Introduction to Bacterial Growth in Batch Culture:
The population of cells nearly always shows four distinct phases of growth when bacteria are cultivated in a fresh liquid medium in a closed environment, such as a test tube, for an extended length of time. This type of culture is known as batch culture.
Plotting the growth of bacteria that reproduce by binary fission against incubation time can be done by taking the logarithm of the number of viable cells. It provides a distinctive growth curve, also referred to as a growth curve.
Role of Oxygen and Enzymes in Bacterial Growth:
Facultative anaerobes use several metabolic methods to grow whether oxygen is present or not.
Because aerobic respiration produces more ATP than fermentation, they primarily congregate at the top during incubation. Bacterial growth is influenced not only by oxygen concentration but also by the growth medium’s oxygen reduction potential.
The presence and distribution of different oxygen-reacting enzymes and oxygen radicals, which are always produced by cells when oxygen is present, determine how an organism reacts to oxygen in its external environment. Example in aerobes and facultative anaerobes, the potential for lethal accumulation of superoxide is prevented by the enzyme superoxide dismutase.
During batch culture in a sterile liquid media, growth is sampled at regular time intervals in the incubation period.
Methods for Determining Bacterial Growth Curves:
Optical Density (OD) Measurement
For determining growth curve of facultative bacteria, using sterile loop, a loopful of bacterial culture is onto the agar plate and incubated at 37 degrees Celsius overnight and pure culture is obtained. Then a single colony of organism is pickup up from the agar plate and inoculated it into a test tube containing autoclaved broth and incubated overnight at 37 degrees Celsius. Then turbidity can be determined at various time intervals by measuring optical density (OD) at appropriate wavelength can be measured using spectrophotometer.
Viable Cell Count
Viable cell count can be performed by serial dilution of samples followed by measuring colony forming units (CFU). The CFU/ml can be measured at various time intervals to determine changes in cell number during incubation so that growth curve can be drawn using log of cell number against the incubation time.
There are two ways to carry out viable count:
Spread plate
Using a sterile spreading tool, facultative bacteria are applied to the agar surface. They grow readily and can be counted after incubation.
Pour plate
Colonies form across the agar once facultative bacteria are combined with melted agar and allowed to cool.
Most Probable Number (MPN) Technique
Another culture-based technique for figuring out the facultative anaerobes’ development curve is the most probable number, or MPN. The quantity of live microorganisms in a sample is estimated using the most probable number (MPN) approach. By monitoring turbidity or color changes brought on by metabolic activity, the MPN technique assesses detectable growth.
Growth Phases in Batch Culture:
By various culture-based techniques, bacterial growth can be measured and all bacteria exhibit sigmoidal curve in batch culture consisting of the typical four phases i.e., lag, log, stationary and decline phases. When facultative anaerobic bacteria grow in a suspension broth in closed system, it takes some time before they start growing.
Lag Phase
The lag phase marks the initial stage of bacterial growth, where the cells are adapting to their new environment and preparing for reproduction, but not multiplying significantly yet.
Log (Exponential) Phase
This is followed by the log (or exponential) phase, during which the bacterial population increases rapidly in a consistent, logarithmic pattern. As the bacteria multiply, they consume nutrients and release waste into their surroundings.
Stationary Phase
Over time, the availability of nutrients decreases, and waste accumulation increases, leading to the stationary phase.
In this phase, the rate of cell division balances out with the rate of cell death, resulting in a stable population size.
Decline (Death) Phase
Eventually, as conditions worsen—due to nutrient exhaustion and toxic buildup—the death rate surpasses the growth rate, and the population enters the decline (or death) phase, where the number of living bacterial cells steadily decreases.
Diauxic growth:
Diauxic growth is the phenomenon whereby a population of microbes, when presented with two carbon sources, shows two log phases intervened by a short lag-phase of minimal growth.
Example: When E. coli is provided with a media containing both glucose and lactose as source of carbon, the population first grows exponentially on glucose until all glucose is exhausted, then stops growing for a considerable amount of time (lag phase) whereby they produce new enzymes for acquisition in new carbon source (lactose) and subsequently resumes exponential growth on lactose. Until switch point, the concentration of glucose is decreasing and the lactose concentration is high and then after switch point, genes switch for utilizing the lactose and then lactose concentration also falls gradually as lactose is utilized for division.
Differences Between Normal and Diauxic Growth Curves:
| S. No | Diauxic growth curve | Normal growth curve |
| 1 | Has two phases of growth response | Has only one growth pattern |
| 2 | Two sources of carbon as limiting nutrients. Example: Glucose and Lactose | Only one source of carbon as limiting nutrient. Example: Glucose |
| 3 | Firstly, the most preferred source of carbon is used. E.g. in E coli, if nutrient media is provided with glucose and lactose, glucose is used first and then uses lactose. So cell number is relatively higher than typical normal growth as two sugars are available. | Growth stops as soon as the source of carbon is exhausted so cell number can be relatively lower in batch culture showing normal growth curve than diauxic growth. |
| 4 | In first phase of growth, the genes for utilizing lactose is turned off by catabolite repression and as glucose is exhausted, lactose utilizing genes are turned on after switch point and then lactose can be utilized. | There is no such gene switch. |
| 5 | Consists of two log phases | Consists of single log phase. |
References:
- Wang, L., Fan, D., Chen, W. et al. Bacterial growth, detachment and cell size control on polyethylene terephthalate surfaces. Sci Rep 5, 15159 (2015). https://doi.org/10.1038/srep15159