techniques involve the culture of infectious agents isolated from a patient. culture allows identification of infectious organisms by examining their microscopic features, by detecting the presence of substances produced by pathogens, and by directly identifying an organism by its genotype. other techniques (such as x-rays, cat scans, pet scans or nmr) are used to produce images of internal abnormalities resulting from the growth of an infectious agent. the images are useful in detection of, for example, a bone abscess or a spongiform encephalopathy produced by a prion. symptomatic diagnostics the diagnosis is aided by the presenting symptoms in any individual with an infectious disease, yet it usually needs additional diagnostic techniques to confirm the suspicion. some signs are specifically characteristic and indicative of a disease and are called pathognomonic signs; but these are rare. not all infections are symptomatic. in children the presence of cyanosis, rapid breathing, poor peripheral perfusion, or a petechial rash increases the risk of a serious infection by greater than 5 fold. other important indicators include parental concern, clinical instinct, and temperature greater than 40 °c. microbial culture four nutrient agar plates growing colonies of common gram negative bacteria many diagnostic approaches depend on microbiological culture to isolate a pathogen from the appropriate clinical specimen. in a microbial culture, a growth medium is provided for a specific agent. a sample taken from potentially diseased tissue or fluid is then tested for the presence of an infectious agent able to grow within that medium. many pathogenic bacteria are easily grown on nutrient agar, a form of solid medium that supplies carbohydrates and proteins necessary for growth, along with copious amounts of water. a single bacterium will grow into a visible mound on the surface of the plate called a colony, which may be separated from other colonies or melded together into a "lawn". the size, color, shape and form of a colony is characteristic of the bacterial species, its specific genetic makeup (its strain), and the environment that supports its growth. other ingredients are often added to the plate to aid in identification. plates may contain substances that permit the growth of some bacteria and not others, or that change color in response to certain bacteria and not others. bacteriological plates such as these are commonly used in the clinical identification of infectious bacterium. microbial culture may also be used in the identification of viruses: the medium, in this case, being cells grown in culture that the virus can infect, and then alter or kill. in the case of viral identification, a region of dead cells results from viral growth, and is called a "plaque". eukaryotic parasites may also be grown in culture as a means of identifying a particular agent. in the absence of suitable plate culture techniques, some microbes require culture within live animals. bacteria such as mycobacterium leprae and treponema pallidum can be grown in animals, although serological and microscopic techniques make the use of live animals unnecessary. viruses are also usually identified using alternatives to growth in culture or animals. some viruses may be grown in embryonated eggs. another useful identification method is xenodiagnosis, or the use of a vector to support the growth of an infectious agent. chagas disease is the most significant example, because it is difficult to directly demonstrate the presence of the causative agent, trypanosoma cruzi in a patient, which therefore makes it difficult to definitively make a diagnosis. in this case, xenodiagnosis involves the use of the vector of the chagas agent t. cruzi, an uninfected triatomine bug, which takes a blood meal from a person suspected of having been infected. the bug is later inspected for growth of t. cruzi within its gut. microscopy another principal tool in the diagnosis of infectious disease is microscopy. virtually all of the culture techniques discussed above rely, at some point, on microscopic examination for definitive identification of the infectious agent. microscopy may be carried out with simple instruments, such as the compound light microscope, or with instruments as complex as an electron microscope. samples obtained from patients may be viewed directly under the light microscope, and can often rapidly lead to identification. microscopy is often also used in conjunction with biochemical staining techniques, and can be made exquisitely specific when used in combination with antibody based techniques. for example, the use of antibodies made artificially fluorescent (fluorescently labeled antibodies) can be directed to bind to and identify a specific antigens present on a pathogen. a fluorescence microscope is then used to detect fluorescently labeled antibodies bound to internalized antigens within clinical samples or cultured cells. this technique is especially useful in the diagnosis of viral diseases, where the light microscope is incapable of identifying a virus directly. other microscopic procedures may also aid in identifying infectious agents. almost all cells readily stain with a number of basic dyes due to the electrostatic attraction between negatively charged cellular molecules and the positive charge on the dye. a cell is normally transparent under a microscope, and using a stain increases the contrast of a cell with its background. staining a cell with a dye such as giemsa stain or crystal violet allows a microscopist to describe its size, shape, internal and external components and its associations with other cells. the response of bacteria to different staining procedures is used in the taxonomic classification of microbes as well. two methods, the gram stain and the acid-fast stain, are the standard approaches used to classify bacteria and to diagnosis of disease. the gram stain identifies the bacterial groups bacillota and actinomycetota, both of which contain many significant human pathogens. the acid-fast staining procedure identifies the actinomycetota genera mycobacterium and nocardia. biochemical tests biochemical tests used in the identification of infectious agents include the detection of metabolic or enzymatic products characteristic of a particular infectious agent. since bacteria ferment carbohydrates in patterns characteristic of their genus and species, the detection of fermentation products is commonly used in bacterial identification. acids, alcohols and gases are usually detected in these tests when bacteria are grown in selective liquid or solid media. the isolation of enzymes from infected tissue can also provide the basis of a biochemical diagnosis of an infectious disease. for example, humans can make neither rna replicases nor reverse transcriptase, and the presence of these enzymes are characteristic., of specific types of viral infections. the ability of the viral protein hemagglutinin to bind red blood cells together into a detectable matrix may also be characterized as a biochemical test for viral infection, although strictly speaking hemagglutinin is not an enzyme and has no metabolic function. serological methods are highly sensitive, specific and often extremely rapid tests used to identify microorganisms. these tests are based upon the ability of an antibody to bind specifically to an antigen. the antigen, usually a protein or carbohydrate made by an infectious agent, is bound by the antibody. this binding then sets off a chain of events that can be visibly obvious in various ways, dependent upon the test. for example, "strep throat" is often diagnosed within minutes, and is based on the appearance of antigens made by the causative agent, s. pyogenes, that is retrieved from a patient's throat with a cotton swab. serological tests, if available, are usually the preferred route of identification, however the tests are costly to develop and the reagents used in the test often require refrigeration. some serological methods are extremely costly, although when commonly used, such as with the "strep test", they can be inexpensive. complex serological techniques have been developed into what are known as immunoassays. immunoassays can use the basic antibody – antigen binding as the basis to produce an electro-magnetic or particle radiation signal, which can be detected by some form of instrumentation. signal of unknowns can be compared to that of standards allowing quantitation of the target antigen. to aid in the diagnosis of infectious diseases, immunoassays can detect or measure antigens from either infectious agents or proteins generated by an infected organism in response to a foreign agent. for example, immunoassay a may detect the presence of a surface protein from a virus particle. immunoassay b on the other hand may detect or measure antibodies produced by an organism's immune system that are made to neutralize and allow the destruction of the virus. instrumentation can be used to read extremely small signals created by secondary reactions linked to the antibody – antigen binding. instrumentation can control sampling, reagent use, reaction times, signal detection, calculation of results, and data management to yield a cost-effective automated process for diagnosis of infectious disease. pcr-based diagnostics nucleic acid testing conducted using an abbott laboratories id now device technologies based upon the polymerase chain reaction (pcr) method will become nearly ubiquitous gold standards of diagnostics of the near future, for several reasons. first, the catalog of infectious agents has grown to the point that virtually all of the significant infectious agents of the human population have been identified. second, an infectious agent must grow within the human body to cause disease; essentially it must amplify its own nucleic acids in order to cause a disease. this amplification of nucleic acid in infected tissue offers an opportunity to detect the infectious agent by using pcr. third, the essential tools for directing pcr, primers, are derived from the genomes of infectious agents, and with time those genomes will be known, if they are not already. thus, the technological ability to detect any infectious agent rapidly and specifically are currently available. the only remaining blockades to the use of pcr as a standard tool of diagnosis are in its cost and application, neither of which is insurmountable. the diagnosis of a few diseases will not benefit from the development of pcr methods, such as some of the clostridial diseases (tetanus and botulism). these diseases are fundamentally biological poisonings by relatively small numbers of infectious bacteria that produce extremely potent neurotoxins. a significant proliferation of the infectious agent does not occur, this limits the ability of pcr to detect the presence of any bacteria. metagenomic sequencing given the wide range of bacterial, viral, fungal, protozoal, and helminthic pathogens that cause debilitating and life-threatening illnesses, the ability to quickly identify the cause of infection is important yet often challenging. for example, more than half of cases of encephalitis, a severe illness affecting the brain, remain undiagnosed, despite extensive testing using the standard of care (microbiological culture) and state-of-the-art clinical laboratory methods. metagenomic sequencing-based diagnostic tests are currently being developed for clinical use and show promise as a sensitive, specific, and rapid way to diagnose infection using a single all-encompassing test. this test is similar to current pcr tests; however, an untargeted whole genome amplification is used rather than primers for a specific infectious agent. this amplification step is followed by next-generation sequencing or third-generation sequencing, alignment comparisons, and taxonomic classification using large databases of thousands of pathogen and commensal reference genomes. simultaneously, antimicrobial resistance genes within pathogen and plasmid genomes are sequenced and aligned to the taxonomically classified pathogen genomes to generate an antimicrobial resistance profile – analogous to antibiotic sensitivity testing – to facilitate antimicrobial stewardship and allow for the optimization of treatment using the most effective drugs for a patient's infection. metagenomic sequencing could prove especially useful for diagnosis when the patient is immunocompromised. an ever-wider array of infectious agents can cause serious harm to individuals with immunosuppression, so clinical screening must often be broader. additionally, the expression of symptoms is often atypical, making a clinical diagnosis based on presentation more difficult. thirdly, diagnostic methods that rely on the detection of antibodies are more likely to fail. a rapid, sensitive, specific, and untargeted test for all known human pathogens that detects the presence of the organism's dna rather than antibodies is therefore highly desirable. indication of tests a temporary drive-in testing site for covid-19 set up with tents in a parking lot there is usually an indication for a specific identification of an infectious agent only when such identification can aid in the treatment or prevention of the disease, or to advance knowledge of the course of an illness prior to the development of effective therapeutic or preventative measures. for example, in the early 1980s, prior to the appearance of azt for the treatment