Sunday, 5 October 2014

CONTROL OF MICROBIAL GROWTH: bacterial cell cycle, growth curve (log phase, exponential phase, stationary phase, senescence & death), measurement of microbial growth, chemostat, turbidostat, influence of environmental factors on growth (solute & water activity, pH, temperature, oxygen concentration, pressure, radiation), pattern of microbial death, physical agents (heat, filtration, radiations), chemical agents (phenols, alcohols, halogens, heavy metals, quaternary ammonium salts, aldehydes & sterilizing gases), biological control, determining the level of antimicrobial activity (dilution susceptibility test, disk diffusion test, the E test), antibacterial drug ( inhibitor of cell wall synthesis, inhibitors of protein synthesis, inhibitor of nucleic acid synthesis), antifungal drugs, antiviral drugs, antiprotozoan drug.

Control of microbial growth
Growth may be defined as an increase in cellular constituents. It leads to a rise in cell number when microorganisms reproduce. Growth also results when cells simply become longer or larger. If the microorganism is coenocytic; that is, a multinucleate organism in which chromosomal replication is not accompanied by cell division— growth results in an increase in cell size but not cell number. It is usually not convenient to investigate the growth and reproduction of individual microorganisms because of their small size. Therefore, when studying growth, microbiologists normally follow changes in the total population number.


 The    cell cycle    is the complete sequence of events extending from the formation of a new cell through the next division. It is of intrinsic interest to microbiologists as a fundamental biological process. However, understanding the cell cycle has practical importance as well. For instance in bacteria, the synthesis of peptidoglycan are the target of numerous antibiotics. Antibacterial drugs: Inhibitors of cell wall synthesis. Although some prokaryotes reproduce by budding, fragmentation, and other means, most prokaryotes reproduce by binary fission. Binary fission is a relatively simple type of cell division: the cell elongates, replicates its chromosome, and separates the newly formed DNA molecules so there is one chromosome in each half of the cell. Finally, a septum (cross wall) is formed at mid cell, dividing the parent cell into two progeny cells, each having its own chromosome and a complement of other cellular constituents. Despite the apparent simplicity of the prokaryotic cell cycle, it is poorly understood. The cell cycles of several bacteria— Escherichia coli, Bacillus subtilis, and the aquatic bacterium Caulobacter crescentus— have been examined extensively, and our understanding of the bacterial cell cycle is based largely on these studies. Two pathways function during the bacterial cell cycle: one pathway replicates and partitions the DNA into the progeny cells, the other carries out cytokinesis—formation of the septum and progeny cells. Although these pathways overlap, it is easiest to consider them separately.

Chromosome Replication and Partitioning
 Recall that most bacterial chromosomes are circular. Each circular chromosome has a single site at which replication starts called the origin of replication or simply the origin.  Replication is completed at the terminus, which is located directly opposite the origin. In a newly formed E. coli cell, the chromosome is compacted and organized so that the origin and terminus are in opposite halves of the cell. Early in the cell cycle, the origin and terminus move to midcell and a group of proteins needed for DNA synthesis assemble at the origin to form the replisome. DNA replication proceeds in both directions from the origin, and the parent DNA is thought to spool through the replisomes. As progeny chromosomes are synthesized, the two newly formed origins move toward opposite ends of the cell, and the rest of the chromosome follows in an orderly fashion. Although the process of DNA synthesis and movement seems rather straightforward, the mechanism by which chromosomes are partitioned to each daughter cell is not well understood. Surprisingly, a picture is emerging in which components of the cytoskeleton are involved. For many years, it was assumed that prokaryotes were too small for eukaryotic, like cytoskeletal structures. However, a protein called MreB, which is similar to eukaryotic actin, seems to be involved in several processes, including determining cell shape and chromosome movement.
MreB polymerizes to form a spiral around the inside periphery of the cell. One model suggests that the origin of each newly replicated chromosome associates with MreB, which then moves them to opposite poles of the cell. The notion that prokaryotic chromosomes may be actively moved to the poles is further suggested by the fact that if MreB is mutated so that it can no longer hydrolyze ATP, its source of energy, chromosomes fail to segregate properly.  Prokaryotic cytoplasm: Prokaryotic cytoskeleton.


    Septation is the process of forming a cross wall between two daughter cells.    Cytokinesis, a term that has traditionally been used to describe the formation of two eukaryotic daughter cells, is now used to describe this process in prokaryotes as well. Septation is divided into several steps: 

(1) Selection of the site where the septum will be formed;
(2) Assembly of a specialized structure called the Z ring, which divides the cell in two by constriction;
(3) Linkage of the Z ring to the plasma membrane and perhaps components of the cell wall;
(4) Assembly of the cell wall-synthesizing machinery (i.e., for synthesis of peptidoglycan and other cell wall constituents); and
 (5) Constriction of the cell and septum formation. Synthesis of sugars and polysaccharides: Synthesis of peptidoglycan. The assembly of the Z ring is a critical step in septation, as it must be formed if subsequent steps are to occur. The FtsZ protein, a tubulin homologue found in most bacteria and many archaea, forms the Z ring. FtsZ, like tubulin, polymerizes to form fi laments, which are thought to create the meshwork that constitutes the Z ring. Numerous studies show that the Z ring is very dynamic, with portions being exchanged constantly with newly formed, short FtsZ polymers from the cytosol. Another protein, called MinCD, is an inhibitor of Z-ring assembly. Like FtsZ, it is very dynamic, oscillating its position from one end of the cell to the other, forcing Z-ring formation only at the center of the cell. Once the Z-ring forms, the rest of the division machinery is constructed. First one or more anchoring proteins link the Z ring to the cell membrane. Then the cell wall-synthesizing machinery is assembled. The final steps in division involve constriction of the cell by the Z ring, accompanied by invagination of the cell membrane and synthesis of the septal wall. Several models for Z-ring function have been proposed. One model holds that the FtsZ filaments are shortened by losing FtsZ subunits (i.e., depolymerization) at sites where the Z ring is anchored to the plasma membrane. This model is supported by the observation that Z rings of cells producing an excessive amount of FtsZ subunits fail to constrict the cell. The preceding discussion of the cell cycle describes what occurs in slowly growing E. coli cells. In these cells, the cell cycle takes approximately 60 minutes to complete. However, E.coli can reproduce at a much more rapid rate, completing the entire cell cycle in about 20 minutes, despite the fact that DNA replication always requires at least 40 minutes.  E. coli accomplishes this by beginning a second round of DNA replication (and sometimes even a third or fourth round) before the first round of replication is completed. Thus the progeny cells receive two or more replication forks, and replication is continuous because the cells are always copying their DNA.    


 Binary fission and other cell division processes bring about an increase in the number of cells in a population. Population growth is studied by analyzing the growth curve of a microbial culture. When microorganisms are cultivated in liquid medium, they usually are grown in a batch culture, i.e.; they are incubated in a closed culture vessel with a single batch of medium. Because no fresh medium is provided during incubation, nutrient concentrations decline and concentrations of wastes increase. The growth of microorganisms reproducing by binary fission can be plotted as the logarithm of the number of viable cells versus the incubation time. The resulting curve has four distinct phases.

 Lag Phase
When microorganisms are introduced into fresh culture medium, usually no immediate increase in cell number occurs. This period is called the lag phase.  However, cells in the culture are synthesizing new components. A lag phase can be necessary for a variety of reasons. The cells may be old and depleted of ATP, essential cofactors, and ribosomes; these must be synthesized before growth can begin. The medium may be different from the one the microorganism was growing in previously. Here new enzymes would be needed to use different nutrients. Possibly the microorganisms have been injured and require time to recover. Whatever the causes, eventually the cells begin to replicate their DNA, increase in mass, and finally divide.   

 Exponential Phase
 During the exponential (log) phase, microorganisms are growing and dividing at the maximal rate possible given their genetic potential, the nature of the medium, and the environmental conditions. Their rate of growth is constant during the exponential phase; that is, they are completing the cell cycle and doubling in number at regular intervals. The population is most uniform in terms of chemical and physiological properties during this phase; therefore exponential phase cultures are usually used in biochemical and physiological studies. Exponential (logarithmic) growth is balanced growth.  That is, all cellular constituents are manufactured at constant rates relative to each other. If nutrient levels or other environmental conditions change, unbalanced growth results. During unbalanced growth, the rates of synthesis of cell components vary relative to one another until a new balanced state is reached. Unbalanced growth is readily observed in two types of experiments: shift-up, where a culture is transferred from a nutritionally poor medium to a richer one; and shift-down, where a culture is transferred from a rich medium to a poor one. In a shift-up experiment, there is a lag while the cells first construct new ribosomes to enhance their capacity for protein synthesis. In a shift-down experiment, there is a lag in growth because cells need time to make the enzymes required for the biosynthesis of unavailable nutrients. Once the cells are able to grow again, balanced growth is resumed and the culture enters the exponential phase. These shift-up and shift-down experiments demonstrate that microbial growth is under precise, coordinated control and responds quickly to changes in environmental conditions. When microbial growth is limited by the low concentration of a required nutrient, the final net growth or yield of cells increases with the initial amount of the limiting nutrient present. The rate of growth also increases with nutrient concentration but in a hyperbolic manner much like that seen with many enzymes. The shape of the curve seems to reflect the rate of nutrient uptake by microbial transport proteins. At sufficiently high nutrient levels, the transport systems are saturated, and the growth rate does not rise further with increasing nutrient concentration. 

 Stationary Phase
 In a closed system such as a batch culture, population growth eventually ceases and the growth curve becomes horizontal. This stationary phase usually is attained by bacteria at a population level of around 10^6 cells per ml. Other microorganisms normally do not reach such high population densities. For instance, protist cultures often have maximum concentrations of about 10^6 cells per ml. Final population size depend on nutrient availability and other factors, as well as the type of microorganism being cultured. In the stationary phase, the total number of viable microorganisms remains constant. This may result from a balance between cell division and cell death or the population may simply cease to divide but remain metabolically active. Microbial populations enter the stationary phase for several reasons. One obvious factor is nutrient limitation; if an essential nutrient is severely depleted, population growth will slow. Aerobic organisms often are limited by O2  availability. Oxygen is not very soluble and may be depleted so quickly that only the surface of a culture will have an O2 concentration adequate for growth. The cells beneath the surface will not be able to grow unless the culture is shaken or aerated in another way. Population growth also may cease due to the accumulation of toxic waste products. This factor seems to limit the growth of many anaerobic cultures (cultures growing in the absence of O2). For example, streptococci can pro- duce so much lactic acid and other organic acids from sugar fermentation that their medium becomes acidic and growth is inhibited. Finally, some evidence exists that growth may cease when a critical population level is reached. Thus entrance into the stationary phase may result from several factors operating in concert.   As we have seen, bacteria in a batch culture may enter stationary phase in response to starvation. This probably occurs often in nature because many environments have low nutrient levels. Prokaryotes have evolved a number of strategies to survive starvation. Some bacteria respond with obvious morphological changes such as endospore formation, but many only decrease somewhat in overall size. This is often accompanied by protoplast shrinkage and nucleoid condensation. The more important changes during starvation are in gene expression and physiology. Starving bacteria frequently produce a variety of starvation proteins, which make the cell much more resistant to damage. Some increase peptidoglycan crosslinking and cell wall strength. The DBP (DNA-binding protein from starved cells) protein protects DNA. Proteins called chaperone proteins prevent protein denaturation and renature damaged proteins. Because of these and many other mechanisms, starved cells become harder to kill and more resistant to starvation, damaging temperature changes, oxidative and osmotic damage, and toxic chemicals such as chlorine. These changes are so effective that some bacteria can survive starvation for years. There is even evidence that Salmonella enterica serovar Typhimurium (S.typhimurium) and some other bacterial pathogens become more virulent when starved. Clearly, these considerations are of great practical importance in medical and industrial microbiology.   

 Senescence and Death
For many years, the decline in viable cells following the stationary phase was described simply as the “death phase.” It was assumed that detrimental environmental changes such as nutrient deprivation and the buildup of toxic wastes caused irreparable harm and loss of viability. That is, even when bacterial cells were transferred to fresh medium, no cellular growth was observed. Because loss of viability was often not accompanied by a loss in total cell number, it was assumed that cells died but did not lyse.   This view is currently under debate. There are two alternative hypotheses. Some microbiologists think starving cells that show an exponential decline in density have not irreversibly lost their ability to reproduce. Rather, they suggest that microbes are temporarily unable to grow, at least under the laboratory conditions used. This phenomenon, in which the cells are called viable but non culturable (VBNC) is thought to be the result of a genetic response triggered in starving, stationary phase cells. Just as some bacteria form endospores as a survival mechanism, it is argued that others are able to become dormant without changes in morphology. Once the appropriate conditions are available (for instance, a change in temperature or passage through an animal), VBNC microbes resume growth. VBNC microorganisms could pose a public health threat, as many assays that test for food and drinking water safety are culture-based. The second alternative to a simple death phase is    programmed cell death. In contrast to the VBNC hypothesis whereby cells are genetically programmed to survive, programmed cell death predicts that a fraction of the microbial population is genetically programmed to die after growth ceases. In this case, some cells die and the nutrients they leak enable the eventual growth of those cells in the population that did not initiate cell death. The dying cells are thus “altruistic”—they sacrifice themselves for the benefit of the larger population. Long-term growth experiments reveal that an exponential decline in viability is sometimes replaced by a gradual decline in the number of culturable cells. This decline can last months to years. During this time, the bacterial population continually evolves so that actively reproducing cells are those best able to use the nutrients released by their dying brethren and best able to tolerate the accumulated toxins. This dynamic process is marked by successive waves of genetically distinct variants. Thus natural selection can be witnessed within a single culture vessel.            


There are many ways to measure microbial growth to determine growth rates and generation times. Either population number or mass may be followed because growth leads to increases in both. Here the most commonly employed techniques for deter- mining population size are examined briefly and the advantages and disadvantages of each noted. No single technique is always best; the most appropriate approach depends on the experimental situation.  

 Measurement of Cell Numbers
 The most obvious way to determine microbial numbers is by direct counts.  Using a counting chamber is easy, inexpensive, and relatively quick; it also gives information about the size and morphology of microorganisms. Petroff-Hausser counting chambers can be used for counting procaryotes; hemocytometers can be used for both procaryotes and eucaryotes. Both of these specially designed slides have chambers of known depth with an etched grid on the chamber bottom. Procaryotes are more easily counted if they are stained or when a phase-contrast or a fluorescence microscope is employed. The number of micro- organisms in a sample can be calculated by taking into account the chamber’s volume and any sample dilutions required. One disadvantage is that to determine population size accurately, the microbial population must be relatively large because only a small volume of the population is sampled.  Larger microorganisms such as protists and yeasts can be directly counted with electronic counters such as the Coulter Counter, although the flow cytometer is increasingly used. In the Coulter Counter, the microbial suspension is forced through a small hole. Electrical current flows through the hole, and electrodes placed on both sides of the whole measure electrical resistance. Every time a microbial cell passes through the hole, electrical resistance increases (i.e., the conductivity drops), and the cell is counted. Flow cytometry gives accurate results with larger cells, and is extensively used in hospital laboratories to count red and white blood cells. It is not as useful in counting bacteria because of interference by small debris p articles, the formation of fi laments, and other problems. The number of bacteria in aquatic samples is frequently determined from direct counts after the bacteria have been trapped on membrane filters. In the membrane filter technique, the sample is first filtered through a black polycarbonate membrane filter. Then the bacteria are stained with a fluorescent dye such as acridine orange or the DNA stain DAPI and observed microscopically. The stained cells are easily observed against the black back- ground of the membrane filter and can be counted when viewed with an epifluorescence microscope. Traditional methods for directly counting microbes in a sample usually yield cell densities that are much higher than the plating methods described next because direct counting procedures do not distinguish dead cells from culturable cells. Newer methods for direct counts avoid this problem. Commercial kits that use fluorescent reagents to stain live and dead cells differently are now available, making it possible to count directly the number of live and dead microorganisms in a sample.

  Several plating methods can be used to determine the number of viable microbes in a sample. These are referred to as    viable counting methods    (plate counts) because they count only those cells that are able to reproduce when cultured. Two commonly used procedures are the spread-plate and the pour-plate techniques. In both of these methods, a diluted sample of microorganisms is dispersed over or within agar. If each cell is far enough away from other cells, then each cell will develop into a distinct colony. The original number of viable microorganisms in the sample can be calculated from the number of colonies formed and the sample dilution. For example, if 1.0 ml of a 1 × 10^6 dilution yielded 150 colonies, the original sample contained around 1.5 × 10^8 cells per ml. usually the count is made more accurate by use of a colony counter. In this way the spread- plate and pour-plate techniques may be used to find the number of microorganisms in a sample. Another commonly used plating method first traps bacteria in aquatic samples on a membrane filter. The filter is then placed on an agar medium or on a pad soaked with liquid media and incubated until each cell forms a separate colony. A colony count gives the number of microorganisms in the filtered sample, and selective media can be used to select for specific microorganisms. This technique is especially useful in analyzing water purity.  Plating techniques are simple, sensitive, and widely used for viable counts of bacteria and other microorganisms in samples of food, water, and soil. Several problems, however, can lead to inaccurate counts. Low counts will result if clumps of cells are not broken up and the microorganisms well dispersed. Because it is not possible to be certain that each colony arose from an individual cell, the results are often expressed in terms of colony forming units (CFU)    rather than the number of microorganisms. The samples should yield between 30 and 300 colonies for most accurate counting (counts of 25 to 250 are used in some applications). Of course the counts will also be low if the medium employed cannot support growth of all the viable microorganisms present. The hot agar used in the pour-plate technique may injure or kill sensitive cells; thus spread plates sometimes give higher counts than pour plates.   

 Measurement of Cell Mass
Techniques for measuring changes in cell mass also can be used to follow growth. The most direct approach is the determination of microbial dry weight. Cells growing in liquid medium are collected by centrifugation, washed, dried in an oven, and weighed. This is an especially useful technique for measuring the growth of filamentous fungi. It is time-consuming, however, and not very sensitive. Because bacteria weigh so little, it may be necessary to centrifuge several hundred milliliters of culture to collect a sufficient quantity.   A more rapid and sensitive method for measuring cell mass is spectrophotometry. Spectrophotometry depends on the fact that microbial cells scatter light that strikes them. Because microbial cells in a population are of roughly constant size, the amount of scattering is directly proportional to the biomass of cells present and indirectly related to cell number. When the concentration of bacteria reaches about a million (10^6) cells per ml, the medium appears slightly cloudy or turbid. Further increases in concentration result in greater turbidity, and less light is transmitted through the medium. The extent of light scattering (i.e., decrease in transmitted light) can be measured by a spectrophotometer and is called the absorbance (optical density) of the medium. Absorbance is almost linearly related to cell concentration at absorbance levels less than about 0.5. If the sample exceeds this value, it must first be diluted and then absorbance measured. Thus population growth can be easily measured as long as the population is high enough to give detectable turbidity. Cell mass can also be estimated by measuring the concentration of some cellular substance, as long as its concentration is constant in each cell. For example, a sample of cells can be analyzed for total protein or nitrogen. An increase in the microbial population will be reflected in higher total protein levels. Similarly, chlorophyll determinations can be used to measure phototrophic protist and cyanobacteria populations, and the quantity of ATP can be used to estimate the amount of living microbial mass.    

Thus far, our focus has been on closed systems called batch cultures in which nutrients are not renewed nor wastes removed. Exponential (logarithmic) growth lasts for only a few generations and soon stationary phase is reached. However, it is possible to grow microorganisms in a system with constant environmental conditions maintained through continual provision of nutrients and removal of wastes. Such a system is called a    continuous culture system.  These systems can maintain a microbial population in exponential growth, growing at a known rate and at a constant biomass concentration for extended periods. Continuous culture systems make possible the study of microbial growth at very low nutrient levels, concentrations close to those present in natural environments. These systems are essential for research in many areas, including ecology. For example, interactions between microbial species in environ- mental conditions resembling those in a freshwater lake or pond can be modeled. Continuous culture systems also are used in food and industrial microbiology. Two major types of continuous culture systems commonly are used: chemostats and turbidostats. 


 A chemostat is constructed so that the rate at which sterile medium is fed into the culture vessel is the same as the rate at which the media containing microorganisms is removed. The culture medium for a chemostat possesses an essential nutrient (e.g., a vitamin) in limiting quantities. Because one nutrient is limiting, growth rate is determined by the rate at which new medium is fed into the growth chamber; the final cell density depends on the concentration of the limiting nutrient. The rate of nutrient exchange is expressed as the dilution rate (D), the rate at which medium flows through the culture vessel relative to the vessel volume, where f is the flow rate (ml/hr.) and V is the vessel volume (ml).         D = f / V 
 For example, if f is 30 ml/hr. and V is 100 ml, the dilution rate is 0.30 hr 1.  Both population size and generation time are related to the dilution rate, and population density remains unchanged over a wide range of dilution rates. The generation time decreases (i.e., the rate of growth increases) as the dilution rate increases. The limiting nutrient will be almost completely depleted under these balanced conditions. If the dilution rate rises too high, microorganisms can actually be washed out of the culture vessel before reproducing because the dilution rate is greater than the maximum growth rate. This occurs because fewer microorganisms are present to consume the limiting nutrient. At very low dilution rates, an increase in D causes a rise in both cell density and the growth rate. This is because of the effect of nutrient concentration on the growth rate, sometimes called the Monod relationship. When dilution rates are low, only a limited supply of nutrient is available and the microbes can conserve only a limited amount of energy. Much of that energy must be used for cell maintenance, not for growth and reproduction. As the dilution rate increases, the amount of nutrients and the resulting cell density rise because energy is available for both maintenance and reproduction. The growth rate increases when the total available energy exceeds the maintenance energy.  


The second type of continuous culture system, the turbidostat, has a photocell that measures the turbidity (absorbance) of the culture in the growth vessel. The flow rate of media through the vessel is automatically regulated to maintain a predetermined turbidity. Because turbidity is related to cell density, the turbidostat maintains a desired cell density. The turbidostat differs from the chemostat in several ways. The dilution rate in a turbidostat varies rather than remaining constant, and a turbidostat’s culture medium contains all nutrients in excess. That is, none of the nutrients is limiting. The turbidostat operates best at high dilution rates; the chemostat is most stable and effective at lower dilution rates.

As we have seen, microorganisms must be able to respond to variations in nutrient levels. Microorganisms also are greatly affected by the chemical and physical nature of their surroundings. An understanding of environmental influences aids in the control of microbial growth and the study of the ecological distribution of microorganisms. The adaptations of some microorganisms to extreme and inhospitable environments are truly remarkable. Microbes are present virtually everywhere on Earth. Many habitats in which microbes thrive would kill most other organisms. Bacteria such as Bacillus infernus  are able to live over 2.4 kilometers below Earth’s surface, without oxygen and at temperatures above 60°C. Other microbes live in acidic hot springs, at great ocean depths, or in lakes such as the Great Salt Lake in Utah (USA) that have high sodium chloride concentrations. Microorganisms that grow in such harsh conditions are called    extremophiles. We briefly review the effects of the most important environmental factors on microbial growth. Major emphasis is given to solutes and water activity, pH, temperature, oxygen level, pressure, and radiation. Microorganisms are categorized in terms of their response to these factors. It is important to note that for most environmental factors, a range of levels supports growth of a microbe. For example, a microbe might exhibit optimum growth at pH 7 but grows, though not optimally, at pH values down to pH 6 (its pH minimum) and up to pH 8 (its pH maximum). Furthermore, outside this range, the microbe might cease reproducing but remain viable for some time. Clearly, each microbe must have evolved adaptations that allow it to adjust its physiology within its preferred range, and it may also have adaptations that protect it in environments outside this range. These adaptations also are discussed in this section.            
 Solutes and Water Activity
Because a selectively permeable plasma membrane separates microorganisms from their environment, they can be affected by changes in the osmotic concentration of their surroundings. If a microorganism is placed in a hypotonic solution (one with a lower osmotic concentration), water will enter the cell and cause it to burst unless something is done to prevent the influx or inhibit plasma membrane expansion. Conversely if it is placed in a hypertonic solution (one with a higher osmotic concentration), water will flow out of the cell. In microbes that have cell walls, the membrane shrinks away from the cell wall a process called plasmolysis. Dehydration of the cell in hypertonic environments may damage the cell membrane and cause the cell to become metabolically inactive.   Clearly it is important that microbes be able to respond to changes in the osmotic concentrations of their environment. Microbes in hypotonic environments can reduce the osmotic con- centration of their cytoplasm. This can be achieved using inclusion bodies or other mechanisms. For example, some prokaryotes have mechanosensitive (MS) channels in their plasma membrane. In a hypotonic environment, the membrane stretches due to an increase in hydrostatic pressure and cellular swelling. MS channels then open and allow solutes to leave. Thus, MS channels act as escape valves to protect cells from bursting. Because many protists do not have a cell wall, they must use contractile vacuoles to expel excess water. Many microorganisms, whether in hypotonic or hypertonic environments, keep the osmotic concentration of their cytoplasm somewhat above that of the habitat by the use of compatible solutes, so that the plasma membrane is always pressed firmly against their cell wall. Compatible solutes are solutes that do not interfere with metabolism and growth when at high intracellular concentrations. Most procaryotes increase their internal osmotic concentration in a hypertonic environment through the synthesis or uptake of choline, betaine, proline, glutamic acid, and other amino acids; elevated levels of potassium ions may also be used. Photosynthetic protists and fungi employ sucrose and polyols for example, arabitol, glycerol, and mannitol for the same purpose. Polyols and amino acids are ideal compatible solutes because they normally do not disrupt enzyme structure and function.

Some microbes are adapted to extreme hypertonic environments. Halophiles    grow optimally in the presence of NaCl or other salts at a concentration above about 0.2 M. Extreme halophiles have adapted so completely to hypertonic, saline conditions that they require high levels of sodium chloride to grow concentrations between about 2 M and saturation (about 6.2 M). The archaeon Halobacterium can be isolated from the Dead Sea (a salt lake between Israel and Jordan), the Great Salt Lake in Utah, and other aquatic habitats with salt concentrations approaching saturation. Halobacterium and other extremely halophilic procaryotes accumulate enormous quantities of potassium in order to remain hypertonic to their environment; the internal potassium concentration may reach 4 to 7 M. Furthermore, their enzymes, ribosomes, and transport proteins require high potassium levels for stability and activity. In addition, the plasma membrane and cell wall of Halobacterium is stabilized by high concentrations of sodium ion. If the sodium concentration decreases too much, the wall and plasma membrane disintegrate. Extreme halophiles have successfully adapted to environmental conditions that would destroy most organisms. In the process, they have become so specialized that they have lost ecological flexibility and can prosper only in a few extreme habitats. Because the osmotic concentration of a habitat has such pro- found effects on microorganisms, it is useful to express quantitatively the degree of water availability. Microbiologists generally use    water activity (a w)     for this purpose (water avail-ability also may be expressed as water potential, which is related to a w). The water activity of a solution is 1/100 the relative humidity of the solution (when expressed as a percent). It is also equivalent to the ratio of the solution’s vapor pressure (P soln) to that of pure water (P water).

  The water activity of a solution or solid can be determined by sealing it in a chamber and measuring the relative humidity after the system has come to equilibrium. Suppose after a sample is treated in this way, the air above it is 95% saturated—that is, the air contains 95% of the moisture it would have when equilibrated at the same temperature with a sample of pure water. The relative humidity would be 95% and the sample’s water activity, 0.95. Water activity is inversely related to osmotic pressure; if a solution has high osmotic pressure, it’s a w is low.   Microorganisms differ greatly in their ability to adapt to habitats with low water activity. A microorganism must expend extra effort to grow in a habitat with a low a w value because it must maintain a high internal solute concentration to retain water. Some microorganisms can do this and are osmo tolerant; they grow over wide ranges of water activity. For example, Staphylococcus aureus is halo tolerant, can be cultured in media containing sodium chloride concentration up to about 3 M, and is well adapted for growth on the skin. The yeast Saccharomyces rouxii grows in sugar solutions with a w values as low as 0.6. The photo- synthetic protist Dunaliella viridis tolerates sodium chloride concentrations from 1.7 M to a saturated solution. Some microbes (e.g., Halobacterium) are true xerophiles. That is, they grow best at low a w. However, most microorganisms only grow well at water activities around 0.98 (the approximate a w for seawater) or higher. This is why drying food or adding large quantities of salt and sugar effectively prevents food spoilage.

pH is a measure of the relative acidity of a solution and is defined as the negative logarithm of the hydrogen ion concentration (expressed in terms of molarity).
 pH = log [H +] = log (1/[H+ ])
 The pH scale extends from pH 0.0 (1.0 M H +) to pH 14.0 (1.0 × 10 14 M H +), and each pH unit represents a tenfold change in hydrogen ion concentration. Microbial habitats vary widely in pH from pH 0 to 2 at the acidic end to alkaline lakes and soil with pH values between 9 and 10. Each species has a definite pH growth range and pH growth optimum.  Acidophiles    have their growth optimum between pH 0 and 5.5; neutrophiles, between pH 5.5 and 8.0; and alkalophiles (alkaliphiles), between pH 8.0 and 11.5. Extreme alkalophiles have growth optima at pH 10 or higher. In general, different microbial groups have characteristic pH preferences. Most bacteria and protists are neutrophiles. Most fungi prefer more acidic surroundings, about pH 4 to 6; photosynthetic protists also seem to favor slight acidity. Many archaea are acidophiles. For example, the archaeon Sulfolobus acidocaldarius is a common inhabitant of acidic hot springs; it grows well from pH 1 to 3 and at high temperatures. The archaea Ferroplasma acidarmanus and Picrophilus oshimae can actually grow very close to pH 0. Alkalophiles are distributed among all three domains of life. They include bacteria belonging to the genera Bacillus, Micrococcus, Pseudomonas, and Streptomyces; yeasts and filamentous fungi; and numerous archaea.  Although microorganisms often grow over wide ranges of pH and far from their optima, there are limits to their tolerance. When the external pH is low, the concentration of H + is greater outside than inside, and H + will move into the cytoplasm and lower the cytoplasmic pH. Drastic variations in cytoplasmic pH can harm microorganisms by disrupting the plasma membrane or inhibiting the activity of enzymes and membrane transport proteins. Most procaryotes die if the internal pH drops much below 5.0 to 5.5. Changes in the external pH also might alter the ionization of nutrient molecules and thus reduce their availability to the organism.  Microorganisms respond to external pH changes using mechanisms that maintain a neutral cytoplasmic pH. Several mechanisms for adjusting to small changes in external pH have been proposed. Neutrophiles appear to exchange potassium for protons using an antiport transport system. Internal buffering also may contribute to pH homeostasis. However, if the external pH becomes too acidic, other mechanisms come into play. When the pH drops below about 5.5 to 6.0, Salmonella enterica serovar Typhimurium and E. coli synthesize an array of new proteins as part of what has been called their acidic tolerance response. A proton-translocating ATPase enzyme contributes to this protective response, either by making more ATP or by pumping protons out of the cell. If the external pH decreases to 4.5 or lower, acid shock proteins and heat shock proteins are synthesized. These prevent the denaturation of proteins and aid in the refolding of denatured proteins in acidic conditions. What about microbes that live at pH extremes? Extreme alkalophiles such as Bacillus alcalophilus maintain their internal pH close to neutrality by exchanging internal sodium ions for external protons. Acidophiles use a variety of measures to maintain a neutral internal pH. These include the transport of cations (e.g., potassium ions) into the cell, thus decreasing the movement of H + into the cell; proton transporters that pump H + out if they get in; and highly impermeable cell membranes.    

 Microorganisms are particularly susceptible to external temperatures because they cannot regulate their internal temperature. An important factor influencing the effect of temperature on growth is the temperature sensitivity of enzyme-catalyzed reactions. Each enzyme has a temperature at which it functions optimally. At some temperature below the optimum, it ceases to be catalytic. As the temperature rises from this low point, the rate of catalysis increases to that observed for the optimal temperature. The velocity of the reaction roughly doubles for every 10°C rise in temperature. When all enzymes in a microbe are considered together, as the rate of each reaction increases, metabolism as a whole becomes more active, and the microorganism grows faster. However, beyond a certain point, further increases actually slow growth, and sufficiently high temperatures are lethal. High temperatures denature enzymes, transport carriers, and other proteins. Temperature also has a significant effect on microbial membranes. At very low temperatures, membranes solidify. At high temperatures, the lipid bilayer simply melts and disintegrates. Thus when organisms are above their optimum temperature, both function and cell structures are affected. If temperatures are very low, function is affected but not necessarily cell chemical composition and structure.  Because of these opposing temperature influences, microbial growth has characteristic temperature dependence with distinct cardinal temperatures minimum, optimum, and maximum growth temperatures. Although the shape of temperature dependence curves varies, the temperature optimum is always closer to the maximum than to the minimum. The cardinal temperatures are not rigidly fixed. Instead they depend to some extent on other environmental factors such as pH and avail- able nutrients. For example, Crithidia fasciculate, a flagellated protist living in the gut of mosquitoes, grows in a simple medium at 22 to 27°C. However, it cannot be cultured at 33 to 34°C without the addition of extra metals, amino acids, vitamins, and lipids. The cardinal temperatures vary greatly among microorganisms. Optima usually range from 0°C to 75°C, whereas microbial growth occurs at temperatures extending from less than 20°C to over 120°C. Some archaea even grow at 121°C (250°F), the temperature normally used in autoclaves. A major factor determining growth range seems to be water. Even at the most extreme temperatures, microorganisms need liquid water to grow. The growth temperature range for a particular microorganism usually spans about 30 degrees. Some species (e.g., Neisseria gonorrhoeae) have a small range; others, such as Enterococcus faecalis, grow over a wide range of temperatures. The major microbial groups differ from one another regarding their maximum growth temperatures. The upper limit for protists is around 50°C. Some fungi grow at temperatures as high as 55 to 60°C. Procaryotes can grow at much higher temperatures than eucaryotes. It has been suggested that eucaryotes are not able to manufacture stable and functional organelles membranes at temperatures above 60°C. The photosynthetic apparatus also appears to be relatively unstable because photosynthetic organisms are not found growing at very high temperatures.   Microorganisms such as those listed in t able 7.4 can be placed in one of five classes based on their temperature ranges for growth.   

   1.     Psychrophiles    grow well at 0°C and have an optimum growth temperature of 10°C or lower; the maximum is around 15°C. They are readily isolated from Arctic and Antarctic habitats. Oceans constitute an enormous habitat for psychrophiles because 90% of ocean water is 5°C or colder. The psychrophilic protist Chlamydomonas nivalis can actually turn a snow field or glacier pink with its bright red spores. Psychrophiles are widespread among bacterial taxa and are found in such genera as Pseudomonas, Vibrio, Alcaligenes, Bacillus, Photobacterium, and Shewanella.  A psychrophilic archaeon, Methanogenium, has been isolated from Ace Lake in Antarctica. Psychrophilic microorganisms have adapted to their environment in several ways. Their enzymes, transport systems, and protein synthetic machinery function well at low temperatures. The cell membranes of psychrophilic microorganisms have high levels of unsaturated fatty acids and remain semifluid when cold. Indeed, many psychrophiles begin to leak cellular constituents at temperatures higher than 20°C because of cell membrane disruption.    

  2.     Psychrotrophs (facultative psychrophiles)    grow at 0 to 7°C even though they have optima between 20 and 30°C, and maxima at about 35°C. Psychrotrophic bacteria and fungi are major causes of refrigerated food spoilage, as described in chapter 34.  
  3.     Mesophiles are microorganisms with growth optima around 20 to 45°C. They often have a temperature minimum of 15 to 20°C, and their maximum is about 45°C or lower. Most microorganisms probably fall within this category. Almost all human pathogens are mesophiles, as might be expected because the human body is a fairly constant 37°C.    

  4.     Thermophiles    grow at temperatures between 55 and 85°C. Their growth minimum is usually around 45°C, and they often have optima between 55 and 65°C. The vast majority are procaryotes, although a few photosynthetic protists and fungi are thermophilic. These organisms flourish in many habitats including composts, self-heating hay stacks, hot water lines, and hot springs.   

   5.     Hyperthermophiles have growth optima between 85°C and about 113°C. They usually do not grow well below 55°C.  Pyrococcus abyssi and Pyrodictium occultum are examples of marine hyperthermophiles found in hot areas of the seafloor. 
 Thermophiles and hyperthermophiles differ from mesophiles in many ways. They have heat-stable enzymes and protein synthesis systems that function properly at high temperatures. These proteins are stable for a variety of reasons. Heat-stable proteins have highly organized hydrophobic interiors and more hydrogen and other non-covalent bonds. Larger quantities of amino acids such as proline also make polypeptide chains less flexible and more heat stable. In addition, the proteins are stabilized and aided in folding by proteins called chaperone proteins. Evidence exists that histone like proteins stabilize the DNA of thermophilic bacteria. The membrane lipids of thermophiles and hyperthermophiles are also quite temperature stable. They tend to be more saturated, more branched, and of higher molecular weight. This increases the melting points of membrane lipids. Archaeal thermophiles have membrane lipids with ether linkages, which protect the lipids from hydrolysis at high temperatures. Sometimes archaeal lipids actually span the membrane to form a rigid, stable monolayer. 

Oxygen Concentration
 The importance of oxygen to the growth of an organism correlates with its metabolism in particular, with the processes it uses to conserve the energy supplied by its energy source. Almost all energy-conserving metabolic processes involve the movement of electrons through a series of membrane-bound electron carriers called the electron transport chain (ETC). For chemotrophs, an externally supplied terminal electron acceptor is critical to the functioning of the ETC. The nature of the terminal electron acceptor is related to an organism’s oxygen requirement.
 An organism able to grow in the presence of atmospheric O2 is an aerobe, whereas one that can grow in its absence is an anaerobe.  Almost all multicellular organisms are completely dependent on atmospheric O2 for growth that is, they are obligate aerobes. Oxygen serves as the terminal electron acceptor for the ETC in the metabolic process called aerobic respiration. In addition, aerobic eucaryotes employ O2 in the synthesis of sterols and unsaturated fatty acids.    Microaerophiles such as Campylobacter are damaged by the normal atmospheric level of O2 (20%) and require O2 levels in the range of 2 to 10% for growth.    Facultative anaerobes do not require O2 for growth but grow better in its presence. In the presence of oxygen, they use O2 as the terminal electron acceptor during aerobic respiration. Aerotolerant anaerobes such as Enterococcus faecalis simply ignore O2 and grow equally well whether it is present or not; chemotrophic aerotolerant anaerobes are often described as having strictly fermentative metabolism. In contrast, strict or obligate anaerobes (e.g., Bacteroides, Clostridium pasteurianum, Methanococcus) are usually killed in the presence of O2. Strict anaerobes cannot generate energy through aerobic respiration and employ other metabolic strategies such as fermentation or anaerobic respiration, neither of which requires O2. The nature of bacterial O2  responses can be readily determined by growing the bacteria in culture tubes filled with a solid culture medium or a medium such as thioglycollate broth, which contains a reducing agent to lower O2levels.  A microbial group may show more than one type of relation- ship to O2. All five types are found among the procaryotes and protists. Fungi are normally aerobic, but a number of species particularly among the yeasts are facultative anaerobes. Photosynthetic protists are usually obligate aerobes. Although obligate anaerobes are killed by O2, they may be recovered from habitats that appear to be toxic. In such cases they associate with facultative anaerobes that use up the available O2and thus make the growth of strict anaerobes possible. For example, the strict anaerobe Bacteroides gingivalis lives in the mouth where it grows in the anoxic crevices around the teeth. Clearly the ability to grow in both toxic and anoxic environments provides considerable flexibility and is an ecological advantage.   The different relationships with O2 are due to several factors, including the inactivation of proteins and the effect of toxic O2 derivatives. Enzymes can be inactivated when sensitive groups such as sulfhydryls are oxidized. A notable example is the nitrogen fixation enzyme nitrogenase, which is very oxygen sensitive. Toxic O2 derivatives are formed when proteins such as flavoproteins promote oxygen reduction. The reduction products are   superoxide radical, hydrogen peroxide,   and hydroxyl radical. 
 O2 + e −→ O2 (superoxide radical)
 O2 + e + 2H + −→ H2 O2 (hydrogen peroxide)
 H2O2+ e + H + −→H 2 O + OH (hydroxyl radical)
 These products are extremely toxic because they oxidize and rapidly destroy cellular constituents. A microorganism must be able to protect itself against such oxygen products or it will be killed. Indeed, neutrophils and macrophages, two important immune system cells, use these toxic oxygen products to destroy invading pathogens.  Many microorganisms possess enzymes that protect against toxic O2 products. Obligate aerobes and facultative anaerobes usually contain the enzymes superoxide dismutase (SOD and catalase, which catalyze the destruction of superoxide radical and hydrogen peroxide, respectively. Peroxidase also can be used to destroy hydrogen peroxide.
Aerotolerant microorganisms may lack catalase but usually have superoxide dismutase. The aerotolerant bacterium Lactobacillus plantarum uses manganous ions instead of superoxide dismutase to destroy the superoxide radical. All strict anaerobes lack both enzymes or have them in very low concentrations and therefore cannot tolerate O2. However, some microaerophilic bacteria and anaerobic archaea protect themselves from the toxic effects of O2 with the enzymes superoxide reductase and peroxidase. Superoxide reductase reduces superoxide to H2O2 without producing O2. The H2O2 is then converted to water by peroxidase.
Because aerobes need O2and anaerobes are killed by it, radically different approaches must be used when they are cultured. When large volumes of aerobic microorganisms are cultured, either they must be shaken to aerate the culture medium or sterile air must be pumped through the culture vessel. Precisely the opposite problem arises with anaerobes all O2 must be excluded. This is accomplished in several ways.
(1) Special anaerobic media containing reducing agents such as thioglycollate or cysteine may be used. The medium is boiled during preparation to dissolve its components and drive off oxygen. The reducing agents eliminate any residual dissolved O2 in the medium so that anaerobes can grow beneath its surface.
(2) Oxygen also may be eliminated by removing air with a vacuum pump and flushing out residual O2with nitrogen gas. Often CO2 as well as nitrogen is added to the chamber since many anaerobes require a small amount of CO2 for best growth.
 (3) One of the most popular ways of culturing small numbers of anaerobes is by use of a GasPak jar, which uses hydrogen and a palladium catalyst to remove O2. (4) A similar approach uses plastic bags or pouches containing calcium carbonate and a catalyst, which produce an anoxic, carbon dioxide rich atmosphere.   
Organisms that spend their lives on land or the surface of water are always subjected to a pressure of 1 atmosphere (atm) and are never affected significantly by pressure. It is thought that high hydrostatic pressure affects membrane fluidity and membrane- associated function. Yet many procaryotes live in the deep sea (ocean depths of 1,000 m or more) where the hydrostatic pressure can reach 600 to 1,100 atm and the temperature is about 2 to 3°C. Many of these procaryotes are barotolerant   : increased pressure adversely affects them but not as much as it does nontolerant microbes. Some procaryotes are truly    barophilic they grow more rapidly at high pressures. A barophile recovered from the Mariana trench near the Philippines (depth about 10,500 m) grows only at pressures between about 400 to 500 atm when incubated at 2°C. Barophiles may play an important role in nutrient recycling in the deep sea. Thus far, they have been found among several bacterial genera (e.g., Photobacterium, Shewanella, and Colwellia). Some archaea are thermobarophiles (e.g., Pyrococcus spp., Methanocaldococcus jannaschii).  
 Our world is bombarded with electromagnetic radiation of various types. Radiation behaves as if it were composed of waves moving through space like waves traveling on the surface of water. The distance between two wave crests or troughs is the wavelength. As the wavelength of electromagnetic radiation decreases, the energy of the radiation increases; gamma rays and X rays are much more energetic than visible light or infrared waves. Electromagnetic radiation also acts like a stream of energy packets called photons, each photon having a quantum of energy whose value depends on the wavelength of the radiation. Sunlight is the major source of radiation on Earth. It includes visible light, ultraviolet (UV) radiation, infrared rays, and radio waves. Visible light is a most conspicuous and important aspect of our environment: most life depends on the ability of photosynthetic organisms to trap the light energy of the sun. Almost 60% of the sun’s radiation is in the infrared region rather than the visible portion of the spectrum. Infrared is the major source of Earth’s heat. At sea level, one finds very little ultraviolet radiation below about 290 to 300 nm. UV radiation of wavelengths shorter than 287 nm is absorbed by O2 in Earth’s atmosphere; this process forms a layer of ozone between 40 and 48 kilometers above Earth’s surface. The ozone layer absorbs somewhat longer UV rays and reforms O2. The even distribution of sunlight throughout the visible spectrum accounts for the fact that sunlight is generally “white.  Many forms of electromagnetic radiation are very harmful to microorganisms. This is particularly true of ionizing radiation, radiation of very short wavelength and high energy, which can cause atoms to lose electrons (ionize). Two major forms of ionizing radiation are
 (1) X rays, which are artificially produced, and
 (2) Gamma rays, which are emitted during radioisotope decay. Low levels of ionizing radiation may pro- duce mutations and may indirectly result in death, whereas higher levels are directly lethal. Although microorganisms are more resistant to ionizing radiation than larger organisms, they are still destroyed by a sufficiently large dose. Ionizing radiation can be used to sterilize items. Some procaryotes (e.g., Deinococcus radiodurans) and bacterial endospores can survive large doses of ionizing radiation.   Ionizing radiation causes a variety of changes in cells. It breaks hydrogen bonds, oxidizes double bonds, destroys ring structures, and polymerizes some molecules. Oxygen enhances these destructive effects, probably through the generation of hydroxyl radicals (OH•). Although many types of constituents can be affected, the destruction of DNA is probably the most important cause of death.      Ultraviolet (UV) radiation    can kill microorganisms due to its short wavelength (approximately from 10 to 400 nm) and high energy. The most lethal UV radiation has a wavelength of 260 nm, the wavelength most effectively absorbed by DNA. The primary mechanism of UV damage is the formation of thymine dimers in DNA. Two adjacent thymines in a DNA strand are covalently joined to inhibit DNA replication and function. The damage caused by UV light can be repaired by several DNA repair mechanisms, as discussed in chapter 14. Excessive expo- sure to UV light outstrips the organism’s ability to repair the damage and death results. Longer wavelengths of UV light (near- UV radiation; 325 to 400 nm) can also harm microorganisms because they induce the breakdown of tryptophan to toxic photo- products. It appears that these toxic tryptophan photoproducts plus the near-UV radiation itself produce breaks in DNA strands. The precise mechanism is not known, although it is different from that seen with 260 nm UV. Even visible light, when present in sufficient intensity, can damage or kill microbial cells. Usually pigments called photo- sensitizers and O2 are involved. Photosensitizers include pigments such as chlorophyll, bacteriochlorophyll, cytochromes, and flavins, which can absorb light energy and become excited or activated. The excited photosensitizer (P) transfers its energy to O2, generating   singlet oxygen    (1 O2).
P  P (activated) light
 P (activated) + O2  P   1 O2
Singlet oxygen is a very reactive, powerful oxidizing agent that quickly destroys a cell.  M any microorganisms that are airborne or live on exposed surfaces use carotenoid pigments for protection against photo- oxidation. Carotenoids effectively quench singlet oxygen that is, they absorb energy from singlet oxygen and convert it back into the unexcited ground state. Both photosynthetic and non-photosynthetic microorganisms employ pigments in this way.
Terminology is especially important when the control of micro- organisms is discussed because words such as disinfectant and antiseptic often are used loosely. The situation is even more con- fusing because a particular treatment can either inhibit growth or kill, depending on the conditions. Sterilization (Latin sterilize, unable to produce offspring or barren) is the process by which all living cells, spores, and acellular entities (e.g., viruses, viroids, and prions) are either destroyed or removed from an object or habitat. A sterile object is totally free of viable microorganisms, spores, and other infectious agents. When sterilization is achieved by a chemical agent, the chemical is called a sterilant. In contrast,    disinfection is the killing, inhibition, or removal of microorganisms that may cause disease; disinfection is the substantial reduction of the total microbial population and the destruction of potential pathogens. Disinfectants are agents, usually chemical, used to carry out dis- infection and normally used only on inanimate objects. A disinfectant does not necessarily sterilize an object because viable spores and a few microorganisms may remain. Sanitization is closely related to disinfection. In sanitization, the microbial population is reduced to levels that are considered safe by public health standards. The inanimate object is usually cleaned as well as partially disinfected. For example, sanitizers are used to clean eating utensils in restaurants. It also is frequently necessary to control microorganisms on or in living tissue with chemical agents.  Antisepsis (Greek anti, against, and sepsis, putrefaction) is the prevention of infection or sepsis and is accomplished with    antiseptics    .  These are chemical agents applied to tissue to prevent infection by killing or inhibiting pathogen growth; they also reduce the total microbial population. Because they must not destroy too much host tissue, antiseptics are generally not as toxic as disinfectants.    Chemotherapy    is the use of chemical agents to kill or inhibit the growth of microorganisms within host tissue. A suffix can be employed to denote the type of antimicrobial agent. Substances that kill organisms often have the suffix –cide (Latin cida, to kill); a    germicide kills pathogens (and many non-pathogens) but not necessarily endospores. A disinfectant or antiseptic can be particularly effective against a specific group, in which case it may be called a bactericide, fungicide or viricide. Other chemicals do not kill but rather prevent growth. If these agents are removed, growth will resume. Their names end in –static (Greek statikos, causing to stand or stopping) for example, bacteriostatic   and   fungistatic.
A microbial population is not killed instantly when exposed to a lethal agent. Population death is generally exponential (logarithmic) that is, the population will be reduced by the same fraction at constant intervals. If the logarithm of the population number remaining is plotted against the time of exposure of the microorganism to the agent, a straight-line plot will result. When the population has been greatly reduced, the rate of killing may slow due to the survival of a more resistant strain of the microorganism. It is essential to have a precise measure of an agent’s killing efficiency. One such measure is the decimal reduction time (D) or D value. The decimal reduction time is the time required to kill 90% of the microorganisms or spores in a sample under specified conditions. For example, in a semilogarithmic plot of the population remaining versus the time of heating, the D value is the time required for the line to drop by one log cycle or tenfold. The D value is usually written with a subscript to indicate the temperature for which it applies. To study the effectiveness of a lethal agent, one must be able to decide when microorganisms are dead, which may pre- sent some challenges. A microbial cell is often defined as dead if it does not grow and reproduce when inoculated into culture medium that would normally support its growth. In like manner, an inactive virus cannot infect a suitable host. This definition has flaws, however. It has been demonstrated that when bacteria are exposed to certain conditions, they can remain alive but are temporarily unable to reproduce. When in this state, they are referred to as viable but non culturable (VBNC). In conventional tests to demonstrate killing by an antimicrobial agent, VBNC bacteria would be thought to be dead. This is a serious problem because after a period of recovery, the bacteria may regain their ability to reproduce and cause infection.
Destruction of microorganisms and inhibition of microbial growth are not simple matters because the efficiency of an antimicrobial agent (an agent that kills microorganisms or inhibits their growth) is affected by at least six factors. 
 1.    Population size.  Because an equal fraction of a microbial population is killed during each interval, a larger population requires a longer time to die than a smaller one.
 2.    Population composition.  The effectiveness of an agent varies greatly with the nature of the organisms being treated because microorganisms differ markedly in susceptibility. Bacterial spores are much more resistant to most antimicrobial agents than are vegetative forms, and younger cells are usually more readily destroyed than mature organisms. Some species are able to withstand adverse conditions better than others. For instance, Mycobacterium tuberculosis, which causes tuberculosis, is much more resistant to antimicrobial agents than most other bacteria.
3.   Concentration or intensity of an antimicrobial agent.  Often, but not always, the more concentrated a chemical agent or intense a physical agent, the more rapidly microorganisms are destroyed. However, agent effectiveness usually is not directly related to concentration or intensity. Over a short range, a small increase in concentration leads to an exponential rise in effectiveness; beyond a certain point, increases may not raise the killing rate much at all. Sometimes an agent is more effective at lower concentrations. For example, 70% ethanol is more bacteriocidal than 95% ethanol because the activity of ethanol is enhanced by the presence of water.
 4.   Duration of exposure.  The longer a population is exposed to a microbicidal agent, the more organisms are killed. To achieve sterilization, exposure should be long enough to reduce the probability of survival to 10^-6 or less.
5.   Temperature.  An increase in the temperature at which a chemical acts often enhances its activity. Frequently a lower concentration of disinfectant or sterilizing agent can be used at a higher temperature.
6.   Local environment.  The population to be controlled is not isolated but surrounded by environmental factors that may either offer protection or aid in its destruction. For example, because heat kills more readily at an acidic pH, acidic foods and beverages such as fruits and tomatoes are easier to pasteurize than more alkaline foods such as milk. A second important environmental factor is organic matter, which can protect microorganisms against physical and chemical disinfecting agents. Biofilms are a good example. The organic matter in a biofilm protects the biofilms microorganisms. Furthermore, it has been clearly documented that bacteria in biofilms are altered physiologically, and this makes them less susceptible to many antimicrobial agents. Because of the impact of organic matter, it may be necessary to clean objects, especially medical and dental equipment, before they are disinfected or sterilized. 
Heat and other physical agents are normally used to control microbial growth and sterilize objects, as can be seen from the continual operation of the autoclave in every microbiology laboratory. The most frequently employed physical agents are heat, filtration, and radiation. 

Moist heat readily destroys viruses, bacteria, and fungi. Moist heat kills by degrading nucleic acids and denaturing enzymes and other essential proteins. It also disrupts cell membranes. Exposure to boiling water for 10 minutes is sufficient to destroy vegetative cells and eukaryotic spores. Unfortunately the temperature of boiling water (100°C or 212°F at sea level) is not sufficient to destroy bacterial spores, which may survive hours of boiling. Therefore boiling can be used for disinfection of drinking water and objects not harmed by water, but boiling does not sterilize to destroy bacterial spores, moist heat sterilization must be carried out at temperatures above 100°C, and this requires the use of saturated steam under pressure. Steam sterilization is carried out with an    autoclave, a device somewhat like a fancy pressure cooker. The development of the autoclave by Chamberland in 1884 tremendously stimulated the growth of microbiology. Water is boiled to produce steam, which is released into the autoclaves chambered. The air initially present in the chamber is forced out until the chamber is filled with saturated steam and the outlets are closed. Hot, saturated steam continues to enter until the chamber reaches the desired temperature and pressure, usually 121°C and 15 pounds of pressure. At this temperature saturated steam destroys all vegetative cells and spores in a small volume of liquid within 10 to 12 minutes. Treatment is continued for at least 15 minutes to provide a margin of safety. Of course larger containers of liquid such as flasks and carboys require much longer treatment times. Autoclaving must be carried out properly or the processed materials will not be sterile. If all air has not been flushed out of the chamber, it will not reach 121°C, even though it may reach a pressure of 15 pounds. The chamber should not be packed too tightly because the steam needs to circulate freely and contact everything in the autoclave. Bacterial spores will be killed only if they are kept at 121°C for 10 to 12 minutes. When a large volume of liquid must be sterilized, an extended sterilization time is needed because it takes longer for the center of the liquid to reach 121°C; 5 liters of liquid may require about 70 minutes. In view of these potential difficulties, a biological indicator is often autoclaved along with other material. This indicator commonly consists of a culture tube containing a sterile ampule of medium and a paper strip covered with spores of Geobacillus stearothermophilus.  After autoclaving, the ampule is aseptically broken and the culture incubated for several days. If the test bacterium does not grow in the medium, the sterilization run has been successful. Sometimes either special indicator tape or paper that changes color upon sufficient heating is autoclaved with a load of material. These approaches are convenient and save time but are not as reliable as the killing of bacterial spores. Any heat-sensitive substances, such as milk, are treated with controlled heating at temperatures well below boiling, a process known as    pasteurization    in honor of its developer, Louis Pasteur. In the 1860s the French wine industry was plagued by the problem of wine spoilage, which made wine storage and shipping difficult. Pasteur examined spoiled wine under the microscope and detected microorganisms that looked like the bacteria responsible for lactic acid and acetic acid fermentations. He then discovered that a brief heating at 55 to 60°C would destroy these microorganisms and preserve wine for long periods. In 1886 the German chemists V. H. and F. Soxhlet adapted the technique for preserving milk and reducing milk- transmissible diseases. Milk pasteurization was introduced into the United States in 1889. Milk, beer, and many other beverages are now pasteurized. Pasteurization does not sterilize a beverage, but it does kill any pathogens present and drastically slows spoilage by reducing the level of nonpathogenic spoilage microorganisms.   Some materials cannot withstand the high temperature of the autoclave, and spore contamination precludes the use of other methods to sterilize them. For these materials, a process of intermittent sterilization, also known as tyndallization (for John Tyndall, the British physicist who used the technique to destroy heat-resistant microorganisms in dust) is used. The process also uses steam (30–60 minutes) to destroy vegetative bacteria. However, steam exposure is repeated for a total of three times with 23 to 24 hour incubations between steam exposures. The incubations permit remaining spores to germinate into heat-sensitive vegetative cells that are then destroyed upon subsequent steam exposures.  Many objects are best sterilized in the absence of water by dry heat sterilization. Some items are sterilized by incineration. For instance, inoculating loops, which are used routinely in the laboratory, can be sterilized in a small, bench-top incinerator. Other items are sterilized in an oven at 160 to 170°C for 2 to 3 hours. Microbial death results from the oxidation of cell constituents and denaturation of proteins. Dry air heat is less effective than moist heat. The spores of Clostridium botulinum, the cause of botulism, are killed in 5 minutes at 121°C by moist heat but only after 2 hours at 160°C with dry heat. However, dry heat has some definite advantages. It does not corrode glassware and metal instruments as moist heat does, and it can be used to sterilize powders, oils, and similar items. Despite these advantages, dry heat sterilization is slow and not suitable for heat-sensitive materials such as many plastic and rubber items. 
Filtration is an excellent way to reduce the microbial population in solutions of heat-sensitive material, and sometimes it can be used to sterilize solutions. Rather than directly destroying contaminating microorganisms, the filter simply removes them. There are two types of filters. Depth filters consist of fibrous or granular materials that have been bonded into a thick layer filled with twisting channels of small diameter. The solution containing microorganisms is sucked through this layer under vacuum, and microbial cells are removed by physical screening or entrapment and by adsorption to the surface of the filter material. Depth filters are made of diatomaceous earth (Berke field filters), unglazed porcelain (Chamberlain filters), asbestos, or other similar materials. Membrane filters have replaced depth filters for many purposes. These circular filters are porous membranes, a little over 0.1 mm thick, made of cellulose acetate, cellulose nitrate, polycarbonate, polyvinylidene fluoride, or other synthetic materials. Although a wide variety of pore sizes are available, membranes with pores about 0.2 μm in diameter are used to remove most vegetative cells, but not viruses, from solutions ranging in volume from less than 1 ml to many liters. The membranes are held in special holders and are often preceded by depth filters made of glass fibers to remove larger particles that might clog the membrane filter. The solution is pulled or forced through the filter with a vacuum or with pressure from a syringe, peristaltic pump, or nitrogen gas, and collected in previously sterilized containers. Membrane filters remove microorganisms by screening them out much as a sieve separates large sand particles from small ones. These filters are used to sterilize pharmaceuticals, ophthalmic solutions, culture media, oils, antibiotics, and other heat-sensitive solutions. Air also can be sterilized by filtration. Two common examples are N-95 disposable masks used in hospitals and labs, and cotton plugs on culture vessels that let air in but keep microorganisms out. N-95 masks exclude 95% of particles that are larger than 0.3 m. Other important examples are laminar flow biological safety cabinets, which employ high efficiency particulate air (HEPA) filters (a type of depth filter) to remove 99.97% of particles 0.3 m or larger. Laminar flow biological safety cabinets or hoods force air through HEPA filters, and then project a vertical curtain of sterile air across the cabinet opening. This protects a worker from microorganisms being handled within the cabinet and prevents contamination of the room. A person uses these cabinets when working with dangerous agents such as M. tuberculosis and tumor viruses. They are also employed in research labs and industries, such as the pharmaceutical industry, when a sterile working environment is needed.            
    Ultraviolet (UV) radiation    around 260 nm is quite lethal. UV radiation causes thiamine-thiamine dimerization of the microbial DNA, preventing polymerase-mediated replication and transcription. However, UV does not penetrate glass, dirt films, water, and other substances very effectively. Because of this disadvantage, UV radiation is used as a sterilizing agent only in a few specific situations. UV lamps are sometimes placed on the ceilings of rooms or in biological safety cabinets to sterilize the air and any exposed surfaces. Because UV radiation burns the skin and damages eyes, people working in such areas must be certain the UV lamps are off when the areas are in use. Commercial UV units are available for water treatment. Pathogens and other microorganisms are destroyed when a thin layer of water is passed under the lamps. Ionizing radiation    is an excellent sterilizing agent and penetrates deep into objects. It will destroy bacterial spores and vegetative cells, both prokaryotic and eukaryotic; however, ionizing radiation is not always effective against viruses. Gamma radiation from a cobalt 60 source and accelerated electrons from high-voltage electricity are used in the cold sterilization of antibiotics, hormones, sutures, and plastic disposable supplies such as syringes. Gamma radiation and electron beams have also been used to sterilize and “pasteurize” meat and other foods. Irradiation can eliminate the threat of such pathogens as Escherichia coli O157:H7, Staphylococcus aureus, and Campylobacter jejuni.  Based on the results of numerous studies, both the U.S. Food and Drug Administration and the World Health Organization have approved irradiated food and declared it safe for human consumption. Currently irradiation is being used to treat poultry, beef, pork, veal, lamb, fruits, vegetables, and spices.

Physical agents are generally used to sterilize objects. Chemicals, on the other hand, are more often employed in disinfection and antisepsis. The proper use of chemical agents is essential to laboratory and hospital safety. Chemicals also are employed to prevent microbial growth in food, and certain chemicals are used to treat infectious disease. Next we discuss chemicals used outside the body. Many different chemicals are available for use as disinfectants, and each has its own advantages and disadvantages. Ideally the disinfectant must be effective against a wide variety of infectious agents (gram-positive and gram-negative bacteria, acid-fast bacteria, bacterial spores, fungi, and viruses) at low concentrations and in the presence of organic matter. Although the chemical must be toxic for infectious agents, it should not be toxic to people or corrosive for common materials. In practice, this balance between effectiveness and low toxicity for animals is hard to achieve. Some chemicals are used despite their low effectiveness because they are relatively nontoxic. The ideal disinfectant should be stable upon storage, odorless or with a pleasant odor, soluble in water and lipids for penetration into microorganisms, have a low surface tension so that it can enter cracks in surfaces, and be relatively inexpensive.  One potentially serious problem is the overuse of antiseptics. For instance, the antibacterial agent triclosan is found in products such as deodorants, mouthwashes, soaps, cutting boards, and baby toys. Unfortunately, the emergence of triclosan-resistant bacteria has become a problem. For example, Pseudomonas aeruginosa actively pumps the antiseptic out of the cell. There is now evidence that extensive use of triclosan also increases the frequency of bacterial resistance to antibiotics. Thus overuse of antiseptics can have unintended harmful consequences. The properties and uses of several groups of common disinfectants and antiseptics are surveyed next. Many of the characteristics of disinfectants and antiseptics.       

Phenol was the first widely used antiseptic and disinfectant. In 1867 Joseph Lister employed it to reduce the risk of infection during surgery. Today phenol and phenolics (phenol derivatives) such as cresols, xylenols, and orthophenylphenol are used as disinfectants in laboratories and hospitals. The commercial disinfectant Lysol is made of a mixture of phenolics. Phenolics act by denaturing proteins and disrupting cell membranes. They have some real advantages as disinfectants: phenolics are tuberculocidal, effective in the presence of organic material, and remain active on surfaces long after application. However, they have a disagreeable odor and can cause skin irritation.  
Alcohols are among the most widely used disinfectants and antiseptics. They are bactericidal and fungicidal but not sporicidal; some lipid-containing viruses are also destroyed. The two most popular alcohol germicides are ethanol and isopropanol, usually used in about 70 to 80% concentration. They act by denaturing proteins and possibly by dissolving membrane lipids. A 10 to 15 minute soaking is sufficient to disinfect small instruments.  
A halogen is any of the five elements (fluorine, chlorine, bromine, iodine, and astatine) in group VIIA of the periodic table. They exist as diatomic molecules in the Free State and form salt like compounds with sodium and most other metals. The halogens iodine and chlorine are important antimicrobial agents. Iodine is used as a skin antiseptic and kills by oxidizing cell constituents and iodinating cell proteins. At higher concentrations, it may even kill some spores. Iodine often has been applied as tincture of iodine, 2% or more iodine in a water-ethanol solution of potassium iodide. Although it is an effective antiseptic, the skin may be damaged, a stain is left, and iodine allergies can result. Iodine has been complexed with an organic carrier to form an iodophor.  Iodophors are water soluble, stable, and non-staining, and release iodine slowly to minimize skin burns and irritation. They are used in hospitals for cleansing preoperative skin and in hospitals and laboratories for disinfecting. Some popular brands are Wesco dyne for skin and laboratory disinfection and Betadine for wounds. Chlorine is the usual disinfectant for municipal water sup- plies and swimming pools and is also employed in the dairy and food industries. It may be applied as chlorine gas (Cl 2), sodium hypochlorite (bleach, NaClO), or calcium hypochlorite [Ca(OCl)2], all of which yield hypochlorous acid (HClO) (see chemical reactions below). The result is oxidation of cellular materials and destruction of vegetative bacteria and fungi.
  Cl 2  + H 2 O HCl + HClO
  NaOCl + H 2 O NaOH + HClO
 Ca(OCl) 2 + 2H 2 O Ca(OH) 2 +  2HClO
  Death of almost all microorganisms usually occurs within 30 minutes. One potential problem is that chlorine reacts with organic compounds to form carcinogenic trihalomethanes, which must be monitored in drinking water. Ozone has been used successfully as an alternative to chlorination in Europe and Canada.
Chlorine is also an excellent disinfectant for individual use because it is effective, inexpensive, and easy to employ. Small quantities of drinking water can be disinfected with halazone tablets. Halazone (parasulfone dichloramidobenzoic acid) slowly releases chloride when added to water and disinfects it in about a half hour. It is frequently used by campers lacking access to uncontaminated drinking water.  
Heavy Metals
F or many years the ions of heavy metals such as mercury, silver, arsenic, zinc, and copper were used as germicides. These have now been superseded by other less toxic and more effective germicides (many heavy metals are more bacteriostatic than bactericidal). There are a few exceptions. In some hospitals, a 1% solution of silver nitrate is added to the eyes of infants to prevent ophthalmic gonorrhea. Silver sulfadiazine is used on burns. Copper sulfate is an effective algicide in lakes and swimming pools. Heavy metals combine with proteins, often with their sulfhydryl groups, and inactivate them. They may also precipitate cell proteins.  
Quaternary Ammonium Compounds
Quaternary ammonium compounds are detergents that have antimicrobial activity and are effective disinfectants.    Detergents (Latin detergere, to wipe away) are organic cleansing agents that are amphipathic, having both polar hydrophilic and nonpolar hydrophobic components. The hydrophilic portion of a quaternary ammonium compound is positively charged quaternary nitrogen; thus quaternary ammonium compounds are cationic detergents. Their antimicrobial activity is the result of their ability to disrupt microbial membranes; they may also denature proteins. Cationic detergents such as benzalkonium chloride and acetyl-pyridinium chloride kill most bacteria but not M. tuberculosis or spores. They have the advantages of being stable and nontoxic, but they are inactivated by hard water and soap. Cationic detergents are often used as disinfectants for food utensils and small instruments and as skin antiseptics.  
Both of the commonly used aldehydes, formaldehyde and glutaraldehyde are highly reactive molecules that combine with nucleic acids and proteins, and inactivate them, probably by cross-linking and alkylating molecules. They are sporicidal and can be used as chemical sterilants. Formaldehyde is usually dissolved in water or alcohol before use. A 2% buffered solution of glutaraldehyde is an effective disinfectant. It is less irritating than formaldehyde and is used to disinfect hospital and laboratory equipment. Glutaraldehyde usually disinfects objects within about 10 minutes but may require as long as 12 hours to destroy all spores.  
 Sterilizing Gases
 Many heat-sensitive items such as disposable plastic petri dishes and syringes, heart-lung machine components, sutures, and catheters are sterilized with ethylene oxide gas. Ethylene oxide (EtO) is both microbicidal and sporicidal. It is a very strong alkylating agent that kills by reacting with functional groups of DNA and proteins to block replication and enzymatic activity. It is a particularly effective sterilizing agent because it rapidly penetrates packing materials, even plastic wraps.  Sterilization is carried out in a special ethylene oxide sterilizer, very much resembling an autoclave in appearance, that controls the EtO concentration, temperature, and humidity. Because pure EtO is explosive, it is usually supplied in a 10 to 20% concentration mixed with either CO2 or dichloro-difluoromethane. The ethylene oxide concentration, humidity, and temperature influence the rate of sterilization. A clean object can be sterilized if treated for 5 to 8 hours at 38°C or 3 to 4 hours at 54°C when the relative humidity is maintained at 40 to 50% and the EtO concentration at 700 mg/l. Because it is so toxic to humans, extensive aeration of the sterilized materials is necessary to remove residual EtO.
Betapropiolactone (BPL) is occasionally employed as a sterilizing gas. In the liquid form it has been used to sterilize vaccines and sera. BPL decomposes to an inactive form after several hours and is therefore not as difficult to eliminate as EtO. It also destroys microorganisms more readily than ethylene oxide but does not penetrate materials well and may be carcinogenic. For these reasons, BPL has not been used as extensively as EtO.   Vaporized hydrogen peroxide can be used to decontaminate biological safety cabinets, operating rooms, and other large facilities. These systems introduce vaporized hydrogen peroxide into the enclosure for some time, depending on the size of the enclosure and the materials within. Hydrogen per- oxide and its oxy-radical byproducts are toxic and kill a wide variety of microorganisms. During the course of the decontamination process, it breaks down to water and oxygen, both of which are harmless. Other advantages of these systems are that they can be used at a wide range of temperatures (4 to 80°C) and they do not damage most materials.
Testing of antimicrobial agents is a complex process regulated by two different federal agencies. The U.S. Environmental Protection Agency regulates disinfectants, whereas agents used on humans and animals are under the control of the Food and Drug Administration. Testing of antimicrobial agents often begins with an initial screening to see if they are effective and at what concentrations. This may be followed by more realistic in-use testing. The best-known disinfectant screening test is the phenol coefficient test in which the potency of a disinfectant is compared with that of phenol. A series of dilutions of phenol and the disinfectant being tested are prepared. Standard amounts of Salmonella enterica Typhi and Staphylococcus aureus  are added to each dilution; the dilutions are then placed in a 20 or 37°C water bath. At 5-minute intervals, samples are withdrawn from each dilution and used to inoculate a growth medium, which is incubated for two or more days and then examined for growth. If there is no growth in the medium, the dilution at that particular time of sampling killed the bacteria. The highest dilution (i.e., the lowest concentration) that kills the bacteria after a 10-minute exposure but not after 5 minutes is used to calculate the phenol coefficient. This is done by dividing the reciprocal of the appropriate dilution for the disinfectant being tested by the reciprocal of the appropriate phenol dilution. For instance, if the phenol dilution was 1/90 and maximum effective dilution for disinfectant X was 1/450, then the phenol coefficient of X would be 5. The higher the phenol coefficient value, the more effective the disinfectant under these test conditions. A value greater than 1 means that the disinfectant is more effective than phenol. The phenol coefficient test is a useful initial screening procedure, but the phenol coefficient can be misleading if taken as a direct indication of disinfectant potency during normal use. This is because the phenol coefficient is determined under carefully controlled conditions with pure bacterial cultures, whereas disinfectants are normally used on complex populations in the presence of organic matter and with significant variations in environmental factors such as pH, temperature, and presence of salts. To more realistically estimate disinfectant effectiveness, other tests are often used. The rates at which selected bacteria are destroyed with various chemical agents may be experimentally determined and compared. A use dilution test can also be carried out. Stainless steel cylinders are contaminated with specific bacterial species under carefully controlled conditions. The cylinders are dried briefly, immersed in the test disinfectants for 10 minutes, transferred to culture media, and incubated for two days. The disinfectant concentration that kills the organisms in the sample with a 95% level of confidence under these conditions is determined. Disinfectants also can be tested under conditions designed to simulate normal in-use situations. In-use testing techniques allow a more accurate determination of the proper disinfectant concentration for a particular situation.    

The emerging field of biological control of microorganisms demonstrates great promise. Scientists are learning to exploit natural control processes such as predation of one microorganism on another, viral-mediated lysis, and toxin-mediated killing. While these control mechanisms occur in nature, their approval and use by humans is relatively new. Studies evaluating control of Salmonella, Shigella, and E. coli by gram-negative predators such as B.dellovibrio  suggest that poultry farms may be sprayed with the predator to reduce potential contamination. The control of human pathogens using bacteriophage is gaining wide support and appears to be effective in the eradication of a number of bacterial species by lysing the pathogenic host. This seems intuitive, knowing that the virus lyses its specific bacterial host, yet unnerving when one thinks about maybe swallowing, injecting, or applying a virus to the human body. The use of microbial toxins (such as bacteriocins) to control susceptible a population suggests yet another method for potential control of other microorganisms.
 As Ehrlich so clearly saw, to be successful a chemotherapeutic agent     must have selective toxicity: it must kill or inhibit the microbial pathogen while damaging the host as little as possible. The degree of selective toxicity may be expressed in terms of (1) the therapeutic dose the drug level required for clinical treatment of a particular infection and (2) the toxic dose the drug level at which the agent becomes too toxic for the host. The therapeutic index is the ratio of the toxic dose to the therapeutic dose. The larger the therapeutic index, the better the chemotherapeutic agent (all other things being equal).  A drug that disrupts a microbial function not found in host animal cells often has a greater selective toxicity and a higher therapeutic index. For example, penicillin inhibits bacterial cell wall peptidoglycan synthesis but has little effect on host cells because they lack cell walls; therefore penicillin’s therapeutic index is high. A drug may have a low therapeutic index because it inhibits the same process in host cells or damages the host in other ways. The undesirable effects on the host, or side effects, are of many kinds and may involve almost any organ system.  Because side effects can be severe, chemotherapeutic agents should be administered with great care.  Some bacteria and fungi are able to naturally produce many of the commonly employed antibiotics. In contrast, several important chemotherapeutic agents, such as sulfonamides, trimethoprim, ciprofloxacin, isoniazid, and dapsone, are synthetic manufactured by chemical procedures independent of microbial activity. Some antibiotics are semisynthetic natural antibiotics that have been structurally modified by the addition of chemical groups to make them less susceptible to inactivation by pathogens (e.g., ampicillin and methicillin). In addition, many semisynthetic drugs have a broader spectrum of antibiotic activity than does their parent molecule. This is particularly true of the semisynthetic penicillins (e.g., ampicillin, amoxycillin) versus the naturally produced penicillin G and penicillin V. D rugs vary considerably in their range of effectiveness. Many are     narrow-spectrum drugs that are; they are effective only against a limited variety of pathogens. Others are broad-spectrum drugs that attack many different kinds of pathogens. Drugs may also be classified based on the general microbial group they act against: antibacterial, antifungal, anti- protozoan, and antiviral. A few agents can be used against more than one group; for example, sulfonamides are active against bacteria and some protozoa. Finally, chemotherapeutic agents can be either cidal or static. Static agents reversibly inhibit growth; if the agent is removed, the microorganisms will recover and grow again. Although a cidal agent kills the target pathogen, it may be static at low levels. The effect of an agent also varies with the target species: an agent may be cidal for one species and static for another. Because static agents do not directly destroy the pathogen, elimination of the infection depends on the host’s own immunity mechanisms. A static agent may not be effective if the host is immunosuppressed.  Some idea of the effectiveness of a chemotherapeutic agent against a pathogen can be obtained from the minimal inhibitory concentration (MIC). The MIC is the lowest concentration of a drug that prevents growth of a particular pathogen. On the other hand, the minimal lethal concentration (MLC) is the lowest drug concentration that kills the pathogen. A cidal drug generally kills pathogens at levels only two to four times the MIC, whereas a static agent kills at much higher concentrations, if at all.   
Determination of antimicrobial effectiveness against specific pathogens is essential for proper therapy. Testing can show which agents are most effective against a pathogen and give an estimate of the proper therapeutic dose. 
 Dilution Susceptibility Tests
Dilution susceptibility tests can be used to determine MIC and MLC values. Antibiotic dilution tests can be done in both agar and broth. In the broth dilution test, a series of broth tubes (usually Mueller-Hinton broth) containing antibiotic concentrations in the range of 0.1 to 128 g/ml (two-fold dilutions) is prepared and inoculated with a standard density of the test organism. The lowest concentration of the antibiotic resulting in no growth after 16 to 20 hours of incubation is the MIC. The MLC can be ascertained if the tubes showing no growth are then cultured into fresh medium lacking antibiotic. The lowest antibiotic concentration from which the microorganisms do not grow when transferred to fresh medium is the MLC. The agar dilution test is very similar to the broth dilution test. Plates containing Mueller-Hinton agar and various amounts of antibiotic are inoculated and examined for growth. Several automated systems for susceptibility testing and MIC determination with broth or agar cultures have been developed. 
Disk Diffusion Tests
 If a rapidly growing microbe such as Staphylococcus or Pseudomonas  is being tested, a disk diffusion technique may be used to save time and media. The principle behind the assay technique is fairly simple. When an antibiotic-impregnated disk is placed on agar previously inoculated with the test bacterium, the antibiotic diffuses radially outward through the agar, producing an antibiotic concentration gradient. The antibiotic is present at high concentrations near the disk and affects even minimally susceptible microorganisms (resistant organisms will grow up to the disk). As the distance from the disk increases, the antibiotic concentration decreases and only more susceptible pathogens are harmed. A clear zone or ring is present around an antibiotic disk after incubation if the agent inhibits bacterial growth. The wider the zone surrounding a disk, the more susceptible the pathogen is. Zone width also is a function of the antibiotic’s initial con- centration, its solubility, and its diffusion rate through agar. Thus zone width cannot be used to compare directly the effectiveness of different antibiotics.  Currently the disk diffusion test most often used is the Kirby- Bauer method, which was developed in the early 1960s at the University of Washington Medical School by William Kirby, A. W. Bauer, and their colleagues. Freshly grown bacteria are used to inoculate the entire surface of a Mueller-Hinton agar plate. After the agar surface has dried for about 5 minutes, the appropriate antibiotic test disks are placed on it, either with sterilized forceps or with a multiple applicator device.  The plate is immediately placed at 35°C. After 16 to 18 hours of incubation, the diameters of the zones of inhibition are measured to the nearest millimeter.   Kirby-Bauer test results are interpreted using a table that relates zone diameter to the degree of microbial resistance. A plot of MIC (on a logarithmic scale) versus zone inhibition diameter (arithmetic scale) is prepared for each antibiotic.  These plots are then used to find the zone diameters corresponding to the drug concentrations actually reached in the body. If the zone diameter for the lowest level reached in the body is smaller than that seen with the test pathogen, the pathogen should have an MIC value low enough to be destroyed by the drug. A pathogen with too high and MIC value (too small a zone diameter) is resistant to the agent at normal body concentrations.      
 The E test   
The E test from AB BIODISK may be used in sensitivity testing under some conditions. It is particularly convenient for use with anaerobic pathogens. Agar medium is streaked in three different directions with the test organism and plastic E test   strips are placed on the surface so that they extend out radially from the center.  Each strip contains a gradient of an antibiotic and is labeled with a scale of MIC values. The lowest concentration in the strip lies at the center of the plate. After 24 to 48 hours of incubation, an elliptical zone of inhibition appears. MICs are determined from the point of intersection between the inhibition zone and the strip’s scale of MIC values. 
Since Fleming’s discovery of penicillin, many antibiotics have been found that can damage pathogens in several ways. A few antibacterial drugs are described here with emphasis on their mechanisms of action. 
 Inhibitors of Cell Wall Synthesis
 The most selective antibiotics are those that interfere with bacterial cell wall synthesis. Drugs such as penicillins, cephalosporins, vancomycin, and bacitracin have a high therapeutic index because they target structures not found in eukaryotic cells.
 Most penicillin (e.g., penicillin G or benzylpenicillin) are derivatives of 6-aminopenicillanic acid and differ from one another with respect to the side chain attached to the amino group.  The most crucial feature of the molecule is the     -lactam ring, which is essential for bioactivity. Many penicillin-resistant bacteria produce penicillinase (also called lactamase), an enzyme that in- activates the antibiotic by hydrolyzing a bond in the lactam ring. The structure of the penicillins resembles the terminal d-alanyl-d-alanine found on the peptide side chain of the peptidoglycan subunit. It is thought that this structural similarity blocks the enzyme catalyzing the transpeptidation reaction that forms the peptidoglycan cross-links. Thus formation of a complete cell wall is blocked, leading to osmotic lysis. This mechanism is consistent with the observation that penicillins act only on growing bacteria that are synthesizing new peptidoglycan. However, the mechanism of penicillin action is actually more complex. It has been discovered that penicillins bind to several periplasmic proteins (penicillin-binding proteins, or PBPs) and may also destroy bacteria by activating their own autolytic enzymes. However, penicillin may kill bacteria even in the absence of autolysins or murein hydrolases. Lysis could occur after bacterial viability has already been lost. Penicillin may stimulate proteins called bacterial holins to form holes or lesions in the plasma membrane, leading directly to membrane leakage and death. Murein hydrolases also could move through the holes, disrupt the peptidoglycan, and lyse the cell. Penicillins differ from each other in several ways. The two naturally occurring penicillins, penicillin G and penicillin V, are narrow-spectrum drugs. Penicillin G is effective against gonococci, meningococci, and several gram-positive pathogens such as streptococci and staphylococci. However, it must be administered by injection (parenterally) because it is destroyed by stomach acid. Penicillin V is similar to penicillin G in spectrum of activity but can be given orally because it is more resistant to acid. The semisynthetic penicillins, on the other hand, have a broader spectrum of activity. Ampicillin can be administered orally and is effective against gram-negative bacteria such as Haemophilus, Salmonella, and Shigella.  Carbenicillin and ticarcillin are potent against Pseudomonas and Proteus. An increasing number of bacteria have become resistant to natural penicillins and many of the semisynthetic analogs. Physicians frequently employ specific semisynthetic penicillins that are not destroyed by -lactamases to combat antibiotic resistant pathogens. These include methicillin, nafcillin, and oxacillin. However, this practice has been confounded by the emergence of methicillin- resistant bacteria. Although penicillins are the least toxic of the antibiotics, about 1 to 5% of the adults in the United States develop allergies to them. Occasionally, a person will die of a violent allergic response; therefore patients should be questioned about penicillin allergies before treatment is begun.
 Cephalosporins Cephalosporins are a family of antibiotics originally isolated in 1948 from the fungus Cephalosporium.  They contain a lactam structure that is very similar to that of the penicillins.  As might be expected from their structural similarities to penicillins, cephalosporins also inhibit the transpeptidation reaction during peptidoglycan synthesis. They are broad-spectrum drugs frequently given to patients with penicillin allergies (although about 10% of patients allergic to penicillin are also allergic to cephalosporins). Cephalosporins are broadly categorized into four generations (groups of drugs that were sequentially developed) based on their spectrum of activity. First-generation cephalosporins are more effective against gram-positive pathogens than gram-negatives. Second-generation drugs, developed after the first generation, have improved effects on gram-negative bacteria with some anaerobe coverage. Third-generation drugs are particularly effective against gram-negative pathogens, and some reach the central nervous system. This is of particular note because many antimicrobial agents do not cross the blood-brain barrier. Finally, fourth-generation cephalosporins are broad spectrum with excellent gram-positive and gram-negative coverage and, like their third-generation predecessors, inhibit the growth of the difficult opportunistic pathogen Pseudomonas aeruginosa.   
 Vancomycin and Teicoplanin Vancomycin is a glycopeptide antibiotic produced by Streptomyces orientalis. It is a cup-shaped molecule composed of a peptide linked to a disaccharide. The peptide portion blocks the transpeptidation reaction by binding specifically to the d-alanine-d-alanine terminal sequence on the pentapeptide portion of peptidoglycan. The antibiotic is bactericidal for Staphylococcus and some members of the genera Clostridium, Bacillus, Streptococcus, and Enterococcus.  It is given both orally and intravenously, and has been particularly important in the treatment of antibiotic-resistant staphylococcal and enterococcal infections. However, vancomycin-resistant strains of Enterococcus have become widespread and cases of resistant Staphylococcus aureus have appeared. In this case, resistance is conferred when bacteria change the terminal d-alanine to either d-lactate or a d-serine residue. Vancomycin resistance poses a serious public health threat: vancomycin has been considered the “drug of last resort” in cases of antibiotic-resistant S. aureus.  Clearly newer drugs must be developed. Teicoplanin, another glycopeptide antibiotic, is produced by the actinomycete Actinoplanes teichomyceticus.  It is similar in structure and mechanism of action to vancomycin but has fewer side effects. It is active against staphylococci, enterococci, streptococci, clostridia, Listeria, and many gram-positive pathogens.   
 Protein Synthesis Inhibitors
 Many antibiotics inhibit protein synthesis by binding with the prokaryotic ribosome and other components of protein synthesis. Because these drugs discriminate between prokaryotic and eukaryotic ribosomes, their therapeutic index is fairly high but not as high as that of cell wall inhibitors. Several different steps in protein synthesis can be affected: aminoacyl-tRNA binding, peptide bond formation, mRNA reading, and translocation.
 Aminoglycosides although considerable variation in structure occurs among several important aminoglycoside antibiotics, all contain a cyclohexane ring and amino sugars. Streptomycin, kanamycin, neomycin, and tobramycin are synthesized by different species of the genus Streptomyces, whereas gentamicin comes from another actinomycete, Micromonospora purpurea.  Aminoglycosides bind to the 30S (small) ribosomal subunit and interfere with protein synthesis by directly inhibiting the synthesis process and causing misreading of the mRNA.     These antibiotics are bactericidal and tend to be most effective against gram-negative pathogens. Streptomycin’s usefulness has decreased greatly due to widespread drug resistance but may still be effective when other aminoglycosides are not tolerated or are contraindicated due to interactions with other drugs (e.g., HIV protease inhibitors). Gentamicin is used to treat Proteus, Escherichia, Klebsiella, and Serratia infections. Aminoglycosides can be quite toxic, however, and can cause deafness, renal dam- age, loss of balance, nausea, and allergic responses.  
 Tetracyclines the tetracyclines are a family of antibiotics with a common four-ring structure to which a variety of side chains are attached.  Oxytetracycline and chlortetracycline are produced naturally by Streptomyces species, whereas others are semisynthetic drugs. These antibiotics are similar to the aminoglycosides and combine with the 30S subunit of the ribosome. This inhibits the binding of aminoacyl-tRNA molecules to the A site of the ribosome. Their action is only bacteriostatic.  Tetracyclines are broad-spectrum antibiotics that are active against most bacteria, including rickettsia, chlamydiae, and mycoplasmas. Although their use has declined in recent years, they are still sometimes used to treat acne.  
 Macrolides The macrolide antibiotics contain 12- to 22-carbon lac- tone rings linked to one or more sugars.  Erythromycin binds to the 23S rRNA of the 50S ribosomal subunit to inhibit peptide chain elongation during protein synthesis. Erythromycin is a relatively broad-spectrum anti- biotic effective against gram-positive bacteria, mycoplasmas, and a few gram-negative bacteria but is usually only bacteriostatic. It is used with patients who are allergic to penicillins and in the treatment of whooping cough, diphtheria, diarrhea caused by Campylobacter, and pneumonia from Legionella or Mycoplasma infections. Clindamycin is effective against a variety of bacteria, including staphylococci, and anaerobes such as Bacteroides.  Azithromycin, which has surpassed erythromycin in use, is particularly effective against many bacteria including Chlamydia trachomatis.    
 Chloramphenicol was first produced from cultures of Streptomyces venezuelae but it is now synthesized chemically. Like erythromycin, this antibiotic binds to 23S rRNA on the 50S ribosomal subunit to inhibit the peptidyl transferase reaction. It has a very broad spectrum of activity but, unfortunately, is quite toxic. The most common side effect is depression of bone mar- row function, leading to aplastic anemia and a decreased number of white blood cells. Consequently, this antibiotic is used only in life-threatening situations when no other drug is adequate.
Metabolic Antagonists
 Several valuable drugs act as antimetabolites: they antagonize, or block, the functioning of metabolic pathways by competitively inhibiting the use of metabolites by key enzymes. These drugs can act as structural analogs, molecules that are structurally similar to naturally occurring metabolic intermediates. These analogs compete with intermediates in metabolic processes because of their similarity but are just different enough that they prevent normal cellular metabolism. By preventing metabolism, they are bacteriostatic but broad spectrum; their removal reestablishes the metabolic activity. 
 Sulfonamides or Sulfa Drugs Sulfonamides    , or sulfa drugs, are structurally related to sulfanilamide, an analog of p-aminobenzoic acid, or PABA.  PABA is used in the synthesis of the cofactor folic acid (folate). When sulfanilamide or another sulfonamide enters a bacterial cell, it competes with PABA for the active site of an enzyme involved in folic acid synthesis, causing a decline in folate concentration. This decline is detrimental to the bacterium because folic acid is a precursor of purines and pyrimidines, the bases used in the construction of DNA, RNA, and other important cell constituents. The resulting inhibition of purine and pyrimidine synthesis leads to cessation of protein synthesis and DNA replication. Sulfonamides are selectively toxic for many bacterial and protozoan pathogens because these microbes manufacture their own folate and cannot effectively take up this cofactor, whereas humans do not synthesize folate (we must obtain it in our diet). Sulfonamides thus have a high therapeutic index. The increasing resistance of many bacteria to sulfa drugs limits their effectiveness. Furthermore, as many as 5% of the patients receiving sulfa drugs experience adverse side effects, chiefly allergic responses such as fever, hives, and rashes.  
Trimethoprim is a synthetic antibiotic that also interferes with the production of folic acid. It does so by binding to dihydrofolate reductase (DHFR), the enzyme responsible for converting dihydrofolic acid to tetrahydrofolic acid, competing against the dihydrofolic acid substrate. It is a broad-spectrum antibiotic often used to treat respiratory and middle ear infections, urinary tract infections, and traveler’s diarrhea. It can be combined with sulfa drugs to increase efficacy of treatment by blocking two key steps in the folic acid pathway.  The inhibition of two successive steps in a single biochemical pathway means that less of each drug is needed in combination than when used alone. This is termed a synergistic drug interaction.       
 Nucleic Acid Synthesis Inhibition
The antibacterial drugs that inhibit nucleic acid synthesis function by inhibiting
(1) DNA polymerase and DNA helicase
(2) RNA polymerase to block replication or transcription, respectively. These drugs are not as selectively toxic as other antibiotics because procaryotes and eucaryotes do not differ greatly with respect to nucleic acid synthesis. 
The quinolones are synthetic drugs that contain the 4-quinolone ring. The quinolones are important antimicrobial agents that inhibit nucleic acid synthesis. They are increasingly used to treat a wide variety of infections. The first quinolone, nalidixic acid was synthesized in 1962. Since that time, generations of fluoroquinolones have been produced. Three of these ciprofloxacin, norfloxacin, and ofloxacin are currently used in the United States, and more fluoroquinolones are being synthesized and tested. Quinolones act by inhibiting the bacterial DNA gyrase and topoisomerase II. DNA gyrase introduces negative twist in DNA and helps separate its strands. Inhibition of DNA gyrase disrupts DNA replication and repair, bacterial chromosome separation during division, and other processes involving DNA.
Fluoroquinolones also inhibit topoisomerase II, another enzyme that untangles DNA during replication. It is not surprising that quinolones are bactericidal.
   The quinolones are broad-spectrum antibiotics. They are highly effective against enteric bacteria such as Escherichia coli and Klebsiella pneumoniae.  They can be used with Haemophilus, Neiserria, P. aeruginosa, and other gram-negative pathogens. The quinolones also are active against gram-positive bacteria such as S. aureus, Streptococcus pyogenes, and  Mycobacterium tuberculosis.  Thus they are used in treating a wide range of infections.     
Treatment of fungal infections generally has been less successful than that of bacterial infections largely because eukaryotic fun- gal cells are much more similar to human cells than are bacteria. Many drugs that inhibit or kill fungi are therefore quite toxic for humans and thus have a low therapeutic index. In addition, most fungi have a detoxification system that modifies many antifungal agents. Therefore antibiotics are fungistatic only as long as repeated application maintains high levels of unmodified antibiotic. Nonetheless, a few drugs are useful in treating many major fungal diseases. Effective antifungal agents frequently either extract membrane sterols or prevent their synthesis. Similarly, because fungi have cell walls made of chitin, the enzyme chitin synthase is the target for antifungals such as polyoxin D and nikkomycin.   Fungal infections are often subdivided into infections called superficial mycoses, subcutaneous mycoses, and systemic mycoses. Treatment for these types of disease is very different. Several drugs are used to treat superficial mycoses. Three drugs containing imidazole miconazole, ketoconazole, and clotrimazole are broad-spectrum agents available as creams and solutions for the treatment of dermatophyte infections such as athlete’s foot, and oral and vaginal candidiasis. They are thought to disrupt fungal membrane permeability and inhibit sterol synthesis. Nystatin, a polyene anti- biotic from Streptomyces, is used to control Candida infections of the skin, vagina, or alimentary tract. It binds to sterols and damages the membrane, leading to fungal membrane leakage. Griseofulvin, an antibiotic formed by Penicillium, is given orally to treat chronic dermatophyte infections. It is thought to disrupt the mitotic spindle and inhibit cell division; it also may inhibit protein and nucleic acid synthesis. Side effects of griseofulvin include headaches, gastrointestinal upset and allergic reactions. Ascomycota Systemic infections are very difficult to control and can be fatal. Three drugs commonly used against systemic mycoses are amphotericin B, 5-flucytosine, and fluconazole. Amphotericin B from Streptomyces spp. binds to the sterols in fungal membranes, disrupting membrane permeability and causing leakage of cell constituents. It is quite toxic to humans and used only for serious, life-threatening infections. The synthetic oral antimycotic agent 5-flucytosine (5-fluorocytosine) is effective against most systemic fungi, although drug resistance often develops rapidly. The drug is converted to 5-fluorouracil by the fungi, incorporated into RNA in place of uracil, and disrupts RNA function. Its side effects include skin rashes, diarrhea, nausea, aplastic anemia, and liver damage. Atovaquone and pentamidine are used to treat Pneumocystis jiroveci (formerly called P.carinii). Some reports indicate that pentamidine interferes with P. jiroveci metabolism, although the drug only moderately inhibits glucose metabolism, protein synthesis, RNA synthesis, and intracellular amino acid transport in vitro. Fluconazole is used in the treatment of candidiasis, cryptococcal meningitis, and coccidioidal meningitis. Because adverse effects of fluconazole are relatively uncommon, it is used prophylactically to prevent life-threatening fungal infections in AIDS patients and other individuals who are severely immunosuppressed. Subcutaneous mycoses, such as mycetoma and sporotrichosis, are typically treated with combinations of therapies that would be used for superficial and systemic mycoses. As with systemic mycoses, strict attention to potential toxic side effects is warranted.    

Because viruses enter host cells and make use of host cell enzymes and constituents, it was long thought that a drug that blocked virus reproduction would be toxic for the host. However, inhibitors of virus specific enzymes and life cycle processes have been discovered, and several drugs are used therapeutically.   Most antiviral drugs disrupt either critical stages in the virus life cycle or the synthesis of virus-specific nucleic acids.  Amantadine and rimantadine can be used to prevent influenza an infections. When given early in the infection (in the first 48 hours), they reduce the incidence of influenza by 50 to 70% in an exposed population. Amantadine blocks the penetration and uncoating of influenza virus particles. Adenine arabinoside or vidarabine disrupts the activity of DNA polymerase and several other enzymes involved in DNA and RNA synthesis and function. It is given intravenously or applied as an ointment to treat herpes infections. A third drug, acyclovir, is also used in the treatment of herpes infections. Upon phosphorylation, acyclovir resembles deoxyGTP and inhibits the viral DNA polymerase. Unfortunately, acyclovir-resistant strains of herpes have developed. Effective acyclovir derivatives and relatives are now available. Valacyclovir is an orally administered prodrug form of acyclovir. Prodrugs are inactive until metabolized. Ganciclovir, penciclovir, and penciclovir’s oral form, famciclovir, are effective in treatment of herpes viruses. Another kind of drug, foscarnet, inhibits the virus DNA polymerase in a different way. Foscarnet is an organic analog of pyrophosphate that binds to the polymerase active site and blocks the cleavage of pyrophosphate from nucleoside triphosphate substrates. It is used in treating herpes and cytomegalovirus infections. Several broad-spectrum anti-DNA virus drugs have been developed. A good example is the drug HPMPC, or cidofovir. It is effective against papovaviruses, adenoviruses, herpes viruses, iridoviruses, and poxviruses. The drug acts on the viral DNA polymerase as a competitive inhibitor and alternative substrate of dCTP. It has been used primarily against cytomegalovirus but also against herpes simplex and human papillomavirus infections. Research on anti-HIV drugs has been particularly active. Many of the first drugs to be developed were reverse transcriptase inhibitors such as azidothymidine (AZT) or zidovudine, lamivudine (3TC), didanosine (ddI), zalcitabine (ddC), and stavudine (d4T). These interfere with reverse transcriptase activity and therefore block HIV reproduction. More recently HIV protease inhibitors have also been developed. Three of the most used are saquinvir, indinavir, and ritonavir. Protease inhibitors are effective because HIV, like many viruses, translates multiple proteins as a single polypeptide. This polypeptide must then be cleaved into individual proteins required for virus replication. Protease inhibitors mimic the pep- tide bond that is normally attacked by the protease. The most successful HIV treatment regimen has been a cocktail of agents given at high dosages to prevent the development of drug resistance. For example, the combination of AZT, 3TC, and ritonavir is very effective in reducing HIV plasma concentrations almost to zero. However, the treatment does not eliminate proviral HIV DNA that still resides in memory T cells and possibly elsewhere.
Probably the most publicized antiviral agent has been Tamiflu (generically, oseltamivir phosphate). Tamiflu is a neuraminidase inhibitor that has received much attention in light of predictions of a twenty-first-century influenza pandemic, including avian influenza (“bird flu”). While Tamiflu is not a cure for neurominidase expressing viruses, two clinical trials showed that patients who took Tamiflu were relieved of flu symptoms 1.3 days faster than patients who did not take Tamiflu. However, prophylactic use has resulted in viral resistance to Tamiflu. Tamiflu is not a substitute for yearly flu vaccination and frequent hand-washing. 

The mechanism of action for most antiprotozoan drugs is not completely understood. Drugs such as chloroquine, atovaquone, mefloquine, iodoquinol, metronidazole, and nitazoxanide, for ex- ample, have potent antiprotozoan action, but a clear mechanism by which protozoan growth is inhibited is unknown. However, most antiprotozoan drugs appear to act on protozoan nucleic acid or some metabolic event. Chloroquine is used to treat malaria. Several mechanisms of action have been reported. It can raise the internal pH, clump the plasmodial pigment, and intercalate into plasmodial DNA. Chloroquine also inhibits heme polymerase, an enzyme that converts toxic heme into nontoxic hemazoin. Inhibition of this enzyme leads to a buildup of toxic heme. Mefl oquine is also used to treat malaria and has been found to swell the Plasmodium falciparum food vacuoles, where it may act by forming toxic complexes that damage membranes and other plasmodial components. Metronidazole is used to treat Entamoeba infections. Anaerobic organisms readily reduce it to the active metabolite within the cytoplasm. Aerobic organisms appear to reduce it using ferrodoxin (a protein of the electron transport system). Reduced metronidazole interacts with DNA, altering its helical structure and causing DNA fragmentation; it prevents normal nucleic acid synthesis, resulting in cell death. A number of antibiotics that inhibit bacterial protein synthesis are also used to treat protozoan infection. These include the aminoglycosides clindamycin and paromomycin. Aminoglycosides can be considered polycationic molecules that have a high affinity for nucleic acids. Specifically, aminoglycosides possess high affinities for RNAs. Different aminoglycoside antibiotics bind to different sites on RNAs. RNA binding interferes with the normal expression and function of the RNA, resulting in cell death.   Interference of eukaryotic electron transport is one common activity of some antiprotozoan drugs. Atovaquone is used to treat Toxoplasma gondii. It is an analog of ubiquinone, an integral component of the eukaryotic electron transport system. As an analog of ubiquinone, atovaquone can act as a competitive inhibitor and thus suppress electron transport. The ultimate metabolic effects of electron transport blockade include inhibited or delayed synthesis of nucleic acids and ATP. Another drug that interferes with electron transport is nitazoxanide, which is used to treat cryptosporidiosis. It appears to exert its effect through interference with the pyruvate: ferredoxin oxidoreductase. It has also been reported to form toxic free radicals once the nitro group is reduced intracellularly.  Pyrimethamine and dapsone, used to treat Toxoplasma infections, appear to act in the same way as trimethoprim interfering with folic acid synthesis by inhibition of dihydrofolate reductase.   As with other antimicrobial therapies, traditional drug development starts by identifying a unique target to which a drug can bind and thus prevent some vital function. A second consideration is often related to drug spectrum: how many different species have that target so that the proposed drug can be used broadly as a chemotherapeutic agent? This is also true for use of agents needed to remove protozoan parasites from their hosts. However, because protozoa are eucaryotes, the potential for drug action on host cells and tissues is greater than it is when targeting procaryotes. Most of the drugs used to treat protozoan infection have signifcant side effects; nonetheless, the side effects are usually acceptable when weighed against the parasitic burden.    






  1. even though it is a very long article, it is very clear and very much informative. almost i can prepare microbio notes for whole year. really happy about ur blog...

  2. This is very powerful I ve been diagnosed with HIV 3years ago and I've become in denial at first for a year but later I accepted it and I'm living with depression so I struggle to keep my cd4 up bit I'm very healthy and I just checked my cd4 was 460 and they advising me to being medication but I'm not ready I wanna take them when at least it 350 I'm only 24years old I have a 6year old healthy boy negative and I've been very stressed lately I slept with a guy a month ago without a condom I always protect my self but this time he took the condom out without me noticing I became so scared knowing my status and I can't live with my self knowing that I infected one another person I told him that he should take p.e.p anti virus he said no he is clean there is no need unless I know I'm not clean I was so scared not ready to tell him following day took me to his doctor for me to come he is clean he tested in front of me n I fogged my result sent him someone else's result with my name I regret it and I'm scared don't know how to tell him I'm scared can't live with my self knowing that I did what I did after protecting all the guys I've been with for so long it;s about a day for me to tell him that I HIV positive that i came across testimony of a lady been cured of HIV from Dr James herbal mix, for me been so desperate I picked up interest and contacted Dr James and told him my problems and he asked me some few questions and then said I should send him money some so he can send me his powerful herbal mixed medicine and I did,2 days later he courier the herbal mixed medicine to me through DHL speed post the medicine got to me in 5 days time and I used it morning and night as he prescribed for me for 3 weeks and I was cured, I couldn't believe it because it was like a magic to me
    then I was bold enough to meet the guy and told him that I had HIV but lucky am cured now, I recommended him to Dr James who cured him and he was cured too today we have been in a good relationship and we have 2years old son now and he is HIV negative Thanks to Dr James herbal mix if you are HIV positive or suffering from
    PID Virus. contact Dr James on


    What's app +2348152855846 

  3. This is very powerful I ve been diagnosed with HIV 3years ago and I've become in denial at first for a year but later I accepted it and I'm living with depression so I struggle to keep my cd4 up bit I'm very healthy and I just checked my cd4 was 460 and they advising me to being medication but I'm not ready I wanna take them when at least it 350 I'm only 24years old I have a 6year old healthy boy negative and I've been very stressed lately I slept with a guy a month ago without a condom I always protect my self but this time he took the condom out without me noticing I became so scared knowing my status and I can't live with my self knowing that I infected one another person I told him that he should take p.e.p anti virus he said no he is clean there is no need unless I know I'm not clean I was so scared not ready to tell him following day took me to his doctor for me to come he is clean he tested in front of me n I fogged my result sent him someone else's result with my name I regret it and I'm scared don't know how to tell him I'm scared can't live with my self knowing that I did what I did after protecting all the guys I've been with for so long it;s about a day for me to tell him that I HIV positive that i came across testimony of a lady been cured of HIV from Dr James herbal mix, for me been so desperate I picked up interest and contacted Dr James and told him my problems and he asked me some few questions and then said I should send him money some so he can send me his powerful herbal mixed medicine and I did,2 days later he courier the herbal mixed medicine to me through DHL speed post the medicine got to me in 5 days time and I used it morning and night as he prescribed for me for 3 weeks and I was cured, I couldn't believe it because it was like a magic to me
    then I was bold enough to meet the guy and told him that I had HIV but lucky am cured now, I recommended him to Dr James who cured him and he was cured too today we have been in a good relationship and we have 2years old son now and he is HIV negative Thanks to Dr James herbal mix if you are HIV positive or suffering from
    PID Virus. contact Dr James on


    What's app +2348152855846