290 Chapter 10 bacterial reproduction and growth of microorganisms

For a growing bacterial culture, the generation time can be expressed as:

where g is the generation time, t is time, and n is the number of generations. The number of cells of a growing culture are expressed by the equation:

Since l/log₁₀2 is equal to 3.3, the original equation for the generation time can be written as:

where N₀ is the number of cells at time 0, N, is the number of cells at any time (t) after time 0, and n is the number of generations. Rearranging this formula:

where g is the generation time, log N, is the logarithm to the base 10 of the number of bacteria at time t, log N₀ is the logarithm to the base 10 of the number of bacteria at the starting time, and t is the time period of growth.

By determining cell numbers during the period of active cell division, the generation time can be esti­mated. A bacterium such as Escherichia coli can have a generation time as short as 20 minutes under opti­mal conditions. Considering a bacterium with a 20-minute generation time, one cell would multiply to 1,000 cells in 3.3 hours and to 1,000,000 cells in 6.6 hours.

Generation time or doubling time is the unit of me sure of bacterial growth; it is the time it takes for the size of a bacterial population to double.

Bacterial Growth Curve

When a bacterium is inoculated into a new culti medium, it exhibits a characteristic pattern or chan in cell numbers. This pattern is called a growth cur (FIG. 10-4). The normal growth curve of bacteria h four phases, the lag phase, the log or exponent growth phase, the stationary phase, and the dea phase. During the lag phase there is no increase cell numbers. Rather, the lag phase is a period adaptation during which bacteria are preparing f reproduction: synthesizing DNA, RNA, other stn tural macromolecules, and the various enzym needed for cell division.

After the lag phase, the bacteria begin to multip by binary fission, doubling in number every tin they divide. This is the log phase of growth, al called the exponential phase. It is so named becau the logarithm of the bacterial cell numbers increas linearly with time. During this phase, bacterial repr duction occurs at a maximal rate for the specific set growth conditions. It is during this period that tl generation time of the bacterium is determined.

After some period of exponential growth, the st tionary growth phase is reached. The stationai phase often occurs when the maximum populatic density that can be supported by the available в sources is reached. Once the stationary growth phas is reached, there is no further net increase in bacterii cell numbers. During the stationary phase the growt

FIG. 10-4 Growth curve for bacteria has four distinct phases: lag, exponential (log), sta­tionary, and death.

BACTERIAL GROWTH 291

rate is exactly equal to the death rate, and cell num­bers therefore remain constant. A bacterial popula­tion may reach stationary growth when a required nutrient is exhausted, when inhibitory end products accumulate, or when physical conditions do not per­mit a further increase in population size. The dura­tion of the stationary phase varies, with some bacte­ria exhibiting a very long stationary phase.

Eventually the number of viable bacterial cells be­gins to decline. This signals the onset of the death phase. During the death phase the number of living bacteria decreases because the rate of cell death ex­ceeds the rate of new cell formation.

A bacterial growth curve has four phases: (1) lag phase during which bacteria prepare to divide, (2) log or exponential growth phase during which cell numbers increase with regular doublings of viable cells, (3) stationary phase during which cell num­bers remain constant, and (4) death phase during which viable cell numbers decline.

Batch and Continuous Growth

The normal bacterial growth curve is characteristic of bacteria in batch culture. In batch culture, growth oc­curs in a closed system with fresh sterile medium simply inoculated with a bacterium to which new materials are not added. A flask containing a liquid nutrient medium inoculated with the bacterium E. coli is an example of such a batch culture. In batch culture, growth nutrients are expended and meta­bolic products accumulate in the closed environment. The batch culture models situations such as occur when a canned food product is contaminated with a bacterium.

Bacteria may also be grown in continuous culture. In continuous culture, nutrients are supplied and end products continuously removed so that the exponen­tial growth phase is maintained. Because end prod­ucts do not accumulate and nutrients are not com­pletely expended, the bacteria never reach the sta­tionary phase. A chemostat is a continuous culture device in which a liquid medium is continuously fed into the bacterial culture (FIG. 10-5). The liquid medium contains some nutrient in growth-limiting concentration, and the concentration of the limiting nutrient in the growth medium determines the rate of bacterial growth. Even though bacteria are contin­uously reproducing, a number of bacterial cells are continuously being washed out and removed from the culture vessel. Thus cell numbers in a chemostat reach a plateau.

Bacterial Growth on Solid Media The development of bacterial colonies on solid growth media follows the basic normal growth

curve. The dividing cells do not disperse and the population is densely packed. Under these condi­tions, nutrients rapidly become limiting at the center of the colony. Microorganisms in this area rapidly reach stationary phase. At the periphery of the colony, cells can continue to grow exponentially even while those at the center of the colony are in the death phase. Bacterial colonies generally do not ex­tend indefinitely across the surface of the media but have a well-defined edge. Therefore individual well-isolated colonies can develop from the growth of in­dividual bacterial cells. The fact that the bacteria have reproduced asexually by binary fission means that all the bacteria in the well-isolated colony should be genetically identical; that is, each colony should contain a clone of identical cells derived from a sin­gle parental cell.

FIG. 10-5 A chemostat continuously provides nutrients with a growth rate limiting factor to a flow-through culture chamber in which bacteria grow.

ENUMERATION OF BACTERIA

To assess rates of bacterial reproduction, it is neces­sary to determine numbers of bacteria. Various meth­ods can be employed for enumerating bacteria. These include viable plate count, direct count, and most probable number (MPN) determinations.