Daniela Gorduza, MD

• Consultant in Pediatric Urology,
• Claude-Bernard University, Lyon I,
• France
• Consultant in Pediatric Urology, H?pital M?re-
• Enfants?GHE,
• Bron, France

Agglutination reactions are sometimes easier to see and interpret with the unaided eye erectile dysfunction medications over the counter red viagra 200mg overnight delivery. Serial dilutions of serum are added to wells containing a constant amount of antigen erectile dysfunction washington dc cheap 200 mg red viagra visa. At lower serum dilutions (higher concentrations of antibody) impotence nitric oxide buy red viagra paypal, agglutination occurs; at higher serum dilutions erectile dysfunction doctor denver buy 200 mg red viagra amex, antibody concentration is too low to produce agglutination encore erectile dysfunction pump cheap red viagra 200 mg overnight delivery. Another use of agglutination is in a type of test that determines the concentration of antibodies in a clinical sample impotence hypothyroidism discount red viagra 200mg with visa. Although the simple detection of antibodies is sufficient for many purposes, it is often more desirable to measure the amount of antibodies in serum. Eventually, the antibodies in the serum become so dilute that they can no longer cause agglutination. The highest dilution of serum giving a positive reaction is a titer, which is expressed as a ratio reflecting the dilution. Where the antibodies react with the surface antigens, the blood cells can be seen to agglutinate, or clump together. What is the blood type of the person whose blood was used in this hemagglutination reaction Neutralization tests work because antibodies can neutralize the biological activity of many pathogens and their toxins. For example, combining antibodies against tetanus toxin with a sample of tetanus toxin renders the sample harmless to mice because the antibodies have reacted with and neutralized the toxin. Next we briefly consider two neutralization tests that, although not simple to perform, effectively reveal the biological activity of antibodies. However, if the viruses are first mixed with specific antibodies against them, their ability to kill cultured cells is neutralized. In a viral neutralization test, a mixture containing serum and a known pathogenic virus is introduced into a cell culture; if there are no cytopathic effects, antibodies against that virus are present in the serum. Viral neutralization tests are sufficiently sensitive and specific to ascertain whether an individual has been exposed to a particular virus or viral strain, which may lead a physician to a diagnosis or treatment or to recommendations to prevent future infection or disease. A different form of serological testing involves labeled (or tagged) antibody tests, so named because these tests use antibody molecules that are linked to some molecular "label" that enables them to be detected easily. For example, gamma radiation detectors can count radioactive chemicals, and fluorescence microscopes can measure fluorescent labels. Labeled antibody tests using radioactive or fluorescent labels can be used to detect either antigens or antibodies. Currently, radioactivity is rarely used, given the regulatory challenges of using and disposing of it. Viral Hemagglutination Inhibition Test Because not all viruses are cytopathic-they do not kill their host cell-a neutralization test cannot be used to identify all viruses. However, many viruses (including influenzaviruses) have surface proteins that naturally clump red blood cells. Such viral hemagglutination inhibition tests can be used to Fluorescent Antibody Tests Fluorescent dyes are used as labels in several serological tests. When exposed to a specific wavelength of light (as in a fluorescence microscope), the fluorescent dye glows. Fluorescently labeled antibodies are used in direct and indirect fluorescent antibody tests. The test is straightforward: A scientist floods a tissue sample suspected of containing the antigen with labeled antibody, waits a short time to allow the antibody to bind to the antigen, washes the preparation to remove any unbound antibody, and examines it with a fluorescence microscope. If the suspected antigen is present, labeled antibody will adhere to it, and the scientist will see fluorescence. This is not a quantitative test-the amount of fluorescence observed is not directly related to the amount of antigen present. Scientists use direct fluorescent antibody tests to identify small numbers of bacteria in patient tissues. This technique has been used to detect Mycobacterium tuberculosis in sputum and rabies viruses infecting a brain. Fluorescent label Anti-IgG (antiglobulin, anti-antibody) An antigen of interest is fixed to a microscope slide. The serum is then washed off, leaving the antibodies bound to the antigen (but not yet visible). After the slide is washed to remove unbound antiantibodies, it is examined with a fluorescence microscope. The presence of antibodies indicates that the patients have been exposed to the pathogen and may need treatment or counseling on steps to take to lower their risk of future infection. Scientists routinely identify and separate types of white blood cells, such as lymphocytes, by using specific monoclonal antibodies produced against each cell type. The researchers can attach differently colored fluorescent dyes to the antibodies, allowing them to differentiate types of lymphocytes by the color of the dye attached to each type of antibody. Excess antigen molecules are washed off, and another protein (such as gelatin) is added to the wells to completely coat any of the surface not coated with antigen. One well is assigned to each serum being tested, and a sample of the serum is added to that well. Whenever a serum sample contains antibodies against the antigen, the antibodies bind to the antigen affixed to the plate. The enzyme and substrate are chosen because their reaction results in products that cause a visible color change. A positive reaction in a well, indicated by the development of color, can occur only if the labeled anti-antibody has bound to antibodies attached to the antigen of interest. The intensity of the color, which can be estimated visually or measured accurately using a spectrophotometer, is proportional to the amount of antibody present in the serum. Enzyme Anti-antibody 4 Enzyme-linked anti-antibody is added and binds to bound antibody. Finally, each well is flooded with enzyme-labeled antibodies specific to the antigen. Such tests can also be used to quantify the amount of antigen in a given sample, such as with tests for illegal drugs. Immunoblots An immunoblot (also called a western blot) is a technique used to detect a specific protein, such as an antibody, in a complex mixture. Immunoblotting tests are used to confirm the presence of proteins, including antibodies against pathogens. Each of the proteins in the solution is resolved into a single band, producing invisible protein bands. This can be done by absorbing the solution into absorbent paper-a process called blotting. Recent years have seen the development of simple immunoassays that give clinicians useful results within minutes. These assays allow point-of-care testing; that is, health care Serological Tests That Use Antigens and Corresponding Antibodies Wells containing proteins 515 Polyacrylamide gel 1 Electrophoresis (not shown) Polyacrylamide gel 2 Blotting Nitrocellulose membrane Filter paper Polyacrylamide gel Absorbent paper Nitrocellulose is removed from blotting stack. Common point-of-care tests include immunofiltration and immunochromatography assays. These tests are not quantitative, but they do rapidly give a positive or negative result, making them very useful in arriving at a quick diagnosis. In these systems, an antigen solution (such as diluted blood or sputum) flowing through a porous material encounters antibody labeled with either pink colloidal3 gold or blue colloidal selenium. Where antigen and antibody bind, colored immune complexes form in the fluid, which then flows through a region where the complexes encounter antibody against them, resulting in a clearly visible pink or blue line, depending on the label used. In one adaptation, the antibodies are coated on membrane strips, which serve as dipsticks. At one end, anti-antibodies are fixed in a line so that they cannot move in the membrane. The lower portion of the membrane is coated with antibodies against the antigen in question. These antibodies are linked to a color indicator in the form of a colloidal metal and are free to move in the membrane by capillary action. A laboratory scientist prepares a solution with a nasal swab from the patient so as to release Streptococcus antigens if they are present. The complexes move up the membrane by capillary action until they reach the line of anti-antibodies, where they bind and must stop because the anti-antibodies are chemically bound to the strip. Previously, the complexes were invisible because they were dilute; now they are concentrated at the line of anti-antibodies and become visible, indicating that this patient has group A Streptococcus in the nose. Knowing that the infection is bacterial and not viral, the physician can prescribe antibacterial drugs. This technique detects specific proteins in a complex mixture, taking advantage of the high affinity and specificity of antibodies against the proteins. The artist has shown the positions of the proteins, but in reality, they would be invisible 1. Connors examines Ryan and collects a nasal wash (saline squirted into and immediately withdrawn from the nostril) to send for lab analysis. Connors admits Ryan to the hospital so he can be monitored until the lab results return. Over the next couple of days, Ryan develops bronchiolitis (inflammation of the bronchioles). He is treated with supplemental oxygen and suctioning of mucus to help him breathe more easily. Immunization is a more general term referring to the use of vaccines against rabies, anthrax, measles, mumps, rubella, polio, and other diseases. Individuals can be protected against many infections by either active immunization or passive immunotherapy. Active immunization involves giving antigen in the form of either attenuated vaccines, inactivated vaccines (killed vaccines), toxoid vaccines, or recombinant gene vaccines. Immunologists can reduce the virulence of microbes used in vaccines so that they are less likely to cause disease, a practice known as attenuation. Pathogens in attenuated vaccines are weakened so that they no longer cause disease, though they are still alive or active and can provide contact immunity in unimmunized individuals who associate with immunized people. Inactivated vaccines are either whole agent or subunit vaccines and often contain adjuvants, which are chemicals added to increase their ability to stimulate active immunity. A combination vaccine is composed of antigens from several pathogens so they can be administered to a patient at once. Vaccine developers can use recombinant gene technology to remove virulence genes from microbes to create attenuated vaccines, to produce large quantities of antigens for use in immunizations, or to alter cells, viruses, or plasmids by introducing genes for antigens into them. Having a large proportion of immunized individuals (>75%) in a population interrupts disease transmission, providing protection to unimmunized individuals. Vaccine developers and administrators monitor vaccine safety by checking for adverse reactions to vaccines, including mild irritation at the site of inoculation, anaphylactic shock, and the possibility of an attenuated vaccine reverting to virulence. Passive immunotherapy (a type of passive immunization) involves administration of an antiserum containing preformed antibodies. The fusion of myelomas (cancerous plasma cells) with plasma cells results in hybridomas, the source of monoclonal antibodies, which can be used in passive immunization. Health providers weigh the advantages and disadvantages of active versus passive immunization. Active immunization, in which patients synthesize their own antibodies and develop immunological memory, is longer lasting but relatively slow to develop. Passive immunity, in which preformed antibodies are administered, is fast acting but does not produce long-term effects. Serology is the study and use of immunological assays on blood serum to diagnose disease or identify antibodies or antigens. Scientists use antibodies to find an antigen in a specimen and use antigen to find antibodies. The simplest of the serological tests is a precipitation test, in which antigen and antibody meet in optimal proportions to form immune complexes, which are often insoluble. Automated light detectors can measure the cloudiness of a solution-an indication of the quantity of protein in the solution. Turbidimetry measures the passage of light through the solution, whereas nephelometry measures the amount of light reflected by protein in the solution. The amount of these antibodies, called a titer is measured by diluting the serum in a process called titration. Antibodies to viruses or toxins can be measured using a neutralization test, such as a viral neutralization test. Infection by viruses that naturally agglutinate red blood cells can be demonstrated using a viral hemagglutination inhibition test. The complement fixation test is a complex assay used to determine the presence of specific antibodies. Fluorescently labeled antibodies-those chemically linked to a fluorescent dye-can be used in a variety of direct fluorescent antibody tests and indirect fluorescent antibody tests. Which of the following is a good test to detect rabies virus in the brain of a dog Induces rapid onset of immunity Induces mainly an antibody response Induces good cell-mediated immunity Increases antigenicity Uses antigen fragments Uses attenuated microbes A. Compare the advantages and disadvantages of passive immunotherapy and active immunization. Is it ethical to approve the use of a vaccine that causes significant illness in 1% of patients if it protects immunized survivors against a serious disease Discuss the importance of costs and technical skill in selecting a practical serological test.

Most viral research and scientific study has focused on bacterial and animal viruses erectile dysfunction doctors augusta ga buy red viagra 200 mg with visa. Scientists have determined that the number of bacteriophages is more than all bacteria impotence herbs buy genuine red viagra on line, archaea erectile dysfunction pump how to use discount 200 mg red viagra, and eukaryotes put together erectile dysfunction gnc purchase generic red viagra on-line. We will return our attention to bacteriophages and animal viruses later in this chapter erectile dysfunction japan generic red viagra 200 mg with mastercard. Viruses of plants are less well known than bacterial and animal viruses erectile dysfunction tucson discount 200 mg red viagra fast delivery, even though viruses were first identified and isolated from tobacco plants. Plant viruses infect many food crops, including corn, beans, sugarcane, tobacco, and potatoes, resulting in billions of dollars in losses each year. Characteristics of Viruses 385 plants are introduced into plant cells either through abrasions of the cell wall or by plant parasites, such as nematodes and aphids. After entry, plant viruses follow the replication cycle discussed below for animal viruses. We do know that fungal viruses are different from animal and bacterial viruses in that fungal viruses appear to exist only within cells; that is, they seemingly have no extracellular state. However, because fusion of cells is typically a part of a fungal life cycle, viral infections can easily be propagated by the fusion of an infected fungal cell with an uninfected one. Sizes of Viruses In the late 1800s, scientists hypothesized that the cause of many diseases, including polio and smallpox, was an agent smaller than a bacterium. Even smaller viruses have diameters as small as 17 nm, while one of the larger viruses, Megavirus, is about 500 nm in diameter- about the diameter of many bacterial cells. He filtered the sap of infected tobacco plants through a porcelain filter fine enough to trap even the smallest of cells. Viruses, however, were not trapped but instead passed through the filter with the liquid, which remained infectious to tobacco plants. This experiment proved the existence of an acellular disease-causing entity smaller than a bacterium. Selected viruses are compared in size to a bacterium, Escherichia coli, and to a human red blood cell. The capsid of a virus is composed of proteinaceous subunits called capsomeres (or capsomers). Some capsomeres are composed of only a single type of protein, whereas others are composed of several different kinds of proteins. The capsid of a helical virus is composed of capsomeres that bond together in a circling fashion to form a tube around the nucleic acid. The capsid of a polyhedral virus is roughly spherical, with a shape similar to a geodesic dome. Complex viruses have capsids of many different shapes that do not readily fit into either of the other two categories. An example of a complex virus is smallpox virus, which has several covering layers (including lipid) and no easily identifiable capsid. The complex shapes of many bacteriophages include icosahedral heads, which contain the genome, attached to helical tails with tail fibers. All viruses lack cell membranes (after all, they are not cells), but some, particularly animal viruses, have an envelope similar in composition to a cell membrane surrounding their capsids. Other viral proteins called matrix proteins fill the region between capsid and envelope. An enveloped virus acquires its envelope from its host cell during viral replication or release (discussed shortly). Indeed, the envelope of a virus is a portion of the membrane system of a host cell. Like a cytoplasmic membrane, a viral envelope is composed of a phospholipid bilayer and proteins. The tubular shape of the capsid results from the tight arrangement of several rows of helical capsomeres. It includes an icosahedral head and an ornate tail that enables viral attachment and penetration. A viral envelope does not perform other physiological roles of a cytoplasmic membrane, such as endocytosis or active transport. An envelope provides some protection to the virus from the immune system; after all, enveloped viruses are carrying membrane from host cells and thus are more chemically similar to the host. However, membranes are more susceptible to detergents, alcohol, and drying out, so enveloped viruses are more fragile than naked ones. Naked viruses are more stable outside a host than are enveloped viruses, but the naked capsid exposes more viral proteins to the environment and are thus more susceptible to recognition and attack by the immune system once inside a host. An algal bloom off the coast of Seattle, cells, do have positive influences, including what appear to be extensive roles in the environment. Viruses, though normally pathogenic to their host Some important ways viruses affect our world: Scientists have found a previously unknown virus that attacks a tiny marine alga that forms algal "blooms" consisting of hundreds of thousands to millions of algal cells per milliliter of water, which are visible from space (see photo). Algal blooms like these can deplete the water of oxygen at night, potentially harming fish and other marine life. The newly discovered virus stops blooms by killing the alga, a result that is good for animal life. The resulting increase in cloudiness noticeably shades the ocean, measurably lowering water temperature. There are up to 10 million of these viruses in a single milliliter of seawater, so the scientists estimate that much of the oxygen we breathe may be attributable to the action of this virus on blue-green bacteria. So far, virologists have established families for all viral genera, but only seven viral orders are described. Taxonomists have not described kingdoms, divisions, and classes for viruses because the relationships among viruses are not well understood. Some researchers consider viruses to be a fourth domain of life (in addition to Bacteria, Archaea, and Eukarya). Family names are typically derived either from special characteristics of viruses within the family or from the name of an important member of the family. Family Herpesviridae is named for herpes simplex, a virus that can cause genital herpes. Specific epithets for viruses are their common English designations written in italics. Viral Replication As previously noted, viruses cannot reproduce themselves because they lack the genes for all the enzymes necessary for replication; in addition, they do not possess functional 1Pico means one-trillionth, 10 -12. Once a host cell falls under control of a viral genome, it is forced to replicate viral genetic material and translate viral proteins, including viral capsomeres and viral enzymes. The replication cycle of a virus usually results in the death and lysis of the host cell. Because the cell undergoes lysis near the end of the cycle, this type of replication is called a lytic replication cycle. In the following sections, we examine the events that occ ur in the replicat ion of bacteriophages and animal viruses. We begin with lytic replication in bacteriophages; Studies of phages revealed the basics of viral biology. Indeed, bacteriophages make excellent tools for the general study of viruses because bacteria are easier and less expensive to culture than animal or human cells. T4 virions are complex, having the polyhedral heads and helical tails seen in many bacteriophages. The circular bacterial chromosome is represented diagrammatically; in reality, it would be much longer. Attachment proteins on the tail fibers precisely fit to complementary receptor proteins on the surface of the cell wall of E. The specificity of the attachment proteins for the receptors ensures that the virus will attach only to E. Phage attachment can be so specific that a phage might infect only one particular strain of a bacterial species. Thus, scientists can use phage attachment for bacterial identification in the lab. Such capsid assembly is a spontaneous process, requiring little or no enzymatic activity. For many years, it was assumed that capsids form around a genome; however, research has shown that for some viruses, enzymes pump the genome into the assembled capsid under high pressure- five times that used in a paintball gun. This process resembles stuffing a strand of cooked spaghetti into a matchbox through a single small hole. The empty capsid, having performed its task, is left on the outside of the cell looking like an abandoned spacecraft. Newly assembled virions are released from the cell as lysozyme completes its work on the cell wall and the bacterium disintegrates. Areas of disintegrating bacterial cells in a lawn of bacteria in a Petri plate look as if the lawn were being eaten, and it was the appearance of these plaques that prompted early scientists to give the name bacteriophage, "bacterial eater," to these viruses. For phage T4, the process of lytic replication takes about 25 minutes and can produce as many as 100 to 200 new virions for each bacterial cell lysed. Scientists do not understand completely how phages are assembled inside a host cell, but it appears that as capsomeres accumulate within the cell, they spontaneously attach to one another to form new capsid heads. Likewise, tails Shown is virion abundance over time for a single lytic replication cycle. New virions are not observed in the culture medium until synthesis, assembly, and release (lysis) are complete, at which time (the burst time) the new virions are released all at once. In fact, half the infected with bacteria on Earth succumb to phages every two bacteriophages. Phage therapy was used in the early 1900s to combat dysentery, typhus, and cholera but was largely abandoned in the 1940s in the United States, eclipsed by the development of antibiotics, such as penicillin. Phage therapy continued in the Soviet Union and Eastern Europe, where research is still centered. A phage reproduces by inserting genetic material into a bacterium, causing the bacterium to build copies of the virus that burst out of the cell to infect other bacteria. Since each type of phage attacks a specific strain of bacteria, phage treatment is effective only if the phages are carefully matched to the disease-causing bacterium. Some bacteriophages have a modified replication cycle in which infected host cells grow and reproduce normally for many generations before they lyse. Here, we examine lysogenic replication as it occurs in a much-studied temperate phage, lambda phage, which is another parasite of E. A prophage remains inactive by coding for a protein that suppresses prophage genes. A side effect of this repressor protein is that it renders the bacterium resistant to additional infection by other viruses of the same type. Every time the cell replicates its infected chromosome, the prophage is also replicated 4. All daughter cells of a lysogenic cell are thus infected with the nearly inactive virus. A prophage and its descendants may remain a part of bacterial chromosomes for generations or even forever. Lysogenic phages can change the phenotype of a bacterium, for example, from a harmless form into a pathogen-a process called lysogenic conversion. Bacteriophage genes are responsible for toxins and other disease-evoking proteins found in the bacterial agents of diphtheria, cholera, rheumatic fever, and certain severe cases of diarrhea caused by E. At some later time, a prophage might be excised from the chromosome by recombination or some other genetic event; it then reenters the lytic phase. The process whereby a prophage is excised from the host chromosome is called induction 5. After induction, the lytic steps of synthesis 6, assembly 7, and release 8 resume from the point at which they stopped. The phage shown in this illustration is phage lambda, and the bacterium shown is E. However, there are significant differences in the replication of animal viruses that result in part from the presence of envelopes around some of the viruses and in part from the eukaryotic nature of animal cells and the lack of an animal cell wall. Unlike the bacteriophages we have examined, animal viruses lack both tails and tail fibers. Instead, animal viruses often have glycoprotein spikes on their capsids or envelopes. Even though entry of animal viruses is not as well understood as entry of bacteriophages, there are at least three different mechanisms: direct penetration, membrane fusion, and endocytosis. With other animal viruses, by contrast, the entire capsid and its contents enter a host cell by either membrane fusion or endocytosis. Most enveloped viruses and some naked viruses enter host cells by triggering endocytosis. For example, adenoviruses (naked) and herpesviruses (enveloped) enter human host cells via endocytosis. For a virus that penetrates a host cell with its capsid intact, the capsid must be removed to release the genome before replication of the virus can continue. Further, in synthesis of enveloped viruses, some viral proteins are inserted into cellular membranes. After penetration, many animal viruses must be uncoated, but bacteriophages need not be. The latter process, which is the reverse of normal transcription, is mediated by a viral enzyme, reverse transcriptase. The number of viruses produced and released depends on both the type of virus and the size and initial health of the host cell.

Using an arithmetic scale for the y-axis makes it difficult to ascertain actual numbers of cells near the beginning and impossible to plot points after only a short time drugs for erectile dysfunction purchase generic red viagra on line. Note that a plot of logarithmic population growth using a logarithmic scale produces a straight line impotence emedicine red viagra 200 mg fast delivery. Why do cells trail behind their optimum reproductive potential during the lag phase Eventually erectile dysfunction treatment rochester ny order red viagra canada, the bacteria synthesize the necessary chemicals for conducting metabolism in their new environment and then enter a phase of rapid chromosome replication long term erectile dysfunction treatment buy discount red viagra 200 mg line, growth erectile dysfunction pump in india buy 200mg red viagra with visa, and reproduction erectile dysfunction treatment by exercise order genuine red viagra online. Populations in log phase are more susceptible to antimicrobial drugs that interfere with metabolism, such as erythromycin, and to drugs that interfere with the formation of cell structures, such as the inhibition of cell wall synthesis by penicillin. Further, because the metabolic rate of individual cells is at a maximum during log phase, this phase may be preferred for industrial and laboratory purposes. For example, bacteria inoculated from a medium containing glucose as a carbon source into a medium containing lactose must synthesize two new types of proteins: membrane proteins to transport lactose into the cell and the enzyme lactase to catabolize lactose. The lag phase can last less than an hour or can last for days, depending on the species and the chemical and physical conditions of the medium. Over 40 athletes have been too sore to play sports, and 13 students have been hospitalized. Nurses take clinical samples of the drainage of the infections from hospitalized students, and medical laboratory scientists culture the samples. Stationary Phase If bacterial growth continued at the exponential rate of the log phase, bacteria would soon overwhelm the Earth. This does not occur because as nutrients are depleted and wastes accumulate, the rate of reproduction decreases. Eventually, the number of dying cells equals the number of cells being produced, and the size of the population remains constant-hence the name stationary phase. Cells in this phase are stress tolerant because they are adapted for challenging conditions, such as heat, high salt concentrations, and the presence of antibiotics. Death Phase If nutrients are not added and wastes are not removed, a population reaches a point at which cells die at a faster rate than Boils in the Locker Room convex, and golden. The bacterium is Gram-positive, mesophilic, and facultatively halophilic and can grow with or without oxygen. If the bacterium divides every 30 minutes under laboratory conditions, how many cells would there be in a colony after 24 hours Bear in mind that during the death phase, some cells remain alive and continue metabolizing and reproducing, but the number of dying cells exceeds the number of new cells produced so that eventually the population decreases to a fraction of its previous abundance. In some cases, all the cells die, whereas in others, a few survivors may remain indefinitely. The latter case is especially true for cultures of bacteria that can develop resting structures called endospores (see Chapter 3). Chemostats make possible the study of microbial population growth at steady but low nutrient levels, such as might be found in biofilms. Such studies are essential to understand interactions of microbial species in nature and can reveal aspects of microbial interactions that are not apparent in a closed system. Scientists also use chemostats to maintain log phase population growth for experimental inquiries into aspects of microbial metabolism, such as enzyme activities. Food and industrial microbiologists use chemostats to maintain constant production of useful microbial products. A chemostat is an open system; that is, fresh medium is continuously supplied while an equal amount of old medium (containing microbes) is removed. As nutrients enter the culture vessel of a chemostat, the cells can metabolize, grow, and reproduce-but only to the extent that a limiting nutrient. By controlling the amount of limiting nutrient entering the culture vessel, a chemostat maintains a culture in a particular phase, typically log phase. This is impossible in a closed system because as nutrients are depleted and wastes accumulate, the culture enters the stationary phase and then the death phase. Fresh medium with a limiting amount of a nutrient Sterile air or other gas Flow-rate regulator We have discussed the concepts of population growth and have seen that large numbers result from logarithmic growth, but we have not discussed practical methods of determining the size of a microbial population. Therefore, laboratory personnel must estimate the number of cells in a population by counting the number in a small, representative sample and then multiplying to estimate the number in the whole specimen. For example, if there are 25 cells in a microliter (ml) sample of urine, then there are approximately 25 million cells in a liter of urine. Estimating the number of microorganisms in a sample is useful for determining such things as the severity of urinary tract infections, the effectiveness of pasteurization and other methods of food preservation, the degree of fecal contamination of water supplies, and the usefulness of particular disinfectants and antibiotics. Microbiologists use either direct or indirect methods to estimate the number of cells. Culture vessel Direct Methods Not Requiring Incubation It is possible to directly count cells without having to incubate cultures. Microscopic Counts Microbiologists can count microorganisms directly through a microscope rather than inoculating them onto the surface of a solid medium. A microbial culture is continuously maintained in a predetermined phase, typically log phase, by controlling the amount and type of fresh medium added while removing an equal amount of old culture. The number of bacteria per milliliter (cm3) can be calculated as follows: mean number of bacteria per square * 25 squares = number of bacteria per 0. Direct microscopic counts are advantageous when there are more than 10 million cells per milliliter or when a speedy estimate of population size is required. However, direct counts can be problematic because it is often difficult to differentiate between living and dead cells and nearly impossible to count rapidly moving microbes. Electronic Counters A Coulter15 counter is a device that directly counts cells as they interrupt an electrical current flowing across a narrow tube held in front of an electronic detector. This device is useful for counting the larger cells of yeasts, unicellular algae, and protozoa; it is less useful for bacterial counts because of debris in the media and the presence of filaments and clumps of cells. A cytometer uses a light-sensitive detector to record changes in light transmission through the tube as cells pass. Scientists use this technique to distinguish among cells that have been differentially stained with fluorescent dyes or tagged with fluorescent antibodies. They can count bacteria in a solution and even count host cells that contain fluorescently stained intracellular parasites. Pipette Bacterial suspension Location of grid Overflow troughs (a) Place under oil immersion Bacterial suspension 1 mm 1 mm (b) Direct Methods Requiring Incubation Among the many direct techniques are techniques requiring incubation-viable plate counts following dilution, membrane filtration, and the most probable number method. Serial Dilution and Viable Plate Counts What if the number of cells in even a very small sample is still too great to count If, for example, a 1-ml sample of milk containing 20,000 bacterial cells per milliliter were plated on a Petri plate, there would be too many colonies to count. In such cases, microbiologists make a serial dilution, which is the stepwise dilution of a liquid culture in which the dilution factor at each step is constant. The scientists plate a set amount of each dilution onto an agar surface and count the number of colonies resulting on a plate from each dilution. A bacterial suspension placed next to the cover slip through a pipette moves under the cover slip and over the grid by capillary action. Each square millimeter of the grid has 25 large squares, each of which is divided into 16 small squares. The calculations involved in estimating the number of bacteria per milliliter (cm3) of suspension are described in the text. Growth of Microbial Populations 183 25 to 250 colonies and multiply the number on each plate by the reciprocal of the dilution used to inoculate that plate to estimate the number of bacteria per milliliter of the original culture. When a plate has fewer than 25 colonies, it is not used to estimate the number of bacteria in the original sample because the chance of underestimating the population increases when the number of colonies is small. In cases where a colonyforming unit consists of more than one cell, a viable plate count underestimates the number of cells present in the sample. The accuracy of a viable plate count also depends on the homogeneity of the dilutions, the ability of the bacteria to grow on the medium used, the number of cell deaths, and the growth phase of the sample population. Thoroughly mixing each dilution, inoculating multiple plates per dilution, and using log-phase cultures minimize errors. In this method, a large sample (perhaps as large as several liters) is poured (or drawn under a vacuum) through a membrane filter with pores small enough to trap the cells. The membrane is then transferred onto a solid medium, and the colonies present after incubation are counted. A researcher inoculates a set of test tubes of a broth medium with a sample of stream water. The researcher also inoculates a set of five tubes with a 1:10 dilution and another set of five tubes with a 1:100 dilution of stream water. After incubation for 48 hours, the researcher counts the number of test tubes in each set that show growth. This generates three numbers in this example-growth occurs in four of the undiluted broth tubes, two of the 1:10 tubes, and in only 1 ml of original culture 1 ml 1 ml 1 ml 1 ml 9 ml of broth + 1 ml of original culture (a) 0. If the results were 5, 3, 1, what would be the most probable number of microorganisms in the original broth The most probable number method is useful for counting microorganisms that do not grow on solid media, when bacterial counts are required routinely, and when samples of wastewater, drinking water, and food samples contain too few organisms to use a viable plate count. The microbial population is estimated by multiplying the number of colonies counted by the volume of sample filtered. Indirect Methods It is not always necessary to count microorganisms to estimate population size or density. The greater the concentration of bacteria within a broth, the more light will be absorbed and scattered, and the less light will pass through and strike a light-sensitive detector. Generally, transmission is inversely proportional to the population size; that is, the larger the population grows, the less light will reach the detector. Scales on the gauge of a spectrophotometer report percentage of transmission and absorbance. These are two ways of looking at the same things; for example, 25% transmission is the same thing as 75% absorbance. Direct counts must be calibrated with transmission and absorbance readings to provide estimates of population size. Once these values are determined, spectrophotometry provides estimates of population size more quickly than any direct method. The benefits of measuring turbidity to estimate population growth include ease of use and speed. After a light beam is passed through an uninoculated sample of the culture medium, the scale is set at 100% transmission. In an inoculated sample, the microbial cells absorb and scatter light, reducing the amount reaching the detector. The percentage of light transmitted is inversely proportional to population density. Further, the technique is accurate only if the cells are suspended uniformly in the medium. If they form either a pellicle (a film of cells at the surface) or a sediment (an accumulation of cells at the bottom), their number will be underestimated. Metabolic Activity Under standard temperature conditions, the rate at which a population of cells utilizes nutrients and produces wastes depends on their number. Dry Weight the abundance of some microorganisms, particularly filamentous microorganisms, is difficult to measure by direct methods. Instead, these organisms are filtered from their culture medium, dried, and weighed. The dry weight method is suitable for broth cultures, but growth cannot be followed over time because the organisms are killed during the process. Molecular Methods the majority of bacteria and archaea have not been grown in the laboratory, and representatives of most species are too few in number to study by direct observation. For example, one study estimated that a single gram of garden soil contains more than 100 billion bacteria and archaea, representing more than 10 million different species. He tells her that he hears a crackling sound in her lungs and that her oxygen levels are slightly lower than normal. Wyatt orders a chest X-ray exam and blood work, and he obtains a sputum (mixture of saliva and mucus) sample from Kalinda by having her cough deeply. This also explains the crackling in her lungs, the pain in her chest, her difficulty in breathing, and the lower oxygen saturation. He adds that, based on her medical history, he thinks her sore throat and cough have progressed to pneumonia. Although they need to wait for the blood and sputum test results to come back from the lab in order to make a definitive diagnosis, Dr. He also prescribes a steroid inhaler to help open up her airways and make breathing easier. He tells her to continue taking the antibiotics and to schedule a follow-up appointment. Her cough tapers off and eventually disappears, and by the time of her follow-up appointment, her right lung is totally clear, and her lung sounds and oxygen levels have returned to normal. Sputum samples are typically plated on blood agar, chocolate agar (blood agar containing lysed red blood cells), and MacConkey agar. Explain why each of these media may be helpful in identifying pathogens in lower respiratory infections. Check your answers to Micro in the Clinic Follow-Up questions in the MasteringMicrobiology Study Area. Visit the MasteringMicrobiology Study Area to challenge your understanding with practice tests, animation quizzes, and clinical case studies! A colony, which is a visible population of microorganisms arising from a single cell or colony-forming unit living in one place, grows in size as the number of cells increases. Most microbes live as biofilms, that is, in association with one another on surfaces. Chemical nutrients such as carbon, hydrogen, oxygen, and nitrogen are required for the growth of microbial populations.

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