9.1 – How Microbes Grow

The bacterial cell cycle involves the formation of new cells through the replication of DNA and partitioning of cellular components into two daughter cells. In prokaryotes, reproduction is always asexual, although extensive genetic recombination in the form of horizontal gene transfer takes place, as will be explored in a different chapter. Most bacteria have a single circular chromosome; however, some exceptions exist. For example, Borrelia burgdorferi, the causative agent of Lyme disease, has a linear chromosome.

Binary Fission

The most common mechanism of cell replication in bacteria is a process called binary fission, which is depicted in Figure 9.2. Before dividing, the cell grows and increases its number of cellular components. Next, the replication of DNA starts at a location on the circular chromosome called the origin of replication, where the chromosome is attached to the inner cell membrane. Replication continues in opposite directions along the chromosome until the terminus is reached.

The center of the enlarged cell constricts until two daughter cells are formed, each offspring receiving a complete copy of the parental genome and a division of the cytoplasm (cytokinesis). This process of cytokinesis and cell division is directed by a protein called FtsZ. FtsZ assembles into a Z ring on the cytoplasmic membrane (Figure 9.3). The Z ring is anchored by FtsZ-binding proteins and defines the division plane between the two daughter cells. Additional proteins required for cell division are added to the Z ring to form a structure called the divisome. The divisome activates to produce a peptidoglycan cell wall and build a septum that divides the two daughter cells. The daughter cells are separated by the division septum, where all of the cells’ outer layers (the cell wall and outer membranes, if present) must be remodeled to complete division. For example, we know that specific enzymes break bonds between the monomers in peptidoglycans and allow addition of new subunits along the division septum.

Generation Time

In eukaryotic organisms, the generation time is the time between the same points of the life cycle in two successive generations. For example, the typical generation time for the human population is 25 years. This definition is not practical for bacteria, which may reproduce rapidly or remain dormant for thousands of years. In prokaryotes (Bacteria and Archaea), the generation time is also called the doubling time and is defined as the time it takes for the population to double through one round of binary fission. Bacterial doubling times vary enormously. Whereas Escherichia coli can double in as little as 20 minutes under optimal growth conditions in the laboratory, bacteria of the same species may need several days to double in especially harsh environments. Most pathogens grow rapidly, like E. coli, but there are exceptions. For example, Mycobacterium tuberculosis, the causative agent of tuberculosis, has a generation time of between 15 and 20 hours. On the other hand, M. leprae, which causes Hansen’s disease (leprosy), grows much more slowly, with a doubling time of 14 days.

The Growth Curve

Microorganisms grown in closed culture (also known as a batch culture), in which no nutrients are added and most waste is not removed, follow a reproducible growth pattern referred to as the growth curve. An example of a batch culture in nature is a pond in which a small number of cells grow in a closed environment. The culture density is defined as the number of cells per unit volume. In a closed environment, the culture density is also a measure of the number of cells in the population. Infections of the body do not always follow the growth curve, but correlations can exist depending upon the site and type of infection. When the number of live cells is plotted against time, distinct phases can be observed in the curve (Figure 9.5).

The Lag Phase

The beginning of the growth curve represents a small number of cells, referred to as an inoculum, that are added to a fresh culture medium, a nutritional broth that supports growth. The initial phase of the growth curve is called the lag phase, during which cells are gearing up for the next phase of growth. The number of cells does not change during the lag phase; however, cells grow larger and are metabolically active, synthesizing proteins needed to grow within the medium. If any cells were damaged or shocked during the transfer to the new medium, repair takes place during the lag phase. The duration of the lag phase is determined by many factors, including the species and genetic make-up of the cells, the composition of the medium, and the size of the original inoculum.

The Log Phase

In the logarithmic (log) growth phase, sometimes called exponential growth phase, the cells are actively dividing by binary fission and their number increases exponentially. For any given bacterial species, the generation time under specific growth conditions (nutrients, temperature, pH, and so forth) is genetically determined, and this generation time is called the intrinsic growth rate. During the log phase, the relationship between time and number of cells is not linear but exponential; however, the growth curve is often plotted on a semilogarithmic graph, as shown in Figure 9.6, which gives the appearance of a linear relationship.

Cells in the log phase show constant growth rate and uniform metabolic activity. For this reason, cells in the log phase are preferentially used for industrial applications and research work. The log phase is also the stage where bacteria are the most susceptible to the action of disinfectants and common antibiotics that affect protein, DNA, and cell-wall synthesis.

Stationary Phase

As the number of cells increases through the log phase, several factors contribute to a slowing of the growth rate. Waste products accumulate and nutrients are gradually used up. In addition, gradual depletion of oxygen begins to limit aerobic cell growth. This combination of unfavorable conditions slows and finally stalls population growth. The total number of live cells reaches a plateau referred to as the stationary phase (Figure 9.5). In this phase, the number of new cells created by cell division is now equivalent to the number of cells dying; thus, the total population of living cells is relatively stagnant. The culture density in a stationary culture is constant. The culture’s carrying capacity, or maximum culture density, depends on the types of microorganisms in the culture and the specific conditions of the culture; however, carrying capacity is constant for a given organism grown under the same conditions.

During the stationary phase, cells switch to a survival mode of metabolism. As growth slows, so too does the synthesis of peptidoglycans, proteins, and nucleic-acids; thus, stationary cultures are less susceptible to antibiotics that disrupt these processes. In bacteria capable of producing endospores, many cells undergo sporulation during the stationary phase. Secondary metabolites, including antibiotics, are synthesized in the stationary phase. In certain pathogenic bacteria, the stationary phase is also associated with the expression of virulence factors, products that contribute to a microbe’s ability to survive, reproduce, and cause disease in a host organism. For example, quorum sensing in Staphylococcus aureus initiates the production of enzymes that can break down human tissue and cellular debris, clearing the way for bacteria to spread to new tissue where nutrients are more plentiful.

The Death Phase

As a culture medium accumulates toxic waste and nutrients are exhausted, cells die in greater and greater numbers. Soon, the number of dying cells exceeds the number of dividing cells, leading to an exponential decrease in the number of cells (Figure 9.5). This is the aptly named death phase, sometimes called the decline phase. Many cells lyse and release nutrients into the medium, allowing surviving cells to maintain viability and form endospores. A few cells, the so-called persisters, are characterized by a slow metabolic rate. Persister cells are medically important because they are associated with certain chronic infections, such as tuberculosis, that do not respond to antibiotic treatment.

Sustaining Microbial Growth

The growth pattern shown in Figure 9.5 takes place in a closed environment; nutrients are not added and waste and dead cells are not removed. In many cases, though, it is advantageous to maintain cells in the logarithmic phase of growth. One example is in industries that harvest microbial products. A chemostat (Figure 9.7) is used to maintain a continuous culture in which nutrients are supplied at a steady rate. A controlled amount of air is mixed in for aerobic processes. Bacterial suspension is removed at the same rate as nutrients flow in to maintain an optimal growth environment.

Measurement of Bacterial Growth

Estimating the number of bacterial cells in a sample, known as a bacterial count, is a common task performed by microbiologists. The number of bacteria in a clinical sample serves as an indication of the extent of an infection. Quality control of drinking water, food, medication, and even cosmetics relies on estimates of bacterial counts to detect contamination and prevent the spread of disease. Two major approaches are used to measure cell number. The direct methods involve counting cells, whereas the indirect methods depend on the measurement of cell presence or activity without actually counting individual cells. Both direct and indirect methods have advantages and disadvantages for specific applications.

Direct Cell Count

Direct cell count refers to counting the cells in a liquid culture or colonies on a plate. It is a direct way of estimating how many organisms are present in a sample. Let’s look first at a simple and fast method that requires only a specialized slide and a compound microscope.

The simplest way to count bacteria is called the direct microscopic cell count, which involves transferring a known volume of a culture to a calibrated slide and counting the cells under a light microscope. The calibrated slide is called a Petroff-Hausser chamber (Figure 9.8) and is similar to a hemocytometer used to count red blood cells. The central area of the counting chamber is etched into squares of various sizes. A sample of the culture suspension is added to the chamber under a coverslip that is placed at a specific height from the surface of the grid. It is possible to estimate the concentration of cells in the original sample by counting individual cells in a number of squares and determining the volume of the sample observed. The area of the squares and the height at which the coverslip is positioned are specified for the chamber. The concentration must be corrected for dilution if the sample was diluted before enumeration.

Cells in several small squares must be counted and the average taken to obtain a reliable measurement. The advantages of the chamber are that the method is easy to use, relatively fast, and inexpensive. On the downside, the counting chamber does not work well with dilute cultures because there may not be enough cells to count.

Using a counting chamber does not necessarily yield an accurate count of the number of live cells because it is not always possible to distinguish between live cells, dead cells, and debris of the same size under the microscope. However, newly developed fluorescence staining techniques make it possible to distinguish viable and dead bacteria. These viability stains (or live stains) bind to nucleic acids, but the primary and secondary stains differ in their ability to cross the cytoplasmic membrane. The primary stain, which fluoresces green, can penetrate intact cytoplasmic membranes, staining both live and dead cells. The secondary stain, which fluoresces red, can stain a cell only if the cytoplasmic membrane is considerably damaged. Thus, live cells fluoresce green because they only absorb the green stain, whereas dead cells appear red because the red stain displaces the green stain on their nucleic acids (Figure 9.9).

Another technique uses an electronic cell counting device (Coulter counter) to detect and count the changes in electrical resistance in a saline solution. A glass tube with a small opening is immersed in an electrolyte solution. A first electrode is suspended in the glass tube. A second electrode is located outside of the tube. As cells are drawn through the small aperture in the glass tube, they briefly change the resistance measured between the two electrodes and the change is recorded by an electronic sensor (Figure 9.10); each resistance change represents a cell. The method is rapid and accurate within a range of concentrations; however, if the culture is too concentrated, more than one cell may pass through the aperture at any given time and skew the results. This method also does not differentiate between live and dead cells.

Direct counts provide an estimate of the total number of cells in a sample. However, in many situations, it is important to know the number of live, or viable, cells. Counts of live cells are needed when assessing the extent of an infection, the effectiveness of antimicrobial compounds and medication, or contamination of food and water.

Plate Count

The viable plate count, or simply plate count, is a count of viable or live cells. It is based on the principle that viable cells replicate and give rise to visible colonies when incubated under suitable conditions for the specimen. The results are usually expressed as colony-forming units per milliliter (CFU/mL) rather than cells per milliliter because more than one cell may have landed on the same spot to give rise to a single colony. Furthermore, samples of bacteria that grow in clusters or chains are difficult to disperse and a single colony may represent several cells. Some cells are described as viable but nonculturable and will not form colonies on solid media. For all these reasons, the viable plate count is considered a low estimate of the actual number of live cells. These limitations do not detract from the usefulness of the method, which provides estimates of live bacterial numbers.

Microbiologists typically count plates with 30–300 colonies. Samples with too few colonies (<30) do not give statistically reliable numbers, and overcrowded plates (>300 colonies) make it difficult to accurately count individual colonies. Also, counts in this range minimize occurrences of more than one bacterial cell forming a single colony. Thus, the calculated CFU is closer to the true number of live bacteria in the population.

There are two common approaches to inoculating plates for viable counts: the pour plate and the spread plate methods. Although the final inoculation procedure differs between these two methods, they both start with a serial dilution of the culture.

Serial Dilution

The serial dilution of a culture is an important first step before proceeding to either the pour plate or spread plate method. The goal of the serial dilution process is to obtain plates with CFUs in the range of 30–300, and the process usually involves several dilutions in multiples of 10 to simplify calculation. The number of serial dilutions is chosen according to a preliminary estimate of the culture density. Figure 9.11 illustrates the serial dilution method.

A fixed volume of the original culture, 1.0 mL, is added to and thoroughly mixed with the first dilution tube solution, which contains 9.0 mL of sterile broth. This step represents a dilution factor of 10, or 1:10, compared with the original culture. From this first dilution, the same volume, 1.0 mL, is withdrawn and mixed with a fresh tube of 9.0 mL of dilution solution. The dilution factor is now 1:100 compared with the original culture. This process continues until a series of dilutions is produced that will bracket the desired cell concentration for accurate counting. From each tube, a sample is plated on solid medium using either the pour plate method (Figure 9.12) or the spread plate method (Figure 9.13). The plates are incubated until colonies appear. Two to three plates are usually prepared from each dilution and the numbers of colonies counted on each plate are averaged. In all cases, thorough mixing of samples with the dilution medium (to ensure the cell distribution in the tube is random) is paramount to obtaining reliable results.

The dilution factor is used to calculate the number of cells in the original cell culture. In our example, an average of 50 colonies was counted on the plates obtained from the 1:10,000 dilution. Because only 0.1 mL of suspension was pipetted on the plate, the multiplier required to reconstitute the original concentration is 10 × 10,000. The number of CFU per mL is equal to 50 × 10 × 10,000 = 5,000,000. The number of bacteria in the culture is estimated as 5 million cells/mL. The colony count obtained from the 1:1000 dilution was 389, well below the expected 500 for a 10-fold difference in dilutions. This highlights the issue of inaccuracy when colony counts are greater than 300 and more than one bacterial cell grows into a single colony.

A very dilute sample—drinking water, for example—may not contain enough organisms to use either of the plate count methods described. In such cases, the original sample must be concentrated rather than diluted before plating. This can be accomplished using a modification of the plate count technique called the membrane filtration technique. Known volumes are vacuum-filtered aseptically through a membrane with a pore size small enough to trap microorganisms. The membrane is transferred to a Petri plate containing an appropriate growth medium. Colonies are counted after incubation. Calculation of the cell density is made by dividing the cell count by the volume of filtered liquid.

The Most Probable Number

The number of microorganisms in dilute samples is usually too low to be detected by the plate count methods described thus far. For these specimens, microbiologists routinely use the most probable number (MPN) method, a statistical procedure for estimating of the number of viable microorganisms in a sample. Often used for water and food samples, the MPN method evaluates detectable growth by observing changes in turbidity or color due to metabolic activity.

A typical application of MPN method is the estimation of the number of coliforms in a sample of pond water. Coliforms are gram-negative rod bacteria that ferment lactose. The presence of coliforms in water is considered a sign of contamination by fecal matter. For the method illustrated in Figure 9.14, a series of three dilutions of the water sample is tested by inoculating five lactose broth tubes with 10 mL of sample, five lactose broth tubes with 1 mL of sample, and five lactose broth tubes with 0.1 mL of sample. The lactose broth tubes contain a pH indicator that changes color from red to yellow when the lactose is fermented. After inoculation and incubation, the tubes are examined for an indication of coliform growth by a color change in media from red to yellow. The first set of tubes (10-mL sample) showed growth in all the tubes; the second set of tubes (1 mL) showed growth in two tubes out of five; in the third set of tubes, no growth is observed in any of the tubes (0.1-mL dilution). The numbers 5, 2, and 0 are compared with Figure B1 in Appendix B, which has been constructed using a probability model of the sampling procedure. From our reading of the table, we conclude that 49 is the most probable number of bacteria per 100 mL of pond water.no lo

1. Reversible attachment of planktonic cells (seconds) 2. First colonizers become irreversibly attached. (seconds, minutes) 3. Growth and cell division. (hours, days) 4. Production of E.P.S. and formation of water channels. (hours, days). 5. Attachment of secondary colonizers and dispersion of microbes to new sites. (days, months)