophotometer (UV–Visible)– It is used to measure optical density (OD) of bacterial culture.
• Incubator– It is used to maintain suitable temperature for bacterial growth.
• Shaker or shaking incubator– It provides continuous mixing and aeration during growth.
• Micropipettes with sterile pipette tips– These are used for accurate transfer of liquids.
• Bunsen burner or alcohol lamp– It is used for maintaining aseptic conditions.
• Sterile inoculating loop– It is used for transferring bacterial culture.
• Metal or glass cell spreader– It is used to spread bacterial culture on agar plates.
• Permanent markers– These are used for labeling culture tubes and plates.
• Lint-free lab wipes – These are used for cleaning laboratory surfaces and instruments.

Protocol for Bacterial Growth Curve

Day 1:– Initial plating

A loopful of bacterial culture is streaked on a sterile agar plate. The plate is incubated at 37°C for about 18–24 hours so that bacterial colonies are produced.

Day 2:– Preparation of starter culture

A single well–isolated colony is selected from the agar plate. The colony is inoculated into a test tube containing 10 ml sterile nutrient broth. The culture is incubated overnight at 37°C to obtain the starter culture.

Day 3:– Monitoring of bacterial growth

About 5 ml of the overnight culture is transferred into a sterile conical flask containing 250 ml fresh sterile broth.

• Immediately 1 ml of culture is taken and the optical density (OD) is measured at 600 nm using a spectrophotometer. This reading is referred to as zero hour reading.
• The flask is placed in an incubator at 37°C for growth of bacteria.
• After this 1 ml sample of culture is removed at regular interval of about 30 minutes.
• The optical density at 600 nm is measured each time using spectrophotometer.
• The readings are continued until the optical density becomes constant and no further increase is observed.
• The obtained values are plotted on a graph with time on X–axis and optical density (OD600) on Y–axis. This graphical representation is referred to as bacterial growth curve.

Result of Bacterial Growth Curve

• A logarithmic growth curve is obtained – The graph represents the change in number of bacterial cells with respect to time.
• The curve shows a hyperbolic pattern – This occurs due to the exponential multiplication of bacterial cells during growth.
• Four distinct growth phases are observed– These are lag phase, log phase, stationary phase and death phase.
• Rapid increase of population is seen in log phase– When the logarithm of living cells is plotted against time it appears as a straight upward line during this phase.
• The growth curve shows complete pattern of bacterial population growth in a closed culture system.

Use of Bacterial Growth Curve

• It is used to determine the rate of multiplication of bacterial population and the generation time (doubling time) of bacteria.
• It helps in determining suitable environmental conditions such as temperature, pH, oxygen supply and nutrient concentration required for bacterial growth.
• It is used in industrial microbiology for the production of microbial products such as enzymes, antibiotics and fermented products. The maximum activity of bacteria is observed during log phase.
• It is used to study the effect of antimicrobial agents on bacterial cells. Bacteria are more sensitive to antibiotics during the log phase.
• It helps in studying the pattern of bacterial growth and infection in host organisms.
• It is used to monitor the growth of microorganisms in food products and pharmaceutical preparations.
• It is used in microbiological research for monitoring bacterial biomass and collection of cells for experimental studies.