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Premium member Presentation Transcript Anaerobic bacteria and Basic culture methods : Anaerobic bacteria and Basic culture methods Dr.T.V.Rao MD Dr.T.V.Rao MD 1What Are Anaerobic Microorganisms: What Are Anaerobic Microorganisms Anaerobic microorganisms are widespread and very important Do not require oxygen for growth - often extremely toxic Dr.T.V.Rao MD 2Defining Anaerobes: Defining Anaerobes Facultative anaerobes - can grow in the presence or absence of oxygen Obtain energy by both respiration and fermentation Oxygen not toxic, some use nitrate (NO 3 - ) or sulphate (SO 4 2- ) as a terminal electron acceptor under anaerobic conditions Dr.T.V.Rao MD 3Strict Anaerobic Bacteria: Strict Anaerobic Bacteria Obligate (strict) anaerobes - oxygen is toxic to these organisms, do not use oxygen as terminal electron acceptor. Archaea such as methanogens and Bacteria, e.g Clostridia, Bacteriodes etc. etc. Dr.T.V.Rao MD 4Oxygen Toxicity: Oxygen Toxicity Oxygen is used by aerobic and facultatively anaerobic organisms as its strong oxidising ability makes it an excellent electron acceptor During the stepwise reduction of oxygen, which takes place in respiration toxic and highly reactive intermediates are produced reactive oxygen species (ROS ). Dr.T.V.Rao MD 5The Requirements for Growth: Related to Oxygen : The Requirements for Growth: Related to Oxygen Oxygen (O 2 ) Table 6.1 Dr.T.V.Rao MD 6Anaerobic and Aerobic Respiration: Anaerobic and Aerobic Respiration Reaction name Reduct. Oxid. Reaction Stoichiometry kcal/mol Aerobic Respiration CHO O 2 C 6 H 12 O 6 + 6O 2 ==> 6CO 2 + 6H 2 O 686 Nitrate Reduction CHO NO 3 - CHO + NO 3 - + H + ==> CO 2 + N 2 + H 2 O 649 Sulfate Reduction CHO SO 4 2- 2CHO + SO 4 2- +2H + => 2CO 2 + H+ 2H 2 O 190 Methanogenesis CHO or H 2 CO 2 4H 2 + CO 2 ==> CH 4 + 2H 2 O 8.3 Dr.T.V.Rao MD 7ROS production during respiration: ROS production during respiration O 2 + e - => O 2 - superoxide anion O 2 - + e - + 2H + => H 2 O 2 hydrogen peroxide H 2 O 2 + e - + H + => H 2 O + OH . Hydroxyl radical OH . + e - + H + => H 2 O water Dr.T.V.Rao MD 8Chemical Dynamics in Anaerobic Bacteria: Chemical Dynamics in Anaerobic Bacteria Organisms that use O 2 have developed defence mechanisms to protect themselves from these toxic forms of oxygen - enzymes Catalase: H 2 O 2 + H 2 O 2 => 2H 2 O + O 2 Peroxidase: H 2 O 2 + NADH + H + => 2H 2 O + NAD + Superoxide dismutase: O 2 - + O 2 - + 2H + => H 2 O 2 + O 2 Dr.T.V.Rao MD 9Anaerobic environments exist in Nature too: Anaerobic environments exist in Nature too Anaerobic environments (low reduction potential) include: Sediments of lakes, rivers and oceans; bogs, marshes, flooded soils, intestinal tract of animals; oral cavity of animals, deep underground areas, e.g. oil packets and some aquifers Anaerobes also important in some infections, e.g. C. tetanii and C. perfringens important in deep puncture wound infections Dr.T.V.Rao MD 10Anaerobic Environments: Anaerobic Environments Dr.T.V.Rao MD 11 Anaerobes and Oxygen: A naerobes and Oxygen Anaerobes generate energy by fermentation Lack the capacity to utilize O2 as a terminal hydrogen acceptor Some are sensitive to O2 concentration as low as 0.5% O2 Most can survive in 3%-5% O2 A few can grow poorly in the presence of air aero tolerant anaerobes Many are members of the normal flora created by presence of facultative anaerobes Dr.T.V.Rao MD 12FACTORS THAT INHIBIT THE GROWTH OF ANAEROBES BY OXYGEN: FACTORS THAT INHIBIT THE GROWTH OF ANAEROBES BY OXYGEN 1. Toxic compounds are produced e.g. H2O2 , Superoxide's 2. Absence of catalase & Superoxide dismutase 3. Oxidation of essential sulfhydryl groups in enzymes without sufficient reducing power to regenerate them Dr.T.V.Rao MD 13ANAEROBES OF CLINICAL IMPORTANCE: ANAEROBES OF CLINICAL IMPORTANCE CLOSTRIDIA C tetani; C perfringens; C difficile; C botulinum BACTEROIDES B fragilis; Prevotella Porphyromonas ACTINOMYCES FUSOBACTERIUM ANAEROBIC STREPTOCOCCI Dr.T.V.Rao MD 14Sites and Infection produced by Anaerobes : Sites and Infection produced by Anaerobes Dr.T.V.Rao MD 15Slide 16: Dr.T.V.Rao MD 16Anaerobic Bacteria of Medical Interest: Anaerobic Bacteria of Medical Interest MORPHOLOGY GRAM STAIN GENUS Spore forming (+) Clostridium Non-spore forming bacilli (+) (-) Actinomycetes, Bifidobacterium,Eubacte-rium,Propionibacerium, Mobilncus,Lactobacillus Bacteroides,Fusobacterium Prevotella,Porphyromonas Non-sporefoming cocci (+) (-) Peptococcus, Pepto-streptococcus Streptococcus Veilonella Dr.T.V.Rao MD 17Gram-positive anaerobes: Gram-positive anaerobes Actinomyces (head, neck, pelvic infections; aspiration pneumonia) Bifid bacterium (ear infections, abdominal infections) Clostridium (gas, gangrene, food poisoning, tetanus, pseudomembranous colitis) Peptostreptococcus (oral, respiratory, and intra-abdominal infections) Propionibacterium (shunt infections) Dr.T.V.Rao MD 18Gram-negative anaerobes: Gram-negative anaerobes Bactericides (the most commonly found anaerobes in cultures; intra-abdominal infections, rectal abscesses, soft tissue infections, liver infection) Fusobacterium (abscesses, wound infections, pulmonary and intracranial infections) Porphyromonas (aspiration pneumonia, periodontitis) Prevotella (intra-abdominal infections, soft tissue infections) Dr.T.V.Rao MD 19ANAEROBIC GRAM NEGATIVE BACILLI: ANAEROBIC GRAM NEGATIVE BACILLI Bactericides, Prevotolla, Porphyromonas and Fusobacterium Present in GI tract -form large component of normal flora >80% of human infections associated with B fragilis virulence factors - capsule, LPS, agglutinins and enzymes Clinical - Endogenous infections Intra-abdominal pyogenic infections pleuro-pulmonary infections genital infection Dr.T.V.Rao MD 20FACTORS RESPONSIBLE FOR THEIR VIRULENCE: FACTORS RESPONSIBLE FOR THEIR VIRULENCE 1 . Lipopolysaccharide - promotes abscess formation, enhanced coagulation 2. Polysaccharide capsule - correlated with abscess production 3. Enzymes a. Collagenase b. Heparinase * develop thrombophlebitis & septic emboli 4. Short chained fatty acids a. Butyrate- seen in dental plaque b. succinic acid – reduces phagocytic killing Dr.T.V.Rao MD 21CLINICAL MANIFESTATION: CLINICAL MANIFESTATION Clinical hints 1. odor 2. tissue 3. location 4. necrotic tissue 5. endocarditis with (-) blood culture 6. infection associated with malignancy 7. black discoloration 8. blood containing exudates 9. associated with sulfur granules 10. Bacteremic feature with jaundice 11. human bites Dr.T.V.Rao MD 22Common Human Anaerobic Infections: Common Human Anaerobic Infections Dr.T.V.Rao MD 23CLOSTRIDIA: CLOSTRIDIA Gram positive spore forming bacilli ubiquitous intestines of man and animals animal and human faeces contaminated soil and water Several species associated with human disease Dr.T.V.Rao MD 24Clostridium perfringens: Clostridium perfringens Large rectangular Gram positive bacillus Spores seldom seen in vivo or in vitro non motile Produces several toxins alpha (lecithinase), beta, epsilon ...... enterotoxin Causes a spectrum of human diseases Bacteraemia Myonecrosis food poisoning enteritis necrotica (pig bel) Dr.T.V.Rao MD 25Clostridium tetani: Clostridium tetani Small motile spore forming gram positive bacillus with round terminal spores Causes tetanus Pathogenesis: produces tetanospasmin during stationary phase which is released when cell lysis occurs heavy chain binds to ganglioside on neuronal membranes toxin internalized and moves from peripheral to central nervous system by retrograde axonal transport crosses synapse and localized within vesicles acts by blocking release of inhibitory neurotransmitters (eg GABA) Dr.T.V.Rao MD 26Clostridium difficile: Clostridium difficile Associated with human disease in mid-1970’s Found in human GIT in small numbers With antibiotic use, increase in number in GIT Clindamycin, ampicillin, cephalosporins ....... Produces 2 entero toxins Toxin A -enterotoxin & Toxin B -cytotoxin Diagnosis Detection of toxins in stools, culture of organism Clinical - AAC Pseudomembranous colitis Treatment omit antibiotic if possible oral vancomycin (125mg qds or metronidazole Dr.T.V.Rao MD 27Clostridium botulinum: Clostridium botulinum Fastidious spore forming anaerobic gram positive bacillus Produces 8 antigenically distinct toxins Human disease described with types A, B & E Heavy chain binds to ganglioside receptor Toxin internalized and prevents release of acetyl choline from vesicles Clinical Food borne botulism (weakness, dizziness, ocular palsy and progressive flaccid paralysis) infant botulism (floppy baby) wound botulism Dr.T.V.Rao MD 28ANAEROBIC GRAM NEGATIVE BACILLI: ANAEROBIC GRAM NEGATIVE BACILLI Bactericides, Prevotolla, Porphyromonas and Fusobacterium Present in GI tract -form large component of normal flora >80% of human infections associated with B fragilis virulence factors - capsule, LPS, agglutinins and enzymes Clinical - Endogenous infections Intra-abdominal pyogenic infections pleuro-pulmonary infections genital infection Dr.T.V.Rao MD 29ACTINOMYCES: ACTINOMYCES Strict anaerobic Gram positive bacilli typically arranged in hyphae which fragment into short bacilli Normal flora of upper respiratory tract, GI tract and female genital tract. Low virulence produce disease when mucosal barrier is breached (eg: following dental trauma or surgery) ENDOGENOUS Establishes chronic infection that spreads through normal anatomical barriers Clinical -cervicofacial, abdominal and thoracic Diagnosis: Gram stain of ‘sulpher’ granules culture Dr.T.V.Rao MD 30Culturing of Anaerobes: Culturing of Anaerobes Dr.T.V.Rao MD 31Culturing of anaerobes need special skills: Culturing of anaerobes need special skills Culture of anaerobes is extremely difficult due to the need to exclude oxygen, slow growth and complex growth requirements Molecular methods based on DNA analysis and direct microscopy have shown that we are largely ignorant of the microbial world and previously unknown diversity has been discovered Dr.T.V.Rao MD 32Culture methods: Culture methods Anaerobes differ in their sensitivity to oxygen and the culture methods employed reflect this - some are simple and suitable for less sensitive organisms, others more complex but necessary for fastidious anaerobes Vessels filled to the top with culture medium can be used for organisms not too sensitive Dr.T.V.Rao MD 33Appropriate Specimens for Anaerobic Cultures: Appropriate Specimens for Anaerobic Cultures The Microbiologists understanding of basic anaerobic bacteriology is critical in the interpretation of an anaerobic culture result for the diagnosis and treatment of anaerobic infection. Since anaerobes from part of the normal bacterial flora of the skin and mucous membrane, proper selection and collection of clinical specimens for the laboratory diagnosis of an anaerobic infection critical factors that will determine the clinical significance of the culture results Dr.T.V.Rao MD 34Acceptable Specimens: Acceptable Specimens Specimens for anaerobic cultures are ideally biopsy samples of needle aspirates. Anaerobic swabs are discouraged but sometimes cannot be avoided. Swabs are the least desirable because of the small amount of the specimen and effect of drying. There is a greater chance of contamination with normal micro flora Dr.T.V.Rao MD 35The accepted specimens for anaerobic processing are as follows:: The accepted specimens for anaerobic processing are as follows: Sites CNS Dental/ENT Acceptable specimen CSF, abscess, tissue Abscess , aspirates, tissues Dr.T.V.Rao MD 36The accepted specimens for anaerobic processing are as follows:: The accepted specimens for anaerobic processing are as follows : Local abscess Pulmonary Needle aspirates Trans tracheal aspirates, lung aspirates, pleural fluid, tissue, Protected bronchial washing Dr.T.V.Rao MD 37The accepted specimens for anaerobic processing are as follows: The accepted specimens for anaerobic processing are as follows Abdominal Urinary tract Genital tract Ulcers/wounds Others Abdominal Abscess aspirate, fluid and tissues Suprapubic bladder aspirate Culdocentesis specimen, endometrial swabs Aspirate/swab pus from deep pockets or from under skin flaps that have been decontaminated Deep tissue or bone lesions, blood, bone marrow, synovial fluid, tissues Dr.T.V.Rao MD 38Handling other Critical Specimens: Handling other Critical Specimens Specimens that are normally sterile, such as blood, CSF and synovial fluid, should be collected aseptically to prevent contamination by skin flora. In general, the best materials for anaerobic cultures are obtained by needle aspiration and able tissue biopsy. Dr.T.V.Rao MD 39Unacceptable Specimens: Unacceptable Specimens Exudates, swabs from burns, wounds and skin abscesses are generally unacceptable for anaerobic cultures. Cysts and abscess are contaminated with normal anaerobic flora. Gastric contents , small bowel contents, feces, colo-cutaneous fistula and colostomy contents should not be cultured for anaerobic bacteria. Voided and catheterized urine are contaminated with distal urethral anaerobes and are therefore unacceptable for anaerobic cultures. Dr.T.V.Rao MD 40Interpretation by Physicians and Microbiologists: Interpretation by Physicians and Microbiologists The physician who collected the specimen can best evaluate the anaerobic culture result. Interpretation of the result should be correlated with the clinical findings and how the specimen was collected. Clinical signs suggesting possible infection with anaerobes include the following: 1. Foul smelling discharge 2. Infection in proximity to a mucosal surface 3. Gas in tissues 4. Negative aerobic cultures of specimens whose gram stains show organisms and pus cells. Dr.T.V.Rao MD 41Limitation with Culturing the Specimens: Limitation with Culturing the Specimens Respiratory specimens that are generally rejected for anaerobic cultures include nasal and throat swabs, sputum and suction specimens; e.g. nasotracheal, tracheal and endotracheal aspirates collected by suction and unprotected bronchial washing. These specimens are contaminated with oral flora anaerobes. Dr.T.V.Rao MD 42Diagnosis: Diagnosis Myonecrosis clinical Gram stain of exudate - typical organisms no pus cells Culture -growth of C perfringens (and/or other clostridia associated with this clinical condition) Food poisoning abdominal pain, diarrhea and vomiting 8-18 hours after a suspect meal. Self limiting Enteritis necroticans severe abdominal pain, bloody diarrhoea , shock and peritonitis ( C perfringens type C) Dr.T.V.Rao MD 43Basic needs in Anaerobic Medium: Basic needs in Anaerobic Medium Most common adaptation of media is the addition of a reducing agent, e.g. Thiglyclolate, cysteine Acts to reduce the oxygen to water, brings down the redox potential -300mV or less. Can add a redox indicator such as rezazurin, pink in the presence of oxygen - colourless in its absence Dr.T.V.Rao MD 44Testing for anaerobes in Routine Practice: Testing for anaerobes in Routine Practice Deep culture tubes can be used to test whether an unknown organism is anaerobic/facultative or aerobic Thiglyclolate added to culture medium, oxygen only found near top where it can diffuse from air -pattern of colony formation characteristic of organisms Dr.T.V.Rao MD 45 LABORATORY DIAGNOSIS: LABORATORY DIAGNOSIS A. COLLECTION Anaerobes are endogenous in nature I. Appropriate specimens for anaerobic culture : 1. pus 2. pleural fluid 3. urine 4. pulmonary secretions 5. uterine secretions or sinus tract material Dr.T.V.Rao MD 46Why Needle Aspiration Preferred for Anaerobic Bacteria: Why Needle Aspiration Preferred for Anaerobic Bacteria II. Collection by needle aspiration is preferable than swab culture because of a. better survival of pathogen b. greater quantity of specimen c. less contamination with extraneous organism are often achieved Dr.T.V.Rao MD 47B. HANDLING: B. HANDLING If a swab must be used, a 2 tube system must be used 1 st tube contains swab in O2 free CO2 2 nd tube contains PRAS (pre-reduced anaerobically sterilized culture media) Specimen should be placed in anaerobic transport device with gas mixture Dr.T.V.Rao MD 48HANDING AND TRANSPORT OF CLINICAL SPECIMENS: HANDING AND TRANSPORT OF CLINICAL SPECIMENS The basic principles to remember are proper collection of specimens to avoid contamination with the normal microbial flora and prompt transport to the laboratory where immediate processing is done. Interpreting anaerobic culture result should be easy if proper collection and transport of the specimens have been assured Dr.T.V.Rao MD 49Transporting: Transporting Anaerobic transport tubes and/or devices should always be available at the OR and ER. Specimens should be placed in leak-proof container with tight fitting caps. Of course, proper label for identification with date and time of collection should accompany all specimens submitted for culture . Put samples in room temperature while waiting for delivery to the laboratory. Some anaerobes are killed by refrigeration. Dr.T.V.Rao MD 50Basic Information with Gram Staining: Basic Information with Gram Staining Gram stain should be done in the laboratory : a choice of appropriate media & methods for culture b. quality control for the types of bacteria that laboratory culture reveal Dr.T.V.Rao MD 51Gram stain can be Guiding factor Interpret with caution and Expertise: Gram stain can be Guiding factor Interpret with caution and Expertise The gram stain result is helpful because bacteria present in the smear should be present in the culture. Specimens from intraabdominal and genital infections usually yield polymicrobial cultures of aerobes and anaerobes. Some aspirates/abscesses may contain more than one anaerobe . These should all be corrected with the gram stain result. Dr.T.V.Rao MD 52Interpretation of Gram Staining: Interpretation of Gram Staining Gram staining is performed on the specimen at the time of culture. While infections can be caused by aerobic or anaerobic bacteria or a mixture of both, some infections have a high probability of being caused by anaerobic bacteria. These infections include brain abscesses, lung abscesses, aspiration pneumonia, and dental infections . Anaerobic organisms can often be suspected because many anaerobes have characteristic microscopic morphology (appearance) Dr.T.V.Rao MD 53Anaerobic culturing Needs Define Chemicals and Environment : Anaerobic culturing Needs Define Chemicals and Environment Pyrogallic acid-sodium hydroxide method can be used, again relies on a chemical reaction to generate an anaerobic environment, but a catalyst rather than a reducing agent Anaerobic jars (GasPak System) are sued to incubate plates in an anaerobic atmosphere, useful if brief exposure to oxygen is not lethal Dr.T.V.Rao MD 54 Anaerobic Culture Methods: Anaerobic Culture Methods Production of a vacuum •Displacement of Oxygen with other gases •Absorption of Oxygen by chemical or biological methods •By using reducing agents Dr.T.V.Rao MD 55Slide 56: P. aeruginosa Strict aerobe Enterococcus Facultative Grows aerobic or anaerobic. Bacteriodes fragilis Dr.T.V.Rao MD 56Obligate Anaerobes needs Optimal Methods: Obligate Anaerobes needs Optimal Methods Obligate anaerobes can be culture in special reducing media such as sodium Thiglyclolate or in anaerobe chambers and handled in anaerobe hoods. Dr.T.V.Rao MD 57 Displacement of Oxygen: Displacement of Oxygen By inert gases like Hydrogen, Nitrogen, Carbon dioxide or Helium •Use of lighted candle - Use up Oxygen, but some Oxygen is left behind Vacuum decicator - Unsatisfactory Dr.T.V.Rao MD 58 McIntosh & Filde’s Jar : McIntosh & Filde’s Jar Hydrogen gas is passed in •Catalyst helps to combine Hydrogen & O2 •Reduced Methylene blue remains colorless if anaerobiosis is achieved Dr.T.V.Rao MD 59 Absorption of O2 by Chemical method: Absorption of O2 by Chemical method Pyrogallol •Chromium and sulphuric acid •Gas-pak -available commercially Dr.T.V.Rao MD 60 By reducing agents: By reducing agents Thiglyclolate broth •Robertson’s Cooked Meat (RCM) broth contains nutrient broth with pieces of fat-free minced cooked meat of ox heart. Dr.T.V.Rao MD 61 McIntosh & Filde’s anaerobic Jar: McIntosh & Filde’s anaerobic Jar Stout glass or metal jar with a lid •Lid has an inlet for gas,outlet&2 terminals •Alumina pellets coated with palladium (catalyst) - under the lid •Inoculated plates kept inside the jar •Lid is clamped tight •Air is evacuated Dr.T.V.Rao MD 62A solid or liquid medium maybe used & must provide an anaerobic environment Anaerobic Culture System: A solid or liquid medium maybe used & must provide an anaerobic environment Anaerobic Culture System ANAEROBIC JAR 1. Candle Jar - reduces O2 environment - only ↑ CO2 tension 2. Gas Pak Jar a. Palladium aluminum coated pellets - catalyst - chemically reduces O2 - reacts with residual O2 in the presence of H2 to form H2O Dr.T.V.Rao MD 63Culture of strict anaerobes: Culture of strict anaerobes For culture of strict anaerobes all traces of oxygen must be removed from medium and for many organisms sample must be kept entirely anaerobic during manipulations Methanogenic archaea from rumen and sewage treatment plants killed by even a brief exposure to O 2 Medium usually boiled during preparation and reducing agent added, stored under O 2 -free atmosphere Dr.T.V.Rao MD 64Choosing the Optimal Media: Choosing the Optimal Media Broth and solid media should both be inoculated. The culture media should include anaerobic blood agar plates enriched with substances such as brain-heart infusion, yeast extract, amino acids, and vitamin K; a selective medium such as kanamycin- vancomycin (KV) blood agar or laked blood agar; and a broth such as brain heart infusion broth with Thiglyclolate or other reducing agent. Dr.T.V.Rao MD 65Media chosen according to our needs: Media chosen according to our needs The choice of media depends upon the type of specimen. Some commonly used media include prereduced peptone-yeast extract-glucose broth which is suitable for analysis of volatile products by gas chromatography; egg yolk agar for detection of lecithinase activity of Clostridium spp . ; cycloserine-cefoxitin-fructose agar (CCFA) for isolation of Clostridium difficile from stool; and Bacteroides bile esculin agar for isolation of the Bacteroides fragilis group. Dr.T.V.Rao MD 66 A skilled plating the Medium is highly essential : A skilled plating the Medium is highly essential Figure 6.10a–b Dr.T.V.Rao MD 67Anaerobic Glove Chamber : Anaerobic Glove Chamber b. Gas Pak envelope - generates CO2 & H2 gases c. Methylene blue strip - indicator blue → (+) O2 white → ( -) O2 II. Anaerobic Glove Chamber - close system - used for premature babies - e.g. incubator III. Roll Tube - has a pedal gas ( CO2 & H2 ) would come out - place test tube directly to the outlet Dr.T.V.Rao MD 68 IDENTIFICATION of ANAEROBES: IDENTIFICATION of ANAEROBES Plates are checked at > 18-24 hours for faster growing species like Cl. Perfringens & B.fragilis & daily thereafter up to > 5-7 days for slowly growing species like Actinomyces, Eubacterium & Propionibacterium Genus is determined by - gram stain, cellular morphology, Gas-liquid chromatography Species determination is based on fermentation of sugars & other biochemical determination Dr.T.V.Rao MD 69Identification of Anaerobes is Complex: Identification of Anaerobes is Complex The identification of anaerobes is highly complex, and laboratories may use different identification systems. Partial identification is often the goal. For example, there are six species of the Bactericides genus that may be identified as the Bactericides fragilis group rather than identified individually. Organisms are identified by their colonial and microscopic morphology, growth on selective media, oxygen tolerance, and biochemical characteristics. Dr.T.V.Rao MD 70 All isolates to the Purified by Sub culturing: All isolates to the Purified by Sub culturing Isolated organisms are always subcultured and the pure culture is tested in order to identify the organism. The identification of anaerobes is highly complex, and laboratories may use different identification systems. Partial identification is often the goal. For example, there are six species of the Bacteroides genus that may be identified as the Bacteroides fragilis group rather than identified individually. Organisms are identified by their colonial and microscopic examination. Dr.T.V.Rao MD 71Needs several Biochemical Tests for Identification: Needs several Biochemical Tests for Identification Organisms are identified by their colonial and microscopic morphology, growth on selective media, oxygen tolerance, and biochemical characteristics. These include sugar fermentation, bile solubility, esculin, starch, and gelatin hydrolysis , casein and gelatin digestion, catalase, lipase, lecithinase, and indole production, nitrate reduction, volatile fatty acids as determined by gas chromatography, and susceptibility to antibiotics . The antibiotic susceptibility profile is determined by the micro tube broth dilution method. Many species of anaerobes are resistant to penicillin, and some are resistant to clindamycin and other commonly used antibiotics Dr.T.V.Rao MD 72Antibiotic Sensitivity Testing: Antibiotic Sensitivity Testing .The antibiotic susceptibility profile is determined by the micro tube broth dilution method. Many species of anaerobes are resistant to penicillin, and some are resistant to clindamycin and other commonly used antibiotics Dr.T.V.Rao MD 73Follow me for More Articles of Interest on Infectious Diseases: Follow me for More Articles of Interest on Infectious Diseases Dr.T.V.Rao MD 74Slide 75: The programme is Created by Dr.T.V.Rao for ‘ e ‘ learning resources to Microbiologists in the Developing World. Email email@example.com Dr.T.V.Rao MD 75 You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.