For example, the Centers for Disease Control and Prevention CDC reference laboratory uses at least 46 tests to identify members of the Enterobacteriaceae, whereas most clinical laboratories, using commercial identification kits or simple rapid tests, identify isolates with far fewer criteria. Particularly for epidemiological purposes, clinical microbiologists must distinguish strains with particular traits from other strains in the same species.
For example, serotype OH7 E. Below the species level, strains are designated as groups or types on the basis of common serologic or biochemical reactions, phage or bacteriocin sensitivity, pathogenicity, or other characteristics.
Many of these characteristics are already used and accepted: serotype, phage type, colicin type, biotype, bioserotype a group of strains from the same species with common biochemical and serologic characteristics that set them apart from other members of the species , and pathotype e.
In addition to species and subspecies designations, clinical microbiologists must be familiar with genera and families. A genus is a group of related species, and a family is a group of related genera. An ideal genus would be composed of species with similar phenotypic and phylogenetic characteristics. Some phenotypically homogeneous genera approach this criterion Citrobacter , Yersinia , and Serratia. More often, however, the phenotypic similarity is present, but the genetic relatedness is not.
Bacillus , Clostridium , and Legionella are examples of accepted phenotypic genera in which genetic relatedness between species is not 50 to 65 percent, but 0 to 65 percent. When phenotypic and genetic similarity are not both present, phenotypic similarity generally should be given priority in establishing genera.
Identification practices are simplified by having the most phenotypically similar species in the same genus. The primary consideration for a genus is that it contain biochemically similar species that are convenient or important to consider as a group separate from other groups of organisms. The sequencing of ribosomal RNA rRNA genes, which have been highly conserved through evolution, allows phylogenetic comparisons to be made between species whose total DNAs are essentially unrelated.
It also allows phylogenetic classification at the genus, family, and higher taxonomic levels. The rRNA sequence data are usually not used to designate genera or families unless supported by similarities in phenotypic tests. Species are named according to principles and rules of nomenclature set forth in the Bacteriological Code. Scientific names are taken from Latin or Greek. The correct name of a species or higher taxon is determined by three criteria: valid publication, legitimacy of the name with regard to the rules of nomenclature, and priority of publication that is, it must be the first validly published name for the taxon.
To be published validly, a new species proposal must contain the species name, a description of the species, and the designation of a type strain for the species, and the name must be published in the International Journal for Systematic Bacteriology IJSB. Once proposed, a name does not go through a formal process to be accepted officially; in fact, the opposite is true—a validly published name is assumed to be correct unless and until it is challenged officially. This occurs only in cases in which the validity of a name is questioned with respect to compliance with the rules of the Bacteriological Code.
A question of classification that is based on scientific data for example, whether a species, on the basis of its biochemical or genetic characteristics, or both, should be placed in a new genus or an existing genus is not settled by the Judicial Commission, but by the preference and usage of the scientific community. More than one name may thus exist for a single organism. This is not, however, restricted to bacterial nomenclature.
Multiple names exist for many antibiotics and other drugs and enzymes. A number of genera have been divided into additional genera and species have been moved to new or existing genera, such as Arcobacter new genus for former members of Campylobacter and Burkholderia species formerly species of Pseudomonas.
Two former Campylobacter species cinaedi and fennelliae have been moved to the existing genus Helicobacter in another example. The best source of information for new species proposals and nomenclatural changes is the IJSB. In addition, the Journal of Clinical Microbiology often publishes descriptions of newly described microorganisms isolated from clinical sources. Information, including biochemical reactions and sources of isolation, about new organisms of clinical importance, disease outbreaks caused by newer species, and reviews of clinical significance of certain organisms may be found in the Annals of Internal Medicine , Journal of Infectious Diseases, Clinical Microbiology Reviews , and Clinical Infectious Diseases.
The data provided in these publications supplement and update Bergey's Manual of Systematic Bacteriology , the definitive taxonomic reference text. Since , the number of genera in the family Enterobacteriaceae has increased from 12 to 28 and the number of species from 42 to more than , some of which have not yet been named. Similar explosions have occurred in other genera. In , five species were listed in the genus Vibrio and four in Campylobacter ; the genus Legionella was unknown.
Today, there are at least 25 species in Vibrio , 12 Campylobacter species, and more than 40 species in Legionella. The total numbers of genera and species continue to increase dramatically. The clinical significance of the agent of legionnaire's disease was well known long before it was isolated, characterized, and classified as Legionella pneumophila. In most cases, little is known about the clinical significance of a new species at the time it is first described.
Assessments of clinical significance begin after clinical laboratories adopt the procedures needed to detect and identify the species and accumulate a body of data. In fact, the detection and even the identification of uncultivatable microbes from different environments are now possible using standard molecular methods.
The agents of cat scratch disease Bartonella henselae and Whipple's disease Tropheryma whippelii were elucidated in this manner. Bartonella henselae has since been cultured from several body sites from numerous patients; T. New species will continue to be described. Many will be able to infect humans and cause disease, especially in those individuals who are immunocompromised, burned, postsurgical, geriatric, and suffering from acquired immunodeficiency syndrome AIDS.
Any organism is capable of causing disease in such patients under the appropriate conditions. Clinical laboratory scientists should be able to isolate, identify, and determine the antimicrobial susceptibility pattern of the vast majority of human disease agents so that physicians can initiate appropriate treatment as soon as possible, and the source and means of transmission of outbreaks can be ascertained to control the disease and prevent its recurrence.
The need to identify clinically relevant microorganisms both quickly and cost-effectively presents a considerable challenge. To be effective, the professional clinical laboratory staff must interact with the infectious diseases staff. Laboratory scientists should attend infectious disease rounds.
They must keep abreast of new technology, equipment, and classification and should communicate this information to their medical colleagues.
They should interpret, qualify, or explain laboratory reports. If a bacterial name is changed or a new species reported, the laboratory should provide background information, including a reference. The clinical laboratory must be efficient. A concerted effort must be made to eliminate or minimize inappropriate and contaminated specimens and the performance of procedures with little or no clinical relevance.
Standards for the selection, collection, and transport of specimens should be developed for both laboratory and nursing procedure manuals and reviewed periodically by a committee composed of medical, nursing, and laboratory staff.
Ongoing dialogues and continuous communication with other health care workers concerning topics such as specimen collection, test selection, results interpretation, and new technology are essential to maintaining high quality microbiological services. Most laboratories today use either commercially available miniaturized biochemical test systems or automated instruments for biochemical tests and for susceptibility testing.
The kits usually contain 10 to 20 tests. The test results are converted to numerical biochemical profiles that are identified by using a codebook or a computer. Carbon source utilization systems with up to 95 tests are also available. Most identification takes 4 to 24 hours. Biochemical and enzymatic test systems for which data bases have not been developed are used by some reference laboratories. Automated instruments can be used to identify most Gram-negative fermenters, nonfermenters, and Gram-positive bacteria, but not for anaerobes.
Antimicrobial susceptibility testing can be performed for some microorganisms with this equipment, with results expressed as approximate minimum inhibitory drug concentrations. Both tasks take 4 to 24 hours. If semiautomated instruments are used, some manipulation is done manually, and the cultures in miniature cards or microdilution plates are incubated outside of the instrument.
The test containers are then read rapidly by the instrument, and the results are generated automatically. Instruments are also available for identification of bacteria by cell wall fatty acid profiles generated with gas-liquid chromatography GLC , analysis of mycolic acids using high performance liquid chromatography HPLC , and by protein-banding patterns generated by polyacrylamide gel electrophoresis PAGE.
Some other instruments designed to speed laboratory diagnosis of bacteria are those that detect but do not identify bacteria in blood cultures, usually faster than manual systems because of continuous monitoring.
Also available are many rapid screening systems for detecting one or a series of specific bacteria, including certain streptococci, N. These screening systems are based on fluorescent antibody, agglutination, or other rapid procedures.
It is important to inform physicians as soon as a presumptive identification of an etiologic agent is obtained so that appropriate therapy can be initiated as quickly as possible.
Gram stain and colony morphology; acid-fast stains; and spot indole, oxidase, and other rapid enzymatic tests may allow presumptive identification of an isolate within minutes. Despite recent advances, the armamentarium of the clinical laboratory is far from complete. Few laboratories can or should conduct the specialized tests that are often essential to distinguish virulent from avirulent strains.
Serotyping is done only for a few species, and phage typing only rarely. Few pathogenicity tests are performed. Not many laboratories can conduct comprehensive biochemical tests on strains that cannot be identified readily by commercially available biochemical systems. Even fewer laboratories are equipped to perform plasmid profiles, gene probes, or DNA hybridization. These and other specialized tests for the serologic or biochemical identification of some exotic bacteria, yeasts, molds, protozoans, and viruses are best done in regional reference laboratories.
It is not cost-effective for smaller laboratories to store and control the quality of reagents and media for tests that are seldom run or quite complex. In addition, it is impossible to maintain proficiency when tests are performed rarely.
Sensitive methods for the epidemiologic subtyping of isolates from disease outbreaks, such as electrophoretic enzyme typing, rRNA fingerprinting, whole-cell protein electrophoretic patterns, and restriction endonuclease analysis of whole-cell or plasmid DNA, are used only in reference laboratories and a few large medical centers.
Specific genetic probes are now available commercially for identifying virulence factors and many bacteria and viruses. Genetic probes are among the most common methods used for identification of Mycobacterium tuberculosis and M. Probes for Neisseria gonorrhoeae and Chlamydia trachomatis are now being used directly on clinical specimens with excellent sensitivity and almost universal specificity with same-day results.
Mycobacterial probes are also being evaluated for direct specimen testing. Hospital and local clinical laboratories interact with district, state, and federal public health laboratories in several important ways Fig.
The clinical laboratories participate in quality control and proficiency testing programs that are conducted by federally regulated agencies. The government reference laboratories supply cultures and often reagents for use in quality control, and they conduct training programs for clinical laboratory personnel.
Pathways for laboratory identification of pathogens and information exchange. All types of laboratories should interact closely to provide diagnostic services and epidemic surveillance. The primary concern of the clinical laboratory is identifying infectious disease agents and studying nosocomial and local outbreaks of disease.
When the situation warrants, the local laboratory may ask the state laboratory for help in identifying an unusual organism, discovering the cause or mode of transmission in a disease outbreak, or performing specialized tests not done routinely in clinical laboratories. Cultures should be pure and should be sent on appropriate media following appropriate procedures for transport of biohazardous materials. Pertinent information, including the type of specimen; patient name or number , date of birth, and sex; clinical diagnosis, associated illness, date of onset, and present condition; specific agent suspected, and any other organisms isolated; relevant epidemiologic and clinical data; treatment of patient; previous laboratory results biochemical or serologic tests ; and necessary information about the submitting party must accompany each request.
These data allow the state laboratory to test the specimen properly and quickly, and they provide information about occurrences within the state. For example, a food-borne outbreak might extend to many parts of the state or beyond its boundaries. The state laboratory can alert local physicians to the possibility of such outbreaks.
Another necessary interaction between local and state laboratories is the reporting of notifiable diseases by the local laboratory. The state laboratory makes available to local laboratories summaries of the incidence of these diseases. The state laboratories also submit the summaries to the CDC weekly or, for some diseases, yearly , and national summaries are published weekly in the Morbidity and Mortality Weekly Report. Interaction between the CDC and state and federal laboratories is very similar to that between local and state laboratories.
The CDC provides quality control cultures and reagents to state laboratories, and serves as a national reference laboratory for diagnostic services and epidemiologic surveillance. Local laboratories, however, must initially send specimens to the local or state public health laboratory, which, when necessary, forwards them to the CDC. The CDC reports its results back to the state laboratory, which then reports to the local laboratory.
Clinical laboratory personnel, including support and clerical employees, are subject to the risk of infection, chemical hazards, and, in some laboratories, radioactive contamination. Such risks can be prevented or minimized by a laboratory safety program. Personnel who work with radioactive materials should have taken a radioactivity safety course; they should wear radiation monitor badges and be aware of the methods for decontaminating hands, clothing, work surfaces, and equipment.
They should wear gloves when working with radioactive compounds. When they work with high-level radiation, they should use a hood and stand behind a radiation shield. Preparative radioactive work should be done in a separate room with access only by personnel who are involved directly in the work. Chemicals can harm laboratory personnel through inhalation or skin absorption of volatile compounds; bodily contact with carcinogens, acids, bases, and other harmful chemicals; or introduction of poisonous or skin-damaging liquids into the mouth.
See Also Pregnancy Reproductive Health. Find an STD testing site near you. ZIP Code:. Links with this icon indicate that you are leaving the CDC website. Linking to a non-federal website does not constitute an endorsement by CDC or any of its employees of the sponsors or the information and products presented on the website. You will be subject to the destination website's privacy policy when you follow the link. Esculetin reacts with ferric citrate in the medium , forming a phenolic iron complex which turns the entire slant dark brown to black.
The tube on the far right was inoculated with E. The tube in the center was inoculated with a bilie esculin negative organism and the tube on the left was uninoculated. It tests the ability of an organism to do several things: reduce sulfur, produce indole and swim through the agar be motile.
SIM is commonly used to differentiate members of Enterobacteriaceae. Sulfur can be reduced to H 2 S hydrogen sulfide either by catabolism of the amino acid cysteine by the enzyme cysteine desulfurase or by reduction of thiosulfate in anaerobic respiration. If hydrogen sulfide is produced, a black color forms in the medium.
Proteus mirabilis is positive for H 2 S production. The organism pictured on the far left is positive for hydrogen sulfide production. Bacteria that have the enzyme tryptophanase, can convert the amino acid, tryptophane to indole. Escherichia coli is indole positive.
The organism pictured second from left is E. SIM tubes are inoculated with a single stab to the bottom of the tube. If an organism is motile than the growth will radiate from the stab mark and make the entire tube appear turbid. Pseudomonas aeruginosa and the strain of Proteus mirabilis that we work with are motile. It also allows for identification of sulfur reducers. This media is commonly used to separate lactose fermenting members of the family Enterobacteriaceae e.
Escherichia coli from members that do not ferment lactose, like Shigella dysenteriae. These lactose nonfermenting enterics generally tend to be the more serious pathogens of the the gastrointestinal tract. The first differential ingredient, glucose, is in very short supply.
Organisms capable of fermenting this sugar will use it up within the first few hours of incubation. Glucose fermentation will create acidic byproducts that will turn the phenol red indicator in the media yelllow. Thus, after the first few hours of incubation, the tube will be entirely yellow.
At this point, when the glucose has been all used up, the organism must choose another food source. If the organism can ferment lactose, this is the sugar it will choose. Lactose fermentation will continue to produce acidic byproducts and the media will remain yellow picture on the far left below.
If gas is produced as a result of glucose or lactose fermentation, then fissures will appear in the agar or the agar will be lifted off the bottom of the tube. The deamination of the amino acids creates NH 3 , a weak base, which causes the medium to become alkaline. The alkaline pH causes the phenol red indicator to begin to turn red.
Since the incubation time is short h , only the slant has a chance to turn red and not the entire tube. Thus an organism that can ferment glucose but not lactose, will produce a red slant and a yellow butt in a KIA tube second from the left below. These organisms are the more serious pathogens of the GIT such as Shigella dysenteriae.
The slant of the tube will be red and the color of the butt will remain unchanged picture on the far right below. Pseudomonas aeruginosa is an example of a nonfermenter. KIA tubes are also capable of detecting the production of H 2 S. It is seen as a black precipitate second picture from the right. Sometimes the black precipitate obscures the butt of the tube. In such cases, the organisms should be considered positive for glucose fermentation yellow butt. Proteus mirabilis pictured here, second from right is a glucose positive, lactose negative, sulfur reducing enteric.
It is used to determine if an organism is capable of reducing nitrate NO 3 - to nitrite NO 2 - or other nitrogenous compounds via the action of the enzyme nitratase also called nitrate reductase. This test is important in the identification of both Gram-positive and Gram-negative species.
After incubation, these tubes are first inspected for the presence of gas in the Durham tube. In the case of nonfermenters, this is indicative of reduction of nitrate to nitrogen gas.
However, in many cases gas is produced by fermentation and further testing is necessary to determine if reduction of nitrate has occurred. This further testing includes the addition of sulfanilic acid often called nitrate I and dimethyl-alpha-napthalamine nitrate II.
If nitrite is present in the media, then it will react with nitrate I and nitrate II to form a red compound. This is considered a positive result. If no red color forms upon addition of nitrate I and II, this indicates that either the NO 3 - has not been converted to NO 2 - a negative result , or that NO 3 - was converted to NO 2 - and then immediately reduced to some other, undetectable form of nitrogen also a positive result.
In order to determine which of the preceding is the case, elemental zinc is added to the broth. If no color change occurs upon addition of zinc then this means that the NO 3 - was converted to NO 2 - and then was converted to some other undetectable form of nitrogen a positive result. If the nitrate broth turns red tubes pictured in the center after nitrate I and nitrate II are added, this color indicates a positive result.
If instead, the tube turns red tube pictured on the left after the addition of Zn, this indicates a negative result. If there is no color change in the tube after the addition of nitrate I and nitrate II, the result is uncertain. If the tube is colorless picture on the right after the addition of Zn this indicates a positive test. This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies hydrogen peroxide by breaking it down into water and oxygen gas.
The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result. The sample on the right below is catalase positive. The Staphylococcus spp. The Streptococcus and Enterococcus spp. This test is used to identify microorganisms containing the enzyme cytochrome oxidase important in the electron transport chain. It is commonly used to distinguish between oxidase negative Enterobacteriaceae and oxidase positive Pseudomadaceae.
Cytochrome oxidase transfers electrons from the electron transport chain to oxygen the final electron acceptor and reduces it to water. In the oxidase test, artificial electron donors and acceptors are provided.
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