Background:
- The growth of microorganisms may be studied by inoculating a culture medium with a small number of microorganisms. When all the nutritional and other factors required for growth are ideal or optimum, the cell continues to multiply. However, growth will be eventually slow if one or the other’s necessary required factors are depleted.
- If during this period, the culture medium is not replaced with fresh culture medium or metabolic waste product are not removed, this type of growth system is known as batch culture. In contrast, if there is continuous addition of fresh growth medium and subsequently withdrawal of equal volume of the bacterial culture from system, it is called continuous culture of microorganism.
- A batch culture is particularly very helpful in studying the growth character of microorganisms. When bacteria are transferred from a slant culture to a known volume of liquid medium, the population undergoes a characteristics sequence in its rate of increase in cell number.
- The increase in cell number of microorganisms may be monitored by counting them in a specific amount at periodic intervals. Thus, when a logarithm of the counted cells is plotted against time a growth curve is generated.
Introduction:
Bacterial growth is a complex process that involves both catabolic (breakdown of cell constituents and metabolites) and anabolic (biosynthesis of cell constituents and metabolites) process.
Basically, bacterial growth curve is sigmoid shows various recognizable stages of the growth of bacteria- lag phase, log phase or exponential growth phase, stationary phase, and the death phase. Since, all the bacterial cells will not be in the same stages of growth, the change from one phase to another phase is gradual and there will be several phases between the four standard phases of growth.
Different phases of Microbial Growth:
These different phases of microbial growth curve are as follows:
- Lag phase or initial slow phase
- Phase of accelerated growth
- Log phase or exponential phase
- Phase of decrease in growth curve
- Stationary phase
- Phase of increase in death rate
- The decline phase
- The survival phase
The lag phase or initial slow phase
In the lag phase, there is no increase in the number of viable cells, but cell growth occurs as indicated by the increase in cell mass. During this period, cells increase in size as a result of extensive synthesis of macromolecular. This stage is characterized by a period of active growth without cell division as the cells prepare for cell division. The length of the lag phase depends on various factors, including the age of the inoculum, the composition of the growth medium, and environmental conditions such as temperature, pH, and aeration.
Phase of accelerated growth
This phase occurs between lag phase and log phase. Once, the cells are sufficiently physiologically mature at the end of lag phase, the cell start dividing. Not all cells divide simultaneously because they do not complete the lag phase at the same time. Therefore, in the phase of accelerated growth, the number of cells dividing will increase gradually.
The log phase or the exponential phase
In the log phase, the cell number increases logarithmically and the cells divide at the maximum rate. The cell number increases progressively during this period of the experiment. Therefore, at this phase, the log of the number of cells plotted against time will show a straight line.
During this phase, cells are actually smaller in size as they are constantly dividing. The log phase is the most suitable period for studying biochemical and physiological properties of cells, because the cells are metabolically more active during this phase. The log phase is also extreme sensitive to the change in environmental factors like pH, temperature, aeration, concentration of nutrients, etc.
Fig: Different phases of bacterial growth curve
Phase of decrease in growth rate
As the microbial cells increase continuously during log phase, it results in gradual decrease in nutrients quantity and also there is accumulation of toxic waste produced by microbial metabolism. These factors will reduce the cell division and the cells do not multiply with the same rate as during the log phase. This phase is the transition phase between log phase and stationary phase.
The Stationary phase: In the stationary phase, the cells can no longer divide as the amount of available nutrients is almost negligible. In the stationary phase, the number of cells is maintained by a balance between cell division and death. The stationary phase is initiated due to many factors like very high concentration of cells, low partial pressure of oxygen and accumulation of toxic waste products. The viable cells count at this stage shows no change.
The length of the stationary phase varies depending on the organism and environmental conditions. Some microorganism’s displays a long stationary phase which lasts for several days, while other microorganisms may show a very short stationary phase of only a few hours before the next phase begins.
Some bacteria, such as the sporulating bacteria, may form endospores as that reach the stationary phase of growth and these would be resistant to lysis or death. In such cases, the number of viable cells will remain stable after reaching the stationary phase, and a decline phase may not occur.
Phase of increasing death rate
This is a transition phase between the stationary phase and the death phase. During this phase, with increase in time. During this phase, with increase in time, there is a gradual decrease in the number of the viable cells. Due to a number of factors the death of the cells gradually increases and reached a maximum at the end of this phase.
The death phase or the decline phase
The death phase or the phase of decline is characterized by an exponential decrease in the number of viable cells. A cell during this phase is considered to have died or to have become inviable when it is no longer capable of multiplying. The death rate of bacteria is not uniform like that of log phase where the increase is number is uniformly exponential, because few species of bacteria (e.g., Neisseria gonorrhea) die rapidly as the cells are very susceptible to autolysis.
The survival phase
During this phase the rate of the cell death is balanced by the rate of cell division, and very less population of cells is maintained. Cell survival varies depending on the organism and environmental conditions. Some microbes perish within days, whereas others can remain viable for months or even years. This is also a stage where the mutant forms induced recently find the favorable condition for rapid growth.
Methods of determining microbial growth:
There are variety of techniques for determining the microbial growth and the selection of the technique depends upon many factors like nature and features of microorganism, aim and objective of the study. Basically, there are two different types
Determination of cell numbers
Total count
As the cell growth is followed by cell division and results in cell number. Therefore, it is very essential to count the number of microbial cells in the respective culture medium in order to determine the microbial growth.
The total count of microorganism can be determined either microscopically or electronically.
In the microscope methos, it is determined by using hemocytometer under microscope. Whereas, a special instrument called as coulter counter is employed for direct counting of cells from the suspension.
Viable count
In the viable count only the living microbial cells are taken in consideration. The principle behind viable count is that all viable cells or spores under suitable conditions, multiply and form a colony. The number of choices is equal to the number of viable cells present in the sample. In viable count method, a know quantity of diluted sample is plated on an appropriate medium and after an incubation period, the number of colonies are determined.
Determination of cell mass
In this procedure, the weight or mass of the cells is calculated as an indicator of increased bulk.
Direct method
Measurement of dry weight of the pellet which is obtained after the suspension of bacterial culture is one of the methods. Moreover, the quantity of nitrogen which is major components of protein is also the other direct method of determination of cell mass.
In-direct method
The use of colorimeter or turbidimeter is the most popular indirect method of determining growth in bacteria and yeast. Likewise, measurement of biochemical activity is also other approach of indirect method in determining microbial growth.