PRINCIPLES OF BACTERIAL GROWTH
Growth may be defined as the orderly increase of all of the chemical constituents of the cell. Bacterial growth involves both an increase in the size of individuals and an increase in the number of individuals. Whatever the balance between these two processes, the net effect is an increase in the total mass (biomass).
A. Bacterial Division
Bacteria divide by binary fission where individual cells enlarge and divide to yield two progeny of approximately equal size. Nuclear division precedes cell division and, therefore, in a growing population, many cells carrying two nuclear bodies can be seen. The cell division occurs by a constrictive or pinching process, or by the ingrowth of a transverse septum across the cell. The daughter cells may remain partially attached after division in some species.
▶Generation Time or Doubling Time
The size of a population of bacteria in a favorable growth medium increases exponentially. The interval
of time between two cell divisions, or the time required for a bacterium to give rise to two daughter cells under optimum conditions is known as the generation time or doubling time.
Examples: In coliform bacilli and many other medically important bacteria, it about 20 minutes, in tubercle bacilli it is about 20 hours and in lepra bacilli, it is about 20 days. It is often difficult to grasp fully the scale of exponential microbial growth.
As bacteria reproduce so rapidly and by geometric progression, a single bacterial cell can theoretically give rise to 1021 progeny in 24 hours, with a mass of approximately 4,000 tonnes. In actual practice, exponential growth cannot be sustained indefinitely in a closed system (batch culture) with limited available nutrients.
Bacteria growing on solid media form colonies. Each colony represents a clone of cells derived from a single parent cell. In contrast to growth in broth, far less is known about the state of the bacteria in a
mature macroscopic colony on an agar plate. It is likely that all phases of growth are represented in colonies, depending on the location of a particular cell and age of the culture. In liquid media, growth is diffuse.
The growth of bacteria is diffuse in liquid media and they form colonies on solid media. Each colony consists of a clone of cells derived from a single parent cell. Bacteria in a culture medium or clinical specimen can be counted by two methods:
1. Total count
This is the total number of bacteria present in a specimen irrespective of whether they are living or dead. Total count is done by counting the bacteria under a microscope using a counting chamber and by comparing the growth with standard opacity tubes.
2. Viable count
This measures only viable (living) cells that are capable of growing and producing a colony on a suitable medium. The viable count measures the number of living cells, that is, cells capable of multiplication.
▶Determination of Viable Counts—Dilution or plating Methods
1. Dilution method
In the dilution method, the suspension is diluted to a point beyond which unit quantities do not yield growth when inoculated into suitable liquid media. With varying dilutions, several tubes are inoculated and the viable counts calculated statistically from the number of tubes showing growth. The method does not give accurate values but is used in the ‘presumptive coliform count’ in drinking water.
2. Plating method
In the plating method, appropriate dilutions are inoculated on solid media by:
i. Pour-plate method
ii. Spread plate method—in which serial dilutions are dropped on the surface of dried plates and colony counts obtained.
Detection and Measurement of Bacterial Growth
1. Direct cell counts
2. Viable cell counts
3. Measuring biomass
4. Measuring cell products
B. Bacterial Growth Curve
If a suitable liquid medium is inoculated with bacterium and incubated, its growth follows a definitive course. Small samples are taken at regular intervals after inoculation and plotted in relation to time. Plotting of the data will yield a characteristic growth curve. The changes of slope on such a graph indicate the transition from one phase of development to another.
Phases of Bacterial Growth Curve
The bacterial growth curve can be divided into four major phases
(i) lag phase
(ii) exponential or log (logarithmic) phase
(iii) stationary phase, and
(iv) decline phase.
These phases reflect the physiologic state of the organisms in the culture at that particular time.
1. Lag phase
When microorganisms are introduced into the fresh culture medium, usually no immediate increase in cell number occurs, and therefore this period is called the lag phase. After inoculation, there is an increase in cell size at a time when little or no cell division is occurring. During this time, however, the cells are not dormant. This initial period is the time required for adaptation to the new environment, during which the necessary enzymes and metabolic intermediates are built up in adequate quantities for multiplication to proceed. The lag phase varies considerably in length with the species, nature of the medium, size of the inoculum, and environmental factors such as temperature and nutrients present in the new medium.
2. Log (logarithmic) or exponential phase
Following the lag phase, the cells start dividing and their numbers increase exponentially or by geometric progression with time. If the logarithm of the viable count is plotted against time, a straight line will be obtained. The population is most uniform in terms of chemical and physiological properties during this phase. Therefore, exponential phase cultures are usually used in biochemical and physiological studies. The exponential phase is of limited duration because of
(i) exhaustion of nutrients;
(ii) accumulation of toxic metabolic end products;
(iii) rise in cell density,
(iv) change in pH; and
(v) decrease in oxygen tension (in the case of aerobic organisms).
The log phase is the time when cells are most active metabolically and are preferred for industrial purposes. However, during their log phase of growth, microorganisms are particularly sensitive to adverse conditions. Radiation and many antimicrobial drugs, e.g. the antibiotic penicillin—exert their effect by interfering with some important step in the growth process and are, therefore, most harmful to cells during this phase.
3. Stationary phase
After a varying period of exponential growth, cell division stops due to depletion of nutrients and accumulation of toxic products. Eventually, growth slows down, and the total bacterial cell number reaches a maximum and stabilizes. The number of progeny cells formed is just enough to replace the number of cells that die. The growth curve becomes horizontal. The viable count remains stationary as an equilibrium exists between the dying cells and the newly formed cells.
4. Decline or death phase
The death phase is the period when the population decreases due to cell death. Eventually, the rate of death exceeds the rate of reproduction, and the number of viable cells declines. Like bacterial growth, death is exponential cell death may also be caused by autolysis besides nutrient deprivation and buildup of toxic wastes. Finally, after a variable period, all the cells die and culture becomes sterile.
When the total count is plotted, it parallels the viable count up to the stationary phase, but it continues steadily without any phase of decline. Even the total count shows a phase of decline with autolytic enzymes.
Association of Growth Curve and Cell Changes
The various stages of the growth curve are associated with morphological and physiological alterations of the cells. It has been possible to define the effect of growth rate on the size and specialized growth techniques.
1. Lag phase: Bacteria have the maximum cell size towards the end of the lag phase.
2. Log phase: Cells are smaller and stain uniformly in the log phase.
3. Stationary phase: In the stationary phase, cells frequently are Gram variable and show irregular
staining due to the presence of intracellular storage granules. Sporulation occurs at this stage and also
many bacteria produce secondary metabolic products such as exotoxins and antibiotics.
4. Decline phase: In the phase of decline, involution forms are common.
Batch Culture or Closed System
In the laboratory, bacteria are typically grown in broth contained in a tube or flask, or on an agar plate. These are considered batch or closed systems.
To maintain cells in a state of continuous growth, nutrients must be continuously added and waste products removed. This is called continuous culture or an open system. Continuous culture is using a chemostat in which cells of a growing culture are continuously harvested and nutrients continuously replenished.
The second type of continuous culture system, the turbidostat, has a photocell that measures the absorbance or turbidity of the culture in the growth vessel. The flow rate of media
through the vessel is automatically regulated to maintain predetermined turbidity or cell density.