In reality, a population of bacteria does not double endlessly, because in most systems numerous factors prevent the cells from continuously dividing at their maximum rate. Quantitative laboratory studies indicate that a population typically displays a predictable pattern, or growth curve, over time. The method traditionally used to observe the population growth pattern is a viable count technique, in which the live cells in a culture are sampled, grown, and counted during a growth period.
The Viable Plate Count: Batch Culture Method
A growing population is established by inoculating a flask containing a known quantity of sterile liquid medium with a few cells of a pure culture. The flask is incubated at that bacterium’s optimum temperature and timed. The population size at any point in the growth cycle is quantified by removing a tiny measured sample of the culture from the growth chamber and plating it out on a solid medium to develop isolated colonies. This procedure is repeated at evenly spaced intervals (i.e., every hour for 24 hours) (figure 1).
Fig1. The general steps in a viable plate count. 1. Place a few cells into a sterile liquid medium. 2. Incubate this culture over a period of several hours. 3. Sample the broth at regular intervals during incubation. 4. Plate each sample onto solid media. 5. Count the number of colonies present after incubation.
Evaluating the samples involves a common and important principle in microbiology: One colony on a plate represents one cell or colony-forming unit (CFU) from the original sample. Because the CFU of some bacteria is actually composed of several cells (consider the clustered arrangement of Staphylococcus, for instance), using a colony count can underestimate the exact population size to an extent. This is not a serious problem, because in such bacteria the CFU is the smallest unit of colony formation and dispersal. Multiplication of the number of colonies in a single sample by the container’s volume gives a fair estimate of the total population size (number of cells) at any given point. The growth curve is determined by graphing the number for each sample in sequence for the whole incubation period (figure2).
Fig2. The growth curve in a bacterial culture. On this graph, the number of viable cells expressed as a logarithm (log) is plotted against time. See text for discussion of the various phases. Note that with a generation time of 30 minutes, the population has risen from 10 (101) cells to 1,000,000,000 (109) cells in only 16 hours. Even though they are not counted, dead cells are indicated to show stages in the cycle where death affects the appearance of the curve.
Because of the scarcity of cells in the early stages of growth, some samples can give a zero reading even if there are viable cells in the culture. The sampling itself can remove enough viable cells to alter the tabulations, but because the purpose is to compare relative trends in growth, these factors do not significantly change the overall pattern.
Stages in the Normal Growth Curve
The system of batch culturing just described is closed, meaning that nutrients and space are finite and there is no mechanism for the removal of waste products. Data from an entire growth period of 3 to 4 days typically produce a curve with a series of phases termed the lag phase, the exponential growth (log) phase, the stationary phase, and the death phase (figure 2).
The lag phase is an early “flat” period on the graph when the population is growing at less than the exponential rate. Growth lags primarily because cells in the newly inoculated culture must adapt to their new environment. Chief among these adaptations is the synthesis of new enzymes required to metabolize nutrients in the culture media.
The length of the lag period varies from one population to another, depending on the condition of the microbes and medium.
The cells reach the maximum rate of cell division during the exponential growth (logarithmic or log) phase, a period during which the curve increases geometrically. This phase will continue as long as cells have adequate nutrients and the environment is favor able. During this phase, cells reach their maximum rate of growth.
At the stationary growth phase, the population reaches a size limit in which some cells divide more slowly or stop dividing entirely and may even have died. The curve levels off because the number of new cells produced equals the number that have died. The number of viable cells has reached maximum and remains relatively constant during this period. The decline in the growth rate is caused by several factors. A common reason is the depletion of nutrients and oxygen. Another is that the increased cell density often causes an accumulation of organic acids and other toxic biochemicals.
As the limiting factors intensify, the population begins to show a decline, seen as a downward slope on the curve. This stage has been called the death phase, but what is actually happening is more complex. Some cells go into dormancy and remain viable but do not grow. Some cells enter a starvation mode that helps them resist the lack of nutrients and other factors. Depending upon the species, some microbes could remain in these states for long periods. It is also apparent that many cells go through a programmed cell death and lyse during this phase, which accounts for the overall reduction in population size. Under these conditions, it is possible for persistent members of the culture to survive by using the nutrients released by the dead cells.
Practical Importance of the Growth Curve The tendency for populations to exhibit phases of rapid growth, slow growth, and death has important implications in microbial control, infection, food microbiology, and culture technology. Antimicrobial agents such as heat and disinfectants rapidly accelerate the death phase in all populations, but microbes in the exponential growth phase are more vulnerable to these agents than are those that have entered the stationary phase. In general, actively growing cells are more vulnerable to conditions that disrupt cell metabolism and binary fission.
Growth patterns in microorganisms can account for the stages of infection. A person shedding bacteria in the early and middle stages of an infection is more likely to spread it to others than is a person in the late stages. The course of an infection is also influenced by the relatively faster rate of multiplication of the microbe, which can overwhelm the slower growth rate of the host’s own cellular defenses.
Understanding the stages of cell growth is crucial for work with cultures. Sometimes a culture that has reached the stationary phase is incubated under the mistaken impression that enough nutrients are present for the culture to continue to multiply. In most cases it is unwise to incubate a culture beyond the stationary phase, because doing so will reduce the number of active cells and the culture could die out completely. It is also preferable to do stains (an exception is the spore stain) and motility tests on young cultures, because the cells will show their natural size and correct reaction, and motile cells will have functioning flagella.
For certain research or industrial applications, closed-batch culturing with its four phases is inefficient. The alternative is an automatic growth chamber called the chemostat, or continuous culture system. This device can admit a steady stream of new nutrients and siphon off used media and old bacterial cells, thereby stabilizing the growth rate and cell number. The chemostat is very similar to industrial fermentors used to produce vitamins and antibiotics. It has the advantage of maintaining the culture in a biochemically active state and preventing it from entering the death phase.
