# Phenotypic Identification of Anaerobes ## Quick Answer - Phenotypic identification of anaerobes relies on observing growth characteristics, biochemical reactions, and metabolic products under strictly oxygen-free conditions. - The practical first step is proper specimen collection and transport in anaerobic containers, followed by culture on prereduced anaerobically sterilized (PRAS) media. - A key limitation is that many anaerobes show weak or variable biochemical reactivity, so phenotypic results often require confirmation through additional methods. ## Specimen Collection and Transport for Anaerobic Culture The success of phenotypic identification begins before the organism reaches the laboratory. Anaerobic bacteria die rapidly when exposed to oxygen, so the way a specimen is collected, transported, and processed determines whether any identification attempt can succeed. ### Appropriate Specimen Types Anaerobic culture is clinically useful only when the specimen is collected from a site that is normally sterile or where anaerobes are the expected pathogens. Acceptable specimens include aspirated pus, tissue biopsies, blood, pleural fluid, peritoneal fluid, joint fluid, and deep wound specimens collected with a syringe and needle. These specimens must be transported in a manner that excludes oxygen. Unacceptable specimens include swabs from surfaces that are normally colonized with anaerobes, such as throat swabs, sputum, stool, vaginal swabs, and skin swabs. These sites contain a complex mixture of anaerobic and aerobic organisms, and culture results from such specimens are difficult to interpret. The presence of anaerobes in these samples does not indicate infection. ### Transport Systems The time between collection and culture inoculation is critical. Anaerobic transport systems contain a reduced environment that maintains viability of anaerobic organisms. These systems typically include a tube or vial with an oxygen-free gas phase and a reducing agent in the transport medium. Specimens should be placed into the transport system immediately after collection and delivered to the laboratory without delay. For blood cultures, the blood is inoculated directly into culture bottles designed to support anaerobic growth. These bottles contain a reduced medium and an anaerobic gas atmosphere. The laboratory should be notified when anaerobic infection is suspected so that the appropriate incubation conditions are used. ### Laboratory Processing Time The laboratory should process anaerobic specimens as soon as they arrive. Delays in processing allow oxygen to diffuse into the specimen and kill fastidious anaerobes. If processing cannot be done immediately, the specimen should be held in an anaerobic environment or in the transport system until it is ready for inoculation. ## Principles of Anaerobic Culture Anaerobic bacteria require an environment with no oxygen for growth. The laboratory must create and maintain this environment throughout the culture and identification process. ### Oxygen Toxicity and Reducing Agents Oxygen is toxic to anaerobic bacteria because these organisms lack the full complement of enzymes needed to neutralize reactive oxygen species. Superoxide dismutase and catalase are present in varying amounts among anaerobes, and some species are more tolerant of oxygen than others. The most oxygen-sensitive organisms, such as those in the genus Fusobacterium, require strict anaerobic conditions. Reducing agents are added to culture media to lower the oxidation-reduction potential of the medium. These agents include cysteine, thioglycolate, and sodium thioglycolate. The reducing agents help maintain a low redox potential that supports the growth of anaerobes. ### Prereduced Anaerobically Sterilized Media PRAS media are prepared and stored under anaerobic conditions. The media are boiled to remove dissolved oxygen, then cooled and sterilized under a stream of oxygen-free gas. The media are stored in an anaerobic environment until they are used. PRAS media are the standard for anaerobic culture because they provide the low redox potential that anaerobes require. The use of PRAS media is a critical step in the phenotypic identification of anaerobes. The media must be used within the expiration date and stored according to the manufacturer instructions. The media should be inspected for signs of oxidation, such as a color change in the indicator, before use. ### Anaerobic Chambers and Jars The laboratory uses an anaerobic chamber or an anaerobic jar to maintain the oxygen-free environment during culture and identification. An anaerobic chamber is a sealed glove box that contains a gas mixture of nitrogen, hydrogen, and carbon dioxide. The chamber has a catalyst that removes oxygen from the atmosphere. The chamber allows the laboratory to manipulate cultures without exposing them to oxygen. An anaerobic jar is a simpler system that uses a gas-generating envelope to create an anaerobic atmosphere. The jar is sealed and the envelope is activated to produce hydrogen and carbon dioxide. The hydrogen reacts with oxygen in the presence of a catalyst to form water, which removes the oxygen from the jar. The choice between a chamber and a jar depends on the workload and the laboratory resources. A chamber is more convenient for handling multiple specimens and for performing biochemical tests. A jar is less expensive and is suitable for smaller workloads. ## Phenotypic Characteristics Used for Identification Phenotypic identification of anaerobes is based on a combination of characteristics that are observed after the organism is grown in pure culture. These characteristics include colony morphology, Gram stain reaction, and biochemical reactions. ### Colony Morphology The appearance of the colony on the agar plate is the first phenotypic characteristic that is observed. The colony size, shape, color, and hemolysis pattern are recorded. The colony morphology can provide a preliminary indication of the genus of the organism. For example, colonies of Bacteroides fragilis are typically gray, smooth, and non-hemolytic. Colonies of Fusobacterium necrophorum are often gray and have a distinctive odor. The colony morphology is not sufficient for a definitive identification, but it helps to narrow the possibilities. ### Gram Stain Reaction The Gram stain is a fundamental test in the identification of anaerobes. The Gram reaction and the morphology of the organism are recorded. The Gram stain is performed on a smear from the colony, and the result is used to select the appropriate biochemical tests. The Gram reaction of anaerobes is variable. Some anaerobes are Gram-positive, such as Clostridium species and Peptostreptococcus species. Others are Gram-negative, such as Bacteroides species and Fusobacterium species. The Gram stain also reveals the shape of the organism, such as rods, cocci, or filaments. ### Growth Characteristics The growth of the organism on different media is also used for identification. The ability to grow on selective media, such as bile esculin agar or kanamycin vancomycin laked blood agar, is recorded. The growth in the presence of bile and the ability to hydrolyze esculin are used to differentiate between genera. The growth of the organism in the presence of oxygen is also tested. An aerotolerance test is performed by subculturing the organism to a plate that is incubated in the presence of oxygen. The organism is considered an obligate anaerobe if it does not grow in the presence of oxygen. ## Biochemical Tests for Anaerobe Identification Biochemical tests are used to identify the genus and species of an anaerobic organism. These tests are based on the ability of the organism to use specific substrates and to produce specific metabolic products. ### Carbohydrate Fermentation Tests Carbohydrate fermentation tests are used to determine the ability of the organism to ferment a specific carbohydrate. The tests are performed in a medium that contains the carbohydrate and a pH indicator. The organism is inoculated into the medium and incubated under anaerobic conditions. The production of acid is detected by a color change in the indicator. The fermentation of glucose, lactose, sucrose, and other carbohydrates is used to differentiate between species. For example, the ability to ferment glucose is used to differentiate between Bacteroides fragilis and other Bacteroides species. ### Enzymatic Tests Enzymatic tests are used to detect the presence of specific enzymes in the organism. These tests are based on the hydrolysis of a substrate that is linked to a chromogenic or fluorogenic compound. The enzyme is detected by the release of the compound, which produces a color or a fluorescent signal. The tests for the enzymes catalase, indole, and urease are commonly used. The catalase test is used to detect the presence of the enzyme catalase, which breaks down hydrogen peroxide. The indole test is used to detect the production of indole from tryptophan. The urease test is used to detect the ability of the organism to hydrolyze urea. ### Preformed Enzyme Tests Preformed enzyme tests are used to detect the presence of enzymes that are already present in the organism. These tests are performed using a substrate that is linked to a chromogenic or fluorogenic compound. The test is performed by adding the substrate to a suspension of the organism and observing the reaction. The preformed enzyme tests are used to identify anaerobes that are difficult to identify by other methods. The tests are available in commercial kits that contain a panel of substrates. The results of the tests are used to generate a profile that is compared to a database for identification. ## Fatty Acid Analysis for Anaerobe Identification Fatty acid analysis is a phenotypic method that is used to identify anaerobes based on the composition of their cellular fatty acids. The method is also known as fatty acid methyl ester (FAME) analysis. ### Principle of Fatty Acid Analysis The cellular fatty acids of bacteria are extracted and converted to methyl esters. The methyl esters are then separated and identified by gas chromatography. The fatty acid profile of the organism is compared to a database of profiles for known species. The fatty acid profile is a stable characteristic of the organism and is used to identify the organism to the genus and species level. The method is particularly useful for the identification of anaerobes that are difficult to identify by biochemical tests. ### Procedure for Fatty Acid Analysis The procedure for fatty acid analysis involves the following steps. The organism is grown on a plate under anaerobic conditions. The cells are harvested and the fatty acids are extracted using a saponification reagent. The fatty acids are then methylated and the methyl esters are extracted into an organic solvent. The methyl esters are analyzed by gas chromatography. The chromatogram is compared to the database of fatty acid profiles. The identification is based on the similarity of the profile to the profiles in the database. ### Limitations of Fatty Acid Analysis Fatty acid analysis requires specialized equipment and expertise. The method is not available in all laboratories. The method is also affected by the growth conditions of the organism. The fatty acid profile can vary with the growth medium and the incubation time. The database of fatty acid profiles is not complete for all species of anaerobes. The identification of an organism that is not in the database may be difficult. The method is best used in combination with other phenotypic methods. ## Commercial Identification Systems Commercial identification systems are available for the identification of anaerobes. These systems use a combination of biochemical tests and are designed to be easy to use and to provide a rapid identification. ### Types of Commercial Systems The commercial systems are available in the form of panels or strips that contain a set of biochemical tests. The organism is inoculated into the panel and the panel is incubated. The reactions are read and the results are used to generate a profile. The profile is compared to a database to identify the organism. The commercial systems are available from several manufacturers. The systems are designed to be used with a standardized inoculum and a standardized incubation time. The systems are used in the clinical laboratory for the identification of anaerobes. ### Use of Commercial Systems The commercial systems are used according to the manufacturer instructions. The organism is grown on a plate of media and the colonies are harvested. The inoculum is prepared in a standardized suspension. The suspension is used to inoculate the panel or strip. The panel or strip is incubated for the recommended time. The reactions are read and the profile is generated. The profile is compared to the database to identify the organism. The identification is reported to the genus and species level. ### Limitations of Commercial Systems The commercial systems have limitations. The systems are not able to identify all species of anaerobes. The systems may not be able to identify organisms that are not in the database. The systems may also be affected by the growth conditions of the organism. The commercial systems are used in combination with other methods. The results of the commercial systems are confirmed by the other methods, such as the Gram stain and the colony morphology. ## Quality Control and Quality Assurance Quality control and quality assurance are essential for the accurate identification of anaerobes. The laboratory must have a quality control program that ensures the accuracy and reliability of the test results. ### Quality Control of Media The media used for the culture of anaerobes must be quality controlled. The media is tested with the control organisms to ensure that the media supports the growth of the anaerobes and that the media is not contaminated. The quality control of the media is performed by the manufacturer and by the laboratory. The laboratory tests the media with the control organisms to ensure that the media is working correctly. The results of the quality control are recorded. ### Quality Control of the Identification Systems The identification systems are quality controlled with the control organisms. The control organisms are used to ensure that the system is working correctly. The results of the quality control are recorded. The quality control of the identification systems is performed according to the manufacturer instructions. The control organisms are selected to represent the range of organisms that the system is designed to identify. ### Biosafety The laboratory must follow the biosafety guidelines for the handling of anaerobic organisms. The organisms are handled in a biosafety cabinet to prevent the exposure of the laboratory personnel to the organisms. The biosafety guidelines are provided by the Centers for Disease Control and Prevention and the National Institutes of Health. The guidelines are used to ensure the safety of the laboratory personnel and the environment. ## Common Failure Patterns and Troubleshooting ### Failure to Grow The most common failure in the identification of anaerobes is the failure of the organism to grow. The failure to grow is caused by the exposure of the organism to oxygen during the collection, transport, or culture. The failure to grow is also caused by the use of the wrong media or the wrong incubation conditions. The media must be PRAS and the incubation must be in an anaerobic environment. ### Contamination of the Culture The contamination of the culture is a common problem. The contamination is caused by the introduction of other organisms into the culture. The contamination is prevented by the use of aseptic technique and the use of a biosafety cabinet. ### Inaccurate Identification The inaccurate identification is caused by the use of the wrong tests or the wrong interpretation of the results. The identification is confirmed by the use of multiple methods. ## Professional Escalation Criteria The laboratory professional should escalate the identification of an anaerobe to a higher level of expertise when the identification is difficult or when the results are inconsistent. The escalation is appropriate when the organism is not identified by the routine methods. The escalation is also appropriate when the organism is a potential pathogen and the identification is important for the treatment of the patient. The escalation is performed by sending the organism to a reference laboratory for further testing. The reference laboratory has the expertise and the equipment to identify the organism. ## At a Glance | Characteristic | Observation | Interpretation | | --- | --- | --- | | Colony morphology | Gray, smooth, non-hemolytic | Suggests Bacteroides species | | Gram stain | Gram-negative rods | Suggests Bacteroides or Fusobacterium | | Growth in oxygen | No growth | Obligate anaerobe | | Fermentation of glucose | Acid produced | Positive for glucose fermentation | | Catalase test | Bubbles produced | Catalase positive | ## A Practical Decision Framework for Anaerobe Identification Workups Phenotypic identification of anaerobes often stalls not because the laboratory lacks the right media or tests, but because the workflow lacks a structured decision framework. Technologists may perform tests in an order that wastes time, use the wrong tests for a given Gram reaction, or interpret results without a clear path to escalation. This section provides a practical decision framework that guides the user from the first observation of a colony to a final identification or a reasoned referral to a reference laboratory. ### The Two-Phase Workflow Model The framework divides the identification process into two distinct phases. The first phase is the preliminary characterization phase. The second phase is the definitive identification phase. This separation prevents the common error of running a full biochemical panel before the organism has been properly categorized. #### Phase One: Preliminary Characterization The preliminary phase answers four questions. What is the Gram reaction and morphology? Does the organism grow in the presence of oxygen? What does the colony look like on the primary isolation plate? What is the organism's behavior on selective media? These four observations are made within the first 24 to 48 hours after the primary culture is read. The results of these observations determine which identification pathway the laboratory will follow. The preliminary phase is not intended to produce a species identification. It is intended to narrow the possibilities to a genus or a small group of genera. The Gram stain is the single most important observation in the preliminary phase. The Gram reaction and the cell morphology are recorded together. For example, a Gram-positive rod that produces spores is almost certainly a Clostridium species. A Gram-negative rod that grows in the presence of bile is likely a Bacteroides species. A Gram-positive coccus in chains is likely a Peptostreptococcus species. The aerotolerance test is the second most important observation. The test is performed by subculturing the organism to two plates. One plate is incubated in an anaerobic environment. The other plate is incubated in the presence of oxygen. The organism is considered an obligate anaerobe if it grows only on the anaerobic plate. The organism is considered a facultative anaerobe if it grows on both plates. The organism is considered an aerobe if it grows only on the aerobic plate. The aerotolerance test is performed on every organism that is suspected to be an anaerobe. The colony morphology is recorded from the primary isolation plate. The colony size, shape, color, and hemolysis pattern are noted. The colony morphology is used to select the appropriate media for the definitive identification phase. The growth on selective media is recorded. The ability to grow on bile esculin agar, kanamycin vancomycin laked blood agar, and other selective media is used to differentiate between genera. The growth on these media is recorded as positive or negative. #### Phase Two: Definitive Identification The definitive phase is entered after the preliminary characterization is complete. The definitive phase uses the results of the preliminary phase to select the appropriate biochemical tests or the appropriate commercial identification system. The definitive phase is performed in a logical order. The first step is to select the appropriate identification system. The second step is to inoculate the system and incubate it under the appropriate conditions. The third step is to read the results and generate a profile. The fourth step is to compare the profile to the database and report the identification. The definitive phase is not a single test. It is a combination of tests that are selected based on the preliminary results. The selection of the tests is the most important decision in the definitive phase. ### The Decision Tree for Gram-Positive Anaerobes The decision tree for Gram-positive anaerobes is based on the morphology of the organism and the presence or absence of spores. #### Gram-Positive Spore-Forming Rods The Gram-positive spore-forming rods are almost always Clostridium species. The identification of Clostridium species is based on the fermentation of carbohydrates, the production of lipase and lecithinase, and the presence of specific enzymes. The first test is the lecithinase test. The organism is inoculated onto an egg yolk agar plate. The plate is incubated anaerobically. The production of a precipitate around the colony indicates the presence of lecithinase. The lecithinase test is positive for Clostridium perfringens and negative for Clostridium tetani. The second test is the lipase test. The organism is inoculated onto an egg yolk agar plate. The plate is incubated anaerobically. The production of an iridescent sheen on the surface of the colony indicates the presence of lipase. The lipase test is positive for Clostridium sporogenes and negative for Clostridium perfringens. The third test is the fermentation of glucose, lactose, and sucrose. The organism is inoculated into a PRAS carbohydrate fermentation broth. The broth is incubated anaerobically. The production of acid is detected by a color change in the pH indicator. The fourth test is the indole test. The organism is inoculated into a PRAS tryptone broth. The broth is incubated anaerobically. The production of indole is detected by the addition of Kovac reagent. #### Gram-Positive Non-Spore-Forming Rods The Gram-positive non-spore-forming rods are a diverse group that includes the genera Actinomyces, Bifidobacterium, Eubacterium, Lactobacillus, and Propionibacterium. The identification of these organisms is determined by the fermentation of carbohydrates, the production of lactic acid, and the presence of specific enzymes. The first test is the catalase test. The organism is mixed with hydrogen peroxide. The production of bubbles indicates the presence of catalase. The catalase test is positive for Propionibacterium and negative for the other genera. The second test is the fermentation of glucose. The organism is inoculated into a PRAS broth containing glucose. The broth is incubated anaerobically. The production of acid is detected by the color change in the broth. The third test is the production of indole. The organism is inoculated into a PRAS tryptone broth. The broth is incubated anaerobically. The production of indole is detected by the addition of Kovac reagent. #### Gram-Positive Cocci The Gram-positive cocci are a small group that includes the genera Peptostreptococcus, Peptococcus, and Finegoldia. The identification of these organisms is determined by the fermentation of carbohydrates and the production of specific enzymes. The first test is the catalase test. The catalase test is negative for the Gram-positive cocci. The second test is the fermentation of glucose. The organism is inoculated into a glucose broth. The broth is incubated anaerobically. The production of acid is indicated by the color change in the broth. The third test is the production of indole. The indole test is positive for Peptostreptococcus indolicus and negative for the other species. ### The Decision Tree for Gram-Negative Anaerobes The decision tree for Gram-negative anaerobes is based on the morphology of the organism and the growth in the presence of bile. #### Gram-Negative Rods That Grow in the Presence of Bile The Gram-negative rods that grow in the presence of bile are the Bacteroides fragilis group and the genus Bilophila. The identification of these organisms is determined by the fermentation of carbohydrates, the hydrolysis of esculin, and the production of specific enzymes. The first test is the growth on bile esculin agar. The organism is inoculated onto a bile esculin agar plate. The plate is incubated anaerobically. The hydrolysis of esculin is indicated by the blackening of the medium. The second test is the fermentation of glucose. The organism is inoculated into a glucose broth. The broth is incubated anaerobically. The production of acid is indicated by the color change in the broth. The third test is the fermentation of lactose. The organism is inoculated into a lactose broth. The broth is incubated anaerobically. The production of acid is indicated by the color change in the broth. #### Gram-Negative Rods That Do Not Grow in the Presence of Bile The Gram-negative rods that do not grow in the presence of bile are the Fusobacterium species and the Porphyromonas species. The identification of these organisms is determined by the fermentation of carbohydrates, the production of butyric acid, and the production of specific enzymes. The first test is the growth on bile esculin agar. The organism is inoculated onto a bile esculin agar plate. The plate is incubated anaerobically. The organism does not grow on the plate. The second test is the fermentation of glucose. The organism is inoculated into a glucose broth. The broth is incubated anaerobically. The production of acid is indicated by the color change in the broth. The third test is the production of indole. The organism is inoculated into a PRAS tryptone broth. The broth is incubated anaerobically. The production of indole is detected by the addition of Kovac reagent. #### Gram-Negative Cocci The Gram-negative cocci are a small group that includes the genus Veillonella. The identification of these organisms is determined by the fermentation of carbohydrates and the production of specific enzymes. The first test is the catalase test. The catalase test is negative for Veillonella. The second test is the fermentation of glucose. The organism is inoculated into a glucose broth. The broth is incubated anaerobically. The production of acid is indicated by the color change in the broth. The third test is the reduction of nitrate. The organism is inoculated into a nitrate broth. The broth is incubated anaerobically. The reduction of nitrate is detected by the addition of the nitrate reagents. ### The Record System for Anaerobic Identification A structured record system is essential for the accurate identification of anaerobes. The record system should capture the results of the preliminary characterization and the definitive identification. The record system should also capture the quality control results and the interpretation of the results. #### The Work Sheet The work sheet is the primary record for the identification of an anaerobe. The work sheet should include the following fields: - The specimen identification number - The date of collection - The date of culture - The date of identification - The Gram stain result - The colony morphology - The aerotolerance result - The growth on selective media - The results of the biochemical tests - The identification result - The interpretation of the result The work sheet is completed by the technologist who performs the identification. The work sheet is reviewed by the supervisor before the result is reported. #### The Quality Control Log The quality control log is a record of the quality control results for the media and the identification systems. The log should include the following fields: - The date of the quality control - The lot number of the media or the system - The control organism used - The expected result - The observed result - The interpretation of the result The quality control log is completed by the technologist who performs the quality control. The log is reviewed by the supervisor on a regular basis. #### The Interpretation Record The interpretation record is a record of the interpretation of the results. The record should include the following fields: - The results of the preliminary characterization - The results of the definitive identification - The interpretation of the results - The confidence level of the identification - The need for further testing The interpretation record is completed by the technologist who performs the identification. The record is reviewed by the supervisor before the result is reported. ### Common Failure Patterns in the Decision Framework The decision framework is designed to reduce the number of failures in the identification of anaerobes. However, failures can still occur. The most common failures are described below. #### Failure to Perform the Aerotolerance Test The aerotolerance test is the most commonly skipped test in the identification of anaerobes. The test is skipped because the organism is assumed to be an anaerobe based on the source of the specimen. The failure to perform the aerotolerance test can lead to the misidentification of a facultative anaerobe as an obligate anaerobe. The aerotolerance test should be performed on every organism that is suspected to be an anaerobe. The test is simple and inexpensive. The test is performed by subculturing the organism to two plates. One plate is incubated in an anaerobic environment. The other plate is incubated in the presence of oxygen. #### Failure to Use the Correct Media The use of the wrong media is a common cause of failure. The media must be PRAS media. The media must be stored under anaerobic conditions. The media must be used within the expiration date. The failure to use the correct media is caused by the use of media that has been exposed to oxygen. The media is exposed to oxygen when the media is stored in the wrong conditions or when the media is used after the expiration date. #### Failure to Interpret the Results Correctly The failure to interpret the results correctly is a common cause of failure. The failure is caused by the use of the wrong interpretation criteria. The failure is also caused by the use of the wrong database for the interpretation. The interpretation of the results should be performed by a technologist who is experienced in the identification of anaerobes. The interpretation should be reviewed by a supervisor before the result is reported. ### Professional Escalation Criteria The decision framework includes a clear path for escalation. The escalation is appropriate when the organism is not identified by the routine methods. The escalation is also appropriate when the organism is a potential pathogen and the identification is important for the treatment of the patient. The escalation is performed by sending the organism to a reference laboratory for further testing. The reference laboratory has the expertise and the equipment to identify the organism. The reference laboratory may use molecular methods, such as 16S rRNA gene sequencing, to identify the organism. The escalation is also appropriate when the results of the identification are inconsistent. The inconsistency is caused by the use of the wrong tests or the wrong interpretation of the results. The escalation is performed by repeating the identification with a different method. ### The Role of the Decision Framework in the Laboratory The decision framework is a practical tool that is used in the laboratory to guide the identification of anaerobes. The framework is used by the technologist to select the appropriate tests and to interpret the results. The framework is also used by the supervisor to review the results and to ensure the accuracy of the identification. The decision framework is not a substitute for the experience of the technologist. The framework is a guide that is used to ensure that the identification is performed in a logical and consistent manner. The framework is also used to ensure that the identification is performed in a timely manner. The decision framework is a valuable tool for the laboratory. The framework is used to reduce the number of failures and to improve the accuracy of the identification. The framework is also used to ensure that the identification is performed in a safe and efficient manner. The decision framework is a practical tool that is used in the laboratory to guide the identification of anaerobes. The framework is used by the technologist to select the appropriate tests and to interpret the results. The framework is also used by the supervisor to review the results and to ensure the accuracy of the identification. The framework is a guide that is used to ensure that the identification is performed in a logical and consistent manner. ## Frequently Asked Questions ### What is the most important step in the identification of anaerobes? The most important step is the collection and transport of the specimen. The specimen must be collected from a site that is normally sterile and transported in an anaerobic transport system to prevent the exposure of the organism to oxygen. ### What is the difference between an anaerobic chamber and an anaerobic jar? An anaerobic chamber is a large glove box that allows the manipulation of the cultures in an oxygen-free environment. An anaerobic jar is a small container that uses a gas-generating packet to create an anaerobic atmosphere. ### What is the role of the Gram stain in the identification of anaerobes? The Gram stain is used to determine the Gram reaction and the morphology of the organism. The Gram stain is used to guide the selection of the biochemical tests. ### What is the fatty acid analysis used for? Fatty acid analysis is used to identify anaerobes based on the composition of their cellular fatty acids. The method is used for the identification of anaerobes that are difficult to identify by other methods. ### What are the limitations of the commercial identification systems? The commercial systems are not able to identify all species of anaerobes. The systems may not be able to identify organisms that are not in the database. ### What is the role of the quality control in the identification of anaerobes? The quality control is used to ensure that the media and the identification systems are working correctly. The quality control is performed with the control organisms. ### When should the identification of an anaerobe be escalated? The identification should be escalated when the organism is not identified by the routine methods or when the organism is a potential pathogen and the identification is not for the treatment of the patient. ### What is the biosafety level for the handling of anaerobes? The biosafety level for the handling of anaerobes is determined by the risk assessment. The organisms are handled in a biosafety cabinet to prevent the exposure of the laboratory personnel. ## Using the Evidence | Source | Best use in this topic | Important limitation | |---|---|---| | [Biosafety in Microbiological and Biomedical Laboratories](https://www.cdc.gov/labs/bmbl/index.html) | official guidance | Check the linked page for current local requirements | | [Microbiology Resources](https://asm.org/) | official guidance | Check the linked page for current local requirements | | [Antimicrobial Resistance](https://www.who.int/health-topics/antimicrobial-resistance) | official guidance | Check the linked page for current local requirements | ## Related Diagnostic Guides - [Microbial Identification Workflows: From Phenotypic to Molecular Methods](/knowledge/diagnostics/molecular/microbial-identification-workflows-from-phenotypic-to-molecular-methods) - [Cell Culture Media: A Guide to Selection and Optimization](/knowledge/diagnostics/microbiology/cell-culture-media-a-guide-to-selection-and-optimization) - [Microbiology in Water Testing: Methods and Significance](/knowledge/diagnostics/microbiology/microbiology-in-water-testing-methods-and-significance) - [Clinical Microbiology: From Specimen Collection to Pathogen Identification](/knowledge/diagnostics/microbiology/clinical-microbiology-from-specimen-collection-to-pathogen-identification) - [Biochemical Tests in Microbiology: A Practical Overview](/knowledge/diagnostics/microbiology/biochemical-tests-in-microbiology-a-practical-overview) ## References and Further Reading - [Biosafety in Microbiological and Biomedical Laboratories](https://www.cdc.gov/labs/bmbl/index.html). CDC and NIH. - [Microbiology Resources](https://asm.org/). American Society for Microbiology. - [Antimicrobial Resistance](https://www.who.int/health-topics/antimicrobial-resistance). World Health Organization. - [Bacteriological Analytical Manual](https://www.fda.gov/food/laboratory-methods-food/bacteriological-analytical-manual-bam). U.S. Food and Drug Administration. - [Antimicrobial Susceptibility Testing](https://clsi.org/standards/products/microbiology). Clinical and Laboratory Standards Institute. - [Culture Collections](https://www.atcc.org/). American Type Culture Collection. - [Laboratory Biosafety Manual](https://www.who.int/publications/i/item/9789240011311). World Health Organization. - [NCBI Literature Resources](https://www.ncbi.nlm.nih.gov/). National Center for Biotechnology Information. - [A metabolomics pipeline for the mechanistic interrogation of the gut microbiome.](https://pubmed.ncbi.nlm.nih.gov/34262212). Nature, 2021. - [Comparison of genotypic and phenotypic methods for species identification of coagulase-negative staphylococcal isolates from blood cultures.](https://pubmed.ncbi.nlm.nih.gov/7833006). APMIS : acta pathologica, microbiologica, et immunologica Scandinavica, 1994. - [Gram-positive anaerobic cocci--commensals and opportunistic pathogens.](https://pubmed.ncbi.nlm.nih.gov/23030831). FEMS microbiology reviews, 2013. > This article is educational and does not replace validated laboratory procedures, institutional biosafety review, manufacturer instructions, or professional interpretation.