Growth can be defined as the orderly increase in cellular constituents and results from the biosynthetic and energy processes. Growth also refers to the increase in mass of a single cell or to the increase in the size of a population of cells.
Bacterial growth kinetics is the relationship between the specific growth rate of a bacteria and the concentration of a substrate.
Objective
Determination of the bacterial growth kinetics.
Principle of Bacterial Growth Kinetics
Depending upon nutritional status, bacteria exhibit different growth patterns. Bacteria take time to adjust to the environment. Freshly inoculated nutrient broth. This gap in time is called the lag phase or the initial stationary phase.
Therefore, it uses the nutrients of the medium and multiplies very fast showing an exponential phase or log phase of growth. The growth becomes stagnant. This stage is called the stationary phase.
After a few days, the nutrients of the medium start diminishing. Therefore, the growth rate retards which is called the deceleration phase. There comes a stage when there is no increase or decrease in the number of bacteria. This phase is called the stationary phase.
Finally, due to the continuous accumulation of toxic metabolites, there occurs death of the bacteria. This phase is known as the death phase.
The latter two phases (stationary and death phase) are accomplished due to the accumulation of toxic inhibitory secondary metabolites of the bacteria. In the laboratory, bacterial growth can be demonstrated by plotting a graph between microbial numbers (measured as optical density by spectrophotometer).
The density of all suspension is expressed as absorbance or optical density which is actually the cell concentration increasing with time duration. Absorbance is a logarithmic value and is used to plot a graph of bacterial growth.
Requirements of Bacterial Growth Kinetics
- Pure culture of Escherichia coli (E. coli)
- Nutritional broth medium
- Erlenmeyer flask (250 ml)
- Spectrophotometer with accessories
- Incubator, inoculating needles, shaker, sterilized pipettes (1 ml)
Procedure of Bacterial Growth Kinetics
- Procure 1 ml or fully grown culture of E. coli from the broth of culture prepared earlier.
- By serial dilution technique dilute the culture to get 1×106 cells/ml (it is approximated when fully grown, culture has at least 1×109 cells/ml).
- Dispense 100 ml of double-strength nutrient broth medium in each 250 ml Erlenmeyer flask and autoclave at 15 psi for 30 minutes.
- Inoculate the sterilized medium by using a calibrated Pasteur pipette for each flask with one drop (about 0.03 ml/drop) of the diluted broth culture.
- Place the flask on a shaker adjusted at 15 rpm at a controlled temperature of 30 ± 1°C for 48 hours.
- Switch on the spectrophotometer at least 20 minutes before taking optical density (OD), so that it can get stabilized.
- Withdraw 2 ml of the broth culture at every 4 hours intervals for 20-24 hours and measure the absorbance (OD using a spectrophotometer at 600 nm wavelength). Un inoculated growth medium is treated as blank.
- Prepare growth curve by plotting graph in terms of absorbance (on Y-axis) against time (hours) [on X-axis].
Result
A sigmoidal growth curve of E. coli is obtained as shown in the given figure:
The optical density(OD) of bacterial suspension increases with increasing time.
