Chapter 4: Growth, Survival, and Death of Microorganisms

The population of microorganisms in the biosphere remains roughly constant because the growth of microorganisms is balanced by the death of these organisms. The survival of any microbial group within an environmental niche is ultimately influenced by successful competition for nutrients and by maintenance of a pool of all living cells, often composed of human cells and a consortium of different microorganisms (referred to as the microbiome or microbiota). Understanding competition for nutritional resources within a given microenvironment is essential to understanding the growth, survival, and death of bacterial species (also known as physiology).

Much of our understanding of microbial physiology has come from the study of isolated cultures grown under optimal conditions in laboratories (nutrient excess). However, most microorganisms compete in the natural environment under nutritional stress. Furthermore, a vacant environmental microbial niche will soon be filled with a different microbiota composition. In the end, appreciating the complex interactions that ensure the survival of a specific microbiome is a balance between availability of nutrients and physiologic efficiency.

Growth is the orderly increase in the sum of all the components of an organism. The increase in size that results when a cell takes up water or deposits lipid or polysaccharide is not true growth. Cell multiplication is a consequence of binary fission that leads to an increase in the number of single bacteria making up a population, referred to as a culture.

The Measurement of Microbial Concentrations

Microbial concentrations can be measured in terms of cell concentration (the number of viable cells per unit volume of culture) or of biomass concentration (dry weight of cells per unit volume of culture). These two parameters are not always equivalent because the average dry weight of the cell varies at different stages of a culture. Nor are they of equal significance: For example, in studies of microbial genetics and the inactivation of microbes, cell concentration is the significant quantity; in studies on microbial biochemistry or nutrition, biomass concentration is the significant quantity.

A. Viable Cell Count

The viable cell count (Table 4-1) is typically considered the measure of cell concentration. For this, a 1-mL volume is removed from a bacterial suspension and serially diluted 10-fold followed by plating 0.1-mL aliquots on an agar medium. Each single invisible bacterium (or clump of bacteria) will grow into a visible colony that can be counted (see Chapter 5). For statistical purposes, plates containing between 30 and 300 colonies give the most accurate data. The plate count × the dilution × 10 will give the number of colony forming units (CFU)/mL in the undiluted bacterial suspension. Using this method, dead bacteria within the suspension do not contribute to the final bacterial count.

B. Turbidity

For most purposes, the turbidity of a culture, measured by photoelectric means, can be related to the viable count using a standard curve. As an alternative a rough visual estimate is sometimes possible: For example, a barely turbid suspension of Escherichia coli contains about 10⁷ cells per milliliter, and a fairly turbid suspension contains about 10⁸ cells per milliliter. The correlation between turbidity and viable count can vary during the growth and death of a culture; cells may lose viability without producing a loss in turbidity of the culture.

C. Biomass Density

In principle, biomass can be measured directly by determining the dry weight of a microbial culture after it has been washed with distilled water. In practice, this procedure is cumbersome, and the investigator customarily prepares a standard curve that correlates dry weight with viable cell count. Alternatively, the concentration of biomass can be estimated indirectly by measuring an important cellular component such as protein or by determining the volume occupied by cells that have settled out of suspension.

The Growth Rate Constant

The growth rate of cells unlimited by nutrient is first order: The rate of growth (measured in grams of biomass produced per hour) is the product of time (t), the growth rate constant (k), and the biomass concentration B:

(1)

Rearrangement of equation (1) demonstrates that the growth rate constant is the rate at which cells are producing more cells:

(2)

A growth rate constant of 4.3 h⁻¹, one of the highest recorded, means that each gram of cells produces 4.3 g of cells per hour during this period of growth. Slower growing organisms may have growth rate constants as low as 0.02 h⁻¹. With this growth rate constant, each gram of cells in the culture produces 0.02 g of cells per hour.

Integration of equation (2) yields

(3)

The natural logarithm of the ratio of B₁ (the biomass at time 1 [t₁]) to B₀ (the biomass at time zero [t₀]) is equal to the product of the growth rate constant (k) and the difference in time (t₁ – t₀). Growth obeying equation (3) is termed exponential because biomass increases exponentially with respect to time. Linear correlations of exponential growth are produced by plotting the logarithm of biomass concentration (B) as a function of time (t).

Calculation of the Growth Rate Constant and Prediction of the Amount of Growth

Bacteria reproduce by binary fission, and the average time required for the population, or the biomass, to double is known as the generation time or doubling time (t_d). Usually the t_d is determined by plotting the amount of growth on a semi-logarithmic scale as a function of time; the time required for doubling the biomass is t_d (Figure 4-1). The growth rate constant can be calculated from the doubling time by substituting the value 2 for B₁/B₀ and t_d for t₁ – t₀ in equation (3), which yields

(4)

A rapid doubling time corresponds to a high growth rate constant. For example, a doubling time of 10 minutes (0.17 hour) corresponds to a growth rate constant of 4.1 h⁻¹. The relatively long doubling time of 35 hours indicates a growth rate constant of 0.02 h⁻¹.

The calculated growth rate constant can be used either to determine the amount of growth that will occur in a specified period of time or to calculate the amount of time required for a specified amount of growth.

The amount of growth within a specified period of time can be predicted on the basis of the following rearrangement of equation (3):

(5)

It is possible to determine the amount of growth that would occur if a culture with a growth rate constant of 4.1 h⁻¹ grew exponentially for 5 hours:

(6)

In this example, the increase in biomass is 10⁻⁹ g; a single bacterial cell with a dry weight of 2 × 10⁻¹³ g would give rise to 2 × 10⁻⁴ g (0.2 mg) of biomass, a quantity that would densely populate a 5 mL culture. Another 5 hours of growth at this rate would produce 200 kg dry weight of biomass, or roughly 1 ton of cells. This assumes that nutrients are unlimited, which is an assumption that does not occur in nature.

Another rearrangement of equation (3) allows calculation of the amount of time required for a specified amount of growth to take place. In equation (7), shown below, N, cell concentration, is substituted for B, biomass concentration, to permit calculation of the time required for a specified increase in cell number.

Using equation (7), it is possible, for example, to determine the time required for a slowly growing organism with a growth rate constant of 0.02 h⁻¹ to grow from a single cell into a barely turbid cell suspension with a concentration of 10⁷ cells per milliliter.

Solving equation (8) reveals that about 800 hours—slightly more than a month—would be required for this amount of growth to occur. The survival of slowly growing organisms implies that the race for biologic survival is not always to the swift—those species flourish that compete successfully for nutrients and avoid annihilation by predators and other environmental hazards.

If a fixed volume of liquid medium is inoculated with microbial cells taken from a culture that has previously been grown to saturation and the number of viable cells per milliliter is determined periodically and plotted, a curve of the type shown in Figure 4-2 is usually obtained. The phases of the bacterial growth curve shown in Figure 4-2 are reflections of the events in a population of cells, not in individual cells. This type of culture is referred to as a batch culture. The typical growth curve may be discussed in terms of four phases (Table 4-2). Batch culture is a closed system with finite resources; this is very different from the environment of the human host where nutrients are metabolized by bacteria and human cells. Nonetheless, understanding growth in batch culture provides fundamental insight into the genetics and physiology of bacterial replication, including the lag, exponential, stationary, and death phases that comprise this process.

Lag Phase

The lag phase represents a period during which cells, depleted of metabolites and enzymes as the result of the unfavorable conditions that existed at the end of their previous culture history, adapt to their new environment. Enzymes and intermediates are formed and accumulate until they are present in concentrations that permit growth to resume.

If the cells are taken from an entirely different medium, it often happens that they are genetically incapable of growth in the new medium. In such cases, a long lag in growth may occur, representing the period necessary for a few variants in the inoculum to multiply sufficiently for a net increase in cell number to be apparent.

Exponential Phase

During the exponential phase, the cells are in a steady state and grow as modeled in equations 5–7. New cell material is being synthesized at a constant rate, but the new material is itself catalytic, and the mass increases in an exponential manner. This continues until one of two things happens: either one or more nutrients in the medium become exhausted or toxic metabolic products accumulate and inhibit growth. For aerobic organisms, the nutrient that becomes limiting is usually oxygen. When the cell concentration exceeds about 1 × 10⁷/mL, the growth rate decreases unless oxygen is forced into the medium by agitation or by bubbling in air. When the bacterial concentration reaches 4–5 × 10⁹/mL, the rate of oxygen diffusion cannot meet the demand even in an aerated medium, and growth is progressively slowed.

Stationary Phase

Eventually, the exhaustion of nutrients or the accumulation of toxic products causes growth to cease completely. In most cases, however, cell turnover takes place in the stationary phase: There is a slow loss of cells through death, which is balanced by the formation of new cells through growth and division. When this occurs, the total cell count slowly increases, although the viable count stays constant.

Death Phase

After a period of time in the stationary phase, cell viability begins to decrease at a defined rate. This varies with the organism and with the culture conditions; the death rate increases until it reaches a steady level. The mathematics of steady-state death is discussed as follows. In most cases, the rate of cell death is much slower than that of exponential growth. Frequently, after the majority of cells have died, the death rate decreases drastically, so that a small number of survivors may persist for months or even years. This persistence may in some cases reflect cell turnover, a few cells growing at the expense of nutrients released from cells that die and lyse.

A bacterial culture phenomenon referred to as viable but not culturable (VBNC) cells, is thought to be the result of a genetic response triggered in starving, stationary phase cells. Just as some bacteria form spores as a survival mechanism, others are able to become dormant without changes in morphology. When the appropriate conditions are available (eg, passage through an animal), VNBC microbes resume growth.

The Chemostat

Cells can be maintained in the exponential phase by transferring them repeatedly into fresh medium of identical composition while they are still growing exponentially. This is referred to as continuous culture; the most common type of continuous culture device used is a chemostat. This device consists of a culture vessel equipped with an overflow siphon and a mechanism for dripping in fresh medium from a reservoir at a regulated rate. The medium in the culture vessel is stirred by a stream of sterile air; each drop of fresh medium that enters causes a drop of culture to siphon out. The medium is prepared such that one nutrient limits growth yield. The vessel is inoculated, and the cells grow until the limiting nutrient is exhausted; fresh medium from the reservoir is then allowed to flow in at such a rate that the cells use up the limiting nutrient as fast as it is supplied. Under these conditions, the cell concentration remains constant, and the growth rate is directly proportionate to the flow rate of the medium. Continuous culture is more similar to conditions that organisms encounter in the real world (eg, the human body), where limiting nutrients are constantly being replaced.

It has been increasingly recognized that many infections are caused by bacteria that do not grow individually (planktonically); rather, they exist in intimate and complex communities. For example, it is routine to debride our teeth every day to remove the bacterial biofilm that accumulates while we sleep. Similarly, biofilms are associated with Streptococcus viridians on heart valves, Pseudomonas aeruginosa lung infections, Staphylococcus aureus on catheters, or Legionella pneumophila colonization of hospital water systems, among many others. Understanding the growth of bacterial biofilms has become an increasingly important aspect of medical microbiology.

Biofilms begin with a single bacterium nucleating on a surface followed by binary fission and ultimately to the formation of an intimate community of progeny bacteria (see Chapter 10). Eventually this bacterial community surrounds itself with a glycocalyx for environmental protection. The glycocalyx also serves to keep the biofilm community intact. Bacteria within a biofilm produce small molecules, such as homoserine lactones, which are taken up by adjacent bacteria and functionally serve as a colony “telecommunication” system, informing individual bacteria to turn on certain genes at a particular time (Quorum Sensing). These signals are known as quorum sensors. One can quickly see that bacterial growth within a biofilm is not unlike the social evolution of higher animals.

Conceptually, the strategy of biofilm formation is logical. It promotes increased metabolic diversity. For example, bacteria on the periphery of the biofilm may have more access to oxygen and other nutrients than organisms on the inner portions of the film. On the other hand, cells on the inner portions may be shielded from predation by immune cells, or from antibiotics. Intimately attached bacteria may be able to efficiently transfer genes that would result in phenotypic versatility when compared to planktonic cells. Because of all these variables it is difficult to mathematically model biofilm growth as compared to growth in batch culture. This is an important area of medical microbiology that needs to be considered in the larger context of infectious disease.

The Meaning of Bacterial Death

For a microbial cell, death means the irreversible loss of the ability to reproduce (grow and divide). Noting the exception of VBMC organisms described previously, the empirical test of death is culture of cells on solid media: A cell is considered dead if it fails to give rise to a colony on appropriate medium. Obviously, then, the reliability of the test depends on the choice of medium and conditions: For example, a culture in which 99% of the cells appear “dead” in terms of the ability to form colonies on one medium may prove to be 100% viable if tested on another medium. Furthermore, the detection of a few viable cells in a large clinical specimen may not be possible by directly plating a sample because the sample fluid itself may be inhibitory to microbial growth. In such cases, the sample may have to be diluted first into liquid medium, permitting the outgrowth of viable cells before plating.

The conditions of incubation in the first hour after treatment are also critical in the determination of “killing.” For example, if bacterial cells are irradiated with ultraviolet light and plated immediately on any medium, it may appear that 99.99% of the cells have been killed. If such irradiated cells are first incubated in a suitable medium for 20 minutes, plating may indicate only 10% killing. In other words, irradiation determines that a cell will “die” if plated immediately but will live if allowed to repair radiation damage before plating. A microbial cell that is not physically disrupted is thus “dead” only in terms of the conditions used to test viability.

The Measurement of Bacterial Death

When dealing with microorganisms, one does not customarily measure the death of an individual cell but the death of a population. This is a statistical problem: Under any condition that may lead to cell death, the probability of a given cell’s dying is constant per unit time. For example, if a condition is used that causes 90% of the cells to die in the first 10 minutes, the probability of any one cell dying in a 10-minute interval is 0.9. Thus, it may be expected that 90% of the surviving cells will die in each succeeding 10-minute interval, and a death curve can be generated. The number of cells dying in each time interval is thus a function of the number of survivors present, so that death of a population proceeds as an exponential process according to the general formula:

(9)

where S₀ is the number of survivors at time zero and S is the number of survivors at any later time t. As in the case of exponential growth, −k represents the rate of exponential death when the fraction ln (S/S₀) is plotted against time.

The kinetics of bacterial cell killing is also a function of the number of targets required to be hit by a particular agent to kill a specific planktonic microbe. For example, a single “hit” could target the haploid chromosome of a bacterium or target its cell membrane. By contrast, a cell that contains several copies of the target to be inactivated exhibits a multihit curve. This analysis is graphically shown in Figure 4-3.

The robust nature of uncontrolled microbial growth clearly presents a conflict with human life. To coexist with bacteria, higher species have to control bacterial growth. We, as humans, do this in a biologic context using an immune system and nutrient limitation. We also use physical methods to prevent exposure to microorganisms. Terms like sterilization, disinfection, pasteurization, and aseptic need to be precisely understood so as to articulate them in a proper sense. A list of these terms and their definitions are provided in Table 4-3.

As an example of the importance of understanding these terms, we speak of sterilization as the process of killing all the organisms, including spores, in a given preparation. Understanding this concept would be particularly important for surgical instruments because one would not want to introduce spores into the surgical site. By contrast, “disinfecting” these instruments may eliminate the bacteria but not the spores. Further, physically “cleaning” the instruments may not remove all of the bacteria and spores but simply decrease the bioburden on the instrument. The point is that an understanding of the terms used in Table 4-3 is critical to controlling the environmental impact of microorganisms in the context of human health.

In medical microbiology one often considers the control of bacteria infecting humans with antibiotics as the gold standard in treating infections. While true, the real first line is to prevent exposure to infectious agents. For example, nearly 240,000 deaths annually occur worldwide as a result of neonatal tetanus. Yet this disease is very rare in developed countries. A major contributing factor is the inability to “sterilize” instruments (in addition to routine immunization with the tetanus vaccine) in many developing countries. If proper practices were used in underdeveloped regions, this disease could be substantially eliminated. Thus, one must understand methods of sterilization, disinfection, and pasteurization, among others. The techniques used to mitigate microbial infection should be understood at the mechanism of action level in order to apply them in the appropriate situation. Table 4-4 represents a nonexhaustive list of routinely used biocides. It is important to understand the terms bacteriostatic and bactericidal as defined in Table 4-4. The general mechanisms by which these biocides accomplish their antimicrobial activity are summarized in the following section.

Disruption of the Cell Membrane or Wall

The cell membrane acts as a selective barrier, allowing some solutes to pass through and excluding others. Many compounds are actively transported through the membrane, becoming concentrated within the cell. The membrane is also the site of enzymes involved in the biosynthesis of components of the cell envelope. Substances that concentrate at the cell surface may alter the physical and chemical properties of the membrane, preventing its normal functions and therefore killing or inhibiting the cell.

The cell wall acts as a corseting structure (best characterized as a fishing net), protecting the cell against osmotic lysis. Thus, agents that destroy the wall (eg, lysozyme, which cleaves the sugar linkages of peptidoglycan) or prevent its normal synthesis (eg, penicillin, which interrupts peptidyl cross-linkages) may bring about lysis of the cell.

Protein Denaturation

Proteins exist in a folded, three-dimensional state determined primarily by intramolecular noncovalent interactions such as ionic, hydrophobic, and hydrogen bonds or covalent disulfide linkages. This state is called the tertiary structure of the protein; it is readily disrupted by a number of physical (eg, heat) or chemical (eg, alcohol) agents, causing the protein to become nonfunctional. The disruption of the tertiary structure of a protein is called protein denaturation.

Disruption of Free Sulfhydryl Groups

Enzymes containing cysteine have side chains terminating in sulfhydryl groups. In addition to these, coenzymes such as coenzyme A and dihydrolipoate contain free sulfhydryl groups. Such enzymes and coenzymes cannot function unless the sulfhydryl groups remain free and reduced. Oxidizing agents thus interfere with metabolism by forming disulfide linkages between neighboring sulfhydryl groups:

Many metals such as mercuric ions likewise interfere by combining with sulfhydryls. There are sulfhydryl-containing enzymes in the cell, so oxidizing agents and heavy metals do widespread damage.

Damage to DNA

A number of physical and chemical agents act by damaging DNA; these include ionizing radiations, ultraviolet light, and DNA-reactive chemicals. Among the last category are alkylating agents and other compounds that react covalently with purine and pyrimidine bases to form DNA adducts or interstrand cross-links. Radiation can damage DNA in several ways: Ultraviolet light, for example, induces cross-linking between adjacent pyrimidines on one or the other of the two polynucleotide strands, forming pyrimidine dimers; ionizing radiations produce breaks in single and double strands. Radiation-induced and chemically-induced DNA lesions kill the cell mainly by interfering with DNA replication. See Chapter 7 for a discussion of DNA repair systems.

Chemical Antagonism

The interference by a chemical agent with the normal reaction between a specific enzyme and its substrate is known as chemical antagonism. The antagonist acts by combining with some part of the holoenzyme (the protein apoenzyme, the mineral activator, or the coenzyme), thereby preventing attachment of the normal substrate. (Substrate here is used in the broad sense to include cases in which the inhibitor combines with the apoenzyme, thereby preventing attachment to it to the coenzyme.)

An antagonist combines with an enzyme because of its chemical affinity for an essential site on that enzyme. Enzymes perform their catalytic function by virtue of their affinity for their natural substrates; hence any compound structurally resembling a substrate in essential aspects may also have an affinity for the enzyme. If this affinity is great enough, the “analog” will displace the normal substrate and prevent the proper reaction from taking place.

Many holoenzymes include a mineral ion as a bridge either between enzyme and coenzyme or between enzyme and substrate. Chemicals that combine readily with these minerals will again prevent attachment of coenzyme or substrate (eg, carbon monoxide and cyanide combine with the iron atom in heme-containing enzymes and prevent their function in respiration).

Chemical antagonists can be conveniently discussed under two headings: (a) antagonists of energy-yielding processes and (b) antagonists of biosynthetic processes. The former include poisons of respiratory enzymes (carbon monoxide, cyanide) and of oxidative phosphorylation (dinitrophenol); the latter include amino acid and nucleic acid analogs. In some cases, the analog simply prevents incorporation of the normal metabolite (eg, 5-methyltryptophan prevents incorporation of tryptophan into proteins), and in other cases, the analog replaces the normal metabolite in the macromolecule, causing it to be nonfunctional. The incorporation of p-fluorophenylalanine in place of phenylalanine in proteins is an example of the latter type of antagonism.

Selected important physical and chemical agents are described in the following sections.

Physical Methods

A. Heat

Application of heat is the simplest means of sterilizing materials, provided the material is itself resistant to heat damage. A temperature of 100°C will kill all but spore forms of eubacteria within 2–3 minutes in laboratory-scale cultures; a temperature of 121°C for 15 minutes is used to kill spores. Steam is generally used, both because bacteria are more quickly killed when moist and because steam provides a means for distributing heat to all parts of the sterilizing vessel. At sea level, steam must be kept at a pressure of 15 lb/sq inches (psi) in excess of atmospheric pressure to obtain a temperature of 121°C; autoclaves or pressure cookers are used for this purpose. At higher altitudes, the pressure would need to be higher than 15 psi to reach 121°C. For sterilizing materials that must remain dry, circulating hot air electric ovens are available; because heat is less effective on dry material, it is customary to apply a temperature of 160–170°C for 1 hour or more. Under these conditions (ie, excessive temperatures applied for long periods of time), heat acts by denaturing cell proteins and nucleic acids and by disrupting cell membranes. This treatment, if performed appropriately, is sporicidal.

B. Radiation

Ultraviolet (UV) radiation that has a wavelength of about 260 nm causes thymidine dimers resulting in the inability of bacterial DNA to be replicated. This is generally bactericidal but may not be sporicidal.

Ionizing radiation of 1 nm or less (gamma or x-ray) causes free radical formation that damage proteins, DNA, and lipids. These treatments are both bactericidal and sporicidal.

C. Chemical Agents

The chemical structures and uses of biocides are shown in Table 4-4; selective activities of these are described in the following sections.

D. Alcohols

These agents effectively remove water from biologic systems. Thus, they functionally act as “liquid desiccants.” Ethyl alcohol, isopropyl alcohol, and n-propanol exhibit rapid, broad-spectrum antimicrobial activity against vegetative bacteria, viruses, and fungi but are not sporicidal. Activity is optimal when they are diluted to a concentration of 60–90% with water. This treatment strategy is generally considered bactericidal but not sporicidal.

E. Aldehydes

Compounds like glutaraldehyde or formaldehyde are used for low-temperature disinfection and sterilization of instruments, endoscopes, and surgical tools. They are normally used as a 2% solution to achieve sporicidal activity. These compounds are generally bactericidal and sporicidal.

F. Biguanides

Chlorhexidine is widely used in hand washing and oral products and as a disinfectant and preservative. These compounds are bactericidal but not sporicidal. The mycobacteria, because of their unique waxy cell envelope, are generally highly resistant to these compounds.

G. Bisphenols

The bisphenols are widely used in antiseptic soaps and hand rinses. In general, they have broad-spectrum microbicidal activity but have little activity against P aeruginosa and molds. Triclosan and hexachlorophene are bactericidal and sporostatic (not sporicidal).

H. Halogen-Releasing Agents

The most important types of chlorine-releasing agents are sodium hypochlorite, chlorine dioxide, and sodium dichloroisocyanurate, which are oxidizing agents that destroy the cellular activity of proteins. Hypochlorous acid is the active compound responsible for the bactericidal effect of these compounds. At higher concentrations, this group is sporicidal. Iodine (I₂) is rapidly bactericidal and sporicidal. Iodophors (eg, povidone-iodine) are complexes of iodine and a solubilizing agent or carrier, which acts as a reservoir of the active I₂.

I. Heavy Metal Derivatives

Silver (Ag⁺) sulfadiazine, a combination of two antibacterial agents, Ag⁺ and sulfadiazine, has a broad spectrum of activity. Binding to cell components such as DNA is principally responsible for its inhibitory properties. These compounds are not sporicidal.

J. Organic Acids

Organic acids are used as preservatives in the pharmaceutical and food industries. Benzoic acid is fungistatic, while propionic acid is both bacteriostatic and fungistatic. Neither is sporicidal.

K. Peroxygens

Hydrogen peroxide (H₂O₂) has broad-spectrum activity against viruses, bacteria, yeasts, and bacterial spores. Sporicidal activity requires higher concentrations (10–30%) of H₂O₂ and longer contact times.

L. Phenols

Phenol and many phenolic compounds have antiseptic, disinfectant, or preservative properties. In general, these are not sporicidal.

M. Quaternary Ammonium Compounds

These compounds have two regions in their molecular structures, one a water-repelling (hydrophobic) group and the other a water-attracting (hydrophilic) group. Cationic detergents, as exemplified by quaternary ammonium compounds (QACs), are useful antiseptics and disinfectants. QACs have been used for a variety of clinical purposes (eg, preoperative disinfection of unbroken skin) as well as for cleaning hard surfaces. They are sporostatic; they inhibit the outgrowth of spores but not the actual germination process. QACs have an effect on enveloped but not nonenveloped viruses. In general, these are not sporicidal.

N. Vapor-Phase Sterilants

Heat-sensitive medical devices and surgical supplies can be effectively sterilized by vapor-phase systems using ethylene oxide, formaldehyde, hydrogen peroxide, or peracetic acid. These are sporicidal.

When biocides described previously are used to effect microbial populations, the variables of time and concentration need to be considered. It is commonly observed that the concentration of the substance used is related to the time required to kill a given fraction of the population by the following expression:

(10)

In this equation, C is the biocide concentration, t is the time required to kill a given fraction of the cells, and n and K are constants.

This expression says that, for example, if n = 6 (as it is for phenol), then doubling the concentration of the drug will reduce the time required to achieve the same extent of inactivation 64-fold. That the effectiveness of a biocide varies with the sixth power of the concentration suggests that six molecules of the drug biocide are required to inactivate a cell, although there is no direct chemical evidence for this conclusion.

To determine the value of n for any biocide, inactivation curves are obtained for each of several concentrations, and the time required at each concentration to inactivate a fixed fraction of the population is determined. For example, let the first concentration used be noted as C₁ and the time required to inactivate 99% of the cells be t₁. Similarly, let C₂ and t₂ be the second concentration and time required to inactivate 99% of the cells. From equation (10), we see that:

(11)

Solving for n gives:

(12)

Thus, n can be determined by measuring the slope of the line that results when log t is plotted against log C (Figure 4-4). If n is experimentally determined in this manner, K can be determined by substituting observed values for C, t, and n in equation (10).

Reversal of Biocide Action

In addition to time- and concentration-dependent kinetics, other considerations of biocide activity involve the ability of antimicrobial activity to be reversed. Table 4-5 summarizes a list of mechanisms that can reverse the activity of biocides. These include agent removal, substrate competition, and agent inactivation. Neutralization of biocides needs to be considered as part of the sterilization/disinfection strategy.

Appreciating the growth and death of bacteria is fundamental to understanding the complex interaction that exists between pathogenic bacteria and their hosts. If unchecked by an intact immune system and nutrient limitation, logarithmic growth of bacteria would quickly outcompete the host for nutrients. The environmental control of microbial growth by biocides limits exposure to potentially pathogenic microorganisms. The concepts of sterilization, disinfection, pasteurization, and others, are central to bacterial control and ultimately to human health. In the end, understanding microbial growth and death is the first step toward effectively managing infectious diseases.

1. Bacteria in humans exist as complex biosystems known as microbiota.

2. Quantification of bacterial cells can be accomplished using viable cell count, turbidity, and biomass.

3. Biomass and generation time are mathematically related.

4. Inoculating a single bacterial colony into a fixed volume of liquid medium is known as batch culture. In this system, bacterial growth exhibits four phases—lag, log, stationary, and death.

5. Some bacteria exist in a state that is defined as viable but not culturable.

6. Growth in continuous culture or as a biofilm more closely approximates bacterial growth within the human host.

7. Sterilization, disinfection, pasteurization, as well as other terms (see Table 4-3) are critical to understanding and communicating the science of microbiology.

8. The general structures of biocides (see Table 4-4) and mechanisms of action should be understood.

9. Depending on the mechanism of action, different biocides are bacteriostatic, bactericidal, and/or sporicidal.

10. Biocide activity is dependent on time and concentration. This activity can be reversed by agent removal, substrate competition, and agent inactivation.