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.

Viable Count Procedures

The viable plate count method is one of the most common procedures for the enumeration of bacteria. In this procedure, serial dilutions of a suspension of bacteria are plated onto a suitable solid growth

medium and after a period of incubation (during which single cells multiply to form visible colonies) the number of colonies are counted or enumerated (FIG. 10-6).

Frequently, the suspension is spread over the sur­face of an agar plate containing growth nutrients (surface spread technique) (FIG. 10-7). Alternatively, it can be mixed with the agar while it is still in a liq- j uid state and poured into the plate (pour plate tech­nique) (FIG. 10-8). The plates are incubated to allow the bacteria to grow and form colonies. The forma­tion of visible colonies generally takes 16 to 24 hours,

FIG. 10-6 A, The plate count procedure is used to determine the viable population in a sample containing bacteria. Dilutions are achieved by adding an aliquot of the specimen to a sterile water dilution tube. If 1 mL of a sample is added to 99 mL of sterile water, the dilu­tion is 1:100 (10~²). (The same dilution could also have been achieved by adding an 0.1 mL sample to 9.9 mL of sterile water). Greater dilutions are achieved by sequentially diluting the sample in series. Adding 1 mL from the first dilution to 9 mL of sterile water achieves an additional tenfold dilution so that the total dilution is 1:1000 (10~³). Adding 1 mL from that second dilution to 9 mL of sterile water achieves a further tenfold dilution so that the total dilution is 1:10000 (10~⁴). Transferring 1 mL samples from each tube to agar media maintains these dilution factors. Transferring 0.1 mL samples increases the dilution by a fac­tor of 10. After incubation the number of colonies are counted. Counts on the plates in the range of 30 to 300 colonies are used to calculate the concentration of bacteria. The standard notation "TNTC" means too numerous to count (greater than 300 colonies). In this example the plate with 61 colonies would be used to calculate the number of bacteria in the original water sample. Because these colonies developed on a plate in which 1 mL from a 1:10000 di­lution was added, the number of bacteria per mL in the original sample is calculated as 6.1 X 10⁵ (61 X 10⁴). B, Colonies of lactose fermenting bacteria growing on MacConkey agar.

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