Dr.T.V.Rao MD   1ANAEROBIC BACTERIA        ANDBASIC CULTURE METHODS          Dr.T.V.Rao MD
What Are Anaerobic Microorganisms2       Anaerobic        microorganisms are        widespread and very        important ...
Defining Anaerobes3       Facultative anaerobes - can grow in the        presence or absence of oxygen       Obtain ener...
Strict Anaerobic Bacteria4       Obligate (strict)        anaerobes - oxygen is        toxic to these organisms,        d...
Oxygen Toxicity5       Oxygen is used by aerobic and facultatively        anaerobic organisms as its strong oxidising    ...
The Requirements for Growth:                    Related to Oxygen6       Oxygen (O2)                               Dr.T.V...
Anaerobic and Aerobic Respiration•Reaction name    •Reduct. •Oxid. •Reaction            •kcal/                            ...
ROS production during respiration8       O2 + e- => O2-                            superoxide                            ...
Chemical Dynamics in Anaerobic Bacteria9       Organisms that use O2 have developed defence        mechanisms to protect ...
Anaerobic environments exist in10               Nature too        Anaerobic environments (low reduction potential)       ...
Anaerobic Environments11                Dr.T.V.Rao MD
Anaerobes and Oxygen12        Anaerobes generate energy by fermentation        Lack the capacity to utilize O2 as a term...
FACTORS THAT INHIBIT THE     GROWTH OF ANAEROBES BY OXYGEN13     1. Toxic compounds are produced          e.g. H2O2 , Supe...
ANAEROBES OF CLINICAL IMPORTANCE14        CLOSTRIDIA            C tetani; C perfringens; C difficile; C botulinum      ...
Sites and Infection produced by                Anaerobes15                     Dr.T.V.Rao MD
16     Dr.T.V.Rao MD
Anaerobic Bacteria of Medical Interest17     MORPHOLOGY             GRAM STAIN                         GENUS      Spore fo...
Gram-positive anaerobes18        Actinomyces (head, neck, pelvic infections; aspiration         pneumonia)        Bifid ...
Gram-negative anaerobes19        Bactericides (the most commonly found anaerobes in         cultures; intra-abdominal inf...
ANAEROBIC GRAM NEGATIVE BACILLI20        Bactericides, Prevotolla, Porphyromonas and         Fusobacterium        Presen...
FACTORS RESPONSIBLE FOR               THEIR VIRULENCE21     1         * develop thrombophlebitis & septic emboli          ...
CLINICAL MANIFESTATION22     Clinical hints      1. odor       2. tissue       3. location       4. necrotic tissue       ...
Dr.T.V.Rao MD   23COMMON HUMANANAEROBIC INFECTIONS
CLOSTRIDIA24        Gram positive spore         forming bacilli        ubiquitous            intestines of man and anim...
Clostridium perfringens25        Large rectangular Gram positive bacillus        Spores seldom seen in vivo or in vitro ...
Clostridium tetani26        Small motile spore forming gram positive bacillus         with round terminal spores        ...
Clostridium difficile27        Associated with human disease in mid-1970’s        Found in human GIT in small numbers   ...
Clostridium botulinum28        Fastidious spore forming anaerobic gram positive         bacillus        Produces 8 antig...
ANAEROBIC GRAM NEGATIVE BACILLI29        Bactericides, Prevotolla, Porphyromonas and         Fusobacterium        Presen...
ACTINOMYCES30    Strict anaerobic Gram positive bacilli typically arranged in hyphae which     fragment into short bacill...
Dr.T.V.Rao MD   31CULTURING OF ANAEROBES
Culturing of anaerobes need32                   special skills        Culture of anaerobes is extremely difficult due to ...
Culture methods33        Anaerobes differ in their         sensitivity to oxygen and         the culture methods         ...
Appropriate Specimens for Anaerobic34                   Cultures        The Microbiologists understanding of basic anaero...
Acceptable Specimens35        Specimens for anaerobic         cultures are ideally biopsy         samples of needle      ...
The accepted specimens for anaerobic36              processing are as follows:        Sites                 Acceptable  ...
The accepted specimens for anaerobic                processing are as follows:37        Local abscess           Needle a...
The accepted specimens for anaerobic                  processing are as follows38        Abdominal                Abdomi...
Handling other Critical Specimens39        Specimens that are         normally sterile, such as         blood, CSF and sy...
Unacceptable Specimens40        Exudates, swabs from burns, wounds and skin         abscesses are generally unacceptable ...
Interpretation by Physicians and41                  Microbiologists        The physician who collected the specimen can b...
Limitation with Culturing the Specimens42        Respiratory specimens that         are generally rejected for         an...
Diagnosis43        Myonecrosis            clinical            Gram stain of exudate - typical organisms                ...
Basic needs in Anaerobic Medium44        Most common adaptation of media is the addition of         a reducing agent, e.g...
Testing for anaerobes in Routine45                      Practice        Deep culture tubes can         be used to test wh...
LABORATORY                  DIAGNOSIS46     A. COLLECTION     Anaerobes are endogenous in nature     I. Appropriate specim...
Why Needle Aspiration Preferred47          for Anaerobic Bacteria     II. Collection by needle       aspiration is       p...
B. HANDLING48     If a swab must be used, a 2 tube system      must be used      1st tube contains swab in O2 free     CO...
HANDING AND TRANSPORT OF49             CLINICAL SPECIMENS        The basic principles to remember are         proper coll...
Transporting50        Anaerobic transport tubes and/or devices should         always be available at the OR and ER.     ...
Basic Information with Gram51               Staining     Gram stain should be     done in the laboratory :      a choice o...
Gram stain can be Guiding factor52     Interpret with caution and Expertise        The gram stain result is helpful becau...
Interpretation of Gram Staining53        Gram staining is performed on the specimen at the         time of culture. While...
Anaerobic culturing Needs Define54       Chemicals and Environment        Pyrogallic acid-sodium hydroxide method can    ...
55     Anaerobic Culture Methods        Production of a vacuum        •Displacement of         Oxygen with other        ...
P. aeruginosa          Strict aerobe       Enterococcus       Facultative       Grows aerobic or anaerobic.56          Dr....
Obligate Anaerobes needs Optimal57                     Methods        Obligate anaerobes         can be culture in       ...
58     Displacement of Oxygen                   By inert gases like                    Hydrogen, Nitrogen,               ...
59            McIntosh & Filde’s Jar        Hydrogen gas is         passed in        •Catalyst helps to         combine ...
Absorption of O2 by Chemical method60                        Pyrogallol                        •Chromium and            ...
61            By reducing agents      Thiglyclolate broth      •Robertson’s       Cooked Meat       (RCM) broth      co...
62         McIntosh & Filde’s anaerobic Jar        Stout glass or metal jar         with a lid        •Lid has an inlet ...
A solid or liquid medium maybe used & must provide an          anaerobic environment Anaerobic Culture System63     A.   ...
Culture of strict anaerobes64        For culture of strict anaerobes all traces of oxygen must         be removed from me...
Choosing the Optimal Media65        Broth and solid media should both be inoculated.         The culture media should inc...
Media chosen according to our needs66        The choice of media depends upon the type of         specimen. Some commonly...
A skilled plating the Medium is67             highly essential                     Dr.T.V.Rao MD                          ...
Anaerobic Glove Chamber68         b. Gas Pak envelope                  - generates CO2 & H2 gases            c. Methylene ...
IDENTIFICATION of ANAEROBES69     Plates are checked at        > 18-24 hours for faster growing species like          Cl. ...
Identification of Anaerobes is70                      Complex        The identification of anaerobes is highly complex,  ...
All isolates to the Purified by Sub culturing71        Isolated organisms are always subcultured and the pure         cul...
Needs several Biochemical Tests for72                   Identification         Organisms are identified by their colonial...
Antibiotic Sensitivity Testing73        .The antibiotic         susceptibility profile is         determined         by t...
Follow me for More Articles of Interest on          Infectious Diseases                      Dr.T.V.Rao MD   74
75        The programme is Created by Dr.T.V.Rao for ‘         e ‘ learning resources to Microbiologists in the          ...
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Anaerobic culture methods

  1. 1. Dr.T.V.Rao MD 1ANAEROBIC BACTERIA ANDBASIC CULTURE METHODS Dr.T.V.Rao MD
  2. 2. What Are Anaerobic Microorganisms2  Anaerobic microorganisms are widespread and very important  Do not require oxygen for growth - often extremely toxic Dr.T.V.Rao MD
  3. 3. Defining Anaerobes3  Facultative anaerobes - can grow in the presence or absence of oxygen  Obtain energy by both respiration and fermentation  Oxygen not toxic, some use nitrate (NO3-) or sulphate (SO42-) as a terminal electron acceptor under anaerobic conditions Dr.T.V.Rao MD
  4. 4. Strict Anaerobic Bacteria4  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
  5. 5. Oxygen Toxicity5  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
  6. 6. The Requirements for Growth: Related to Oxygen6  Oxygen (O2) Dr.T.V.Rao MD Table 6.1
  7. 7. Anaerobic and Aerobic Respiration•Reaction name •Reduct. •Oxid. •Reaction •kcal/ Stoichiometry mol•Aerobic •CHO •O2 •C6H12O6 + 6O2 ==> •686Respiration 6CO2 + 6H2O•Nitrate Reduction •CHO •NO3- •CHO + NO3- + H+ ==> •649 CO2+ N2+ H2O•Sulfate Reduction •CHO •SO42- •2CHO + SO42-+2H+ •190 => 2CO2+ H+ 2H2O•Methanogenesis •CHO •CO2 •4H2 + CO2 ==> CH4 + •8.3 or H2 2H2O MD Dr.T.V.Rao 7
  8. 8. ROS production during respiration8  O2 + e- => O2- superoxide anion  O2- + e- + 2H+ => H2O2 hydrogen peroxide  H2O2 + e- + H+ => H2O + OH. Hydroxyl radical  OH. + e- + H+ => H2O water Dr.T.V.Rao MD
  9. 9. Chemical Dynamics in Anaerobic Bacteria9  Organisms that use O2 have developed defence mechanisms to protect themselves from these toxic forms of oxygen - enzymes  Catalase: H2O2 + H2O2 => 2H2O + O2  Peroxidase: H2O2 + NADH + H+ => 2H2O + NAD+  Superoxide dismutase: O2- + O2- + 2H+ => H2O2 + O2 Dr.T.V.Rao MD
  10. 10. Anaerobic environments exist in10 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
  11. 11. Anaerobic Environments11 Dr.T.V.Rao MD
  12. 12. Anaerobes and Oxygen12  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
  13. 13. FACTORS THAT INHIBIT THE GROWTH OF ANAEROBES BY OXYGEN13 1. Toxic compounds are produced e.g. H2O2 , Superoxides 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
  14. 14. ANAEROBES OF CLINICAL IMPORTANCE14  CLOSTRIDIA  C tetani; C perfringens; C difficile; C botulinum  BACTEROIDES B fragilis;  Prevotella  Porphyromonas  ACTINOMYCES  FUSOBACTERIUM  ANAEROBIC STREPTOCOCCI Dr.T.V.Rao MD
  15. 15. Sites and Infection produced by Anaerobes15 Dr.T.V.Rao MD
  16. 16. 16 Dr.T.V.Rao MD
  17. 17. Anaerobic Bacteria of Medical Interest17 MORPHOLOGY GRAM STAIN GENUS Spore forming (+) ClostridiumNon-spore forming bacilli Actinomycetes, Bifidobacterium,Eubacte- (+) rium,Propionibacerium, Mobilncus,Lactobacillus (-) Bacteroides,Fusobacterium Prevotella,PorphyromonasNon-sporefoming cocci Peptococcus, (+) Pepto-streptococcus Streptococcus (-) Veilonella Dr.T.V.Rao MD
  18. 18. Gram-positive anaerobes18  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
  19. 19. Gram-negative anaerobes19  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
  20. 20. ANAEROBIC GRAM NEGATIVE BACILLI20  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
  21. 21. FACTORS RESPONSIBLE FOR THEIR VIRULENCE21 1 * develop thrombophlebitis & septic emboli Dr.T.V.Rao MD
  22. 22. CLINICAL MANIFESTATION22 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
  23. 23. Dr.T.V.Rao MD 23COMMON HUMANANAEROBIC INFECTIONS
  24. 24. CLOSTRIDIA24  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
  25. 25. Clostridium perfringens25  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
  26. 26. Clostridium tetani26  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
  27. 27. Clostridium difficile27  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
  28. 28. Clostridium botulinum28  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
  29. 29. ANAEROBIC GRAM NEGATIVE BACILLI29  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
  30. 30. ACTINOMYCES30 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
  31. 31. Dr.T.V.Rao MD 31CULTURING OF ANAEROBES
  32. 32. Culturing of anaerobes need32 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
  33. 33. Culture methods33  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
  34. 34. Appropriate Specimens for Anaerobic34 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
  35. 35. Acceptable Specimens35  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
  36. 36. The accepted specimens for anaerobic36 processing are as follows:  Sites  Acceptable specimen  CNS  CSF, abscess, tissue   Dental/ENT Abscess, aspirates, tissues Dr.T.V.Rao MD
  37. 37. The accepted specimens for anaerobic processing are as follows:37  Local abscess  Needle aspirates  Pulmonary  Trans tracheal aspirates, lung aspirates, pleural fluid, tissue,  Protected bronchial washing Dr.T.V.Rao MD
  38. 38. The accepted specimens for anaerobic processing are as follows38  Abdominal  Abdominal Abscess  Urinary tract aspirate, fluid and tissues  Genital tract  Suprapubic bladder aspirate  Ulcers/wounds  Culdocentesis specimen, endometrial swabs  Others  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
  39. 39. Handling other Critical Specimens39  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
  40. 40. Unacceptable Specimens40  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
  41. 41. Interpretation by Physicians and41 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 Dr.T.V.Rao MD  pus cells.
  42. 42. Limitation with Culturing the Specimens42  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
  43. 43. Diagnosis43  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
  44. 44. Basic needs in Anaerobic Medium44  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
  45. 45. Testing for anaerobes in Routine45 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
  46. 46. LABORATORY DIAGNOSIS46 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
  47. 47. Why Needle Aspiration Preferred47 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
  48. 48. B. HANDLING48 If a swab must be used, a 2 tube system must be used  1st tube contains swab in O2 free CO2  2nd 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
  49. 49. HANDING AND TRANSPORT OF49 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
  50. 50. Transporting50  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
  51. 51. Basic Information with Gram51 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
  52. 52. Gram stain can be Guiding factor52 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
  53. 53. Interpretation of Gram Staining53  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
  54. 54. Anaerobic culturing Needs Define54 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
  55. 55. 55 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
  56. 56. P. aeruginosa Strict aerobe Enterococcus Facultative Grows aerobic or anaerobic.56 Dr.T.V.Rao MD Bacteriodes fragilis
  57. 57. Obligate Anaerobes needs Optimal57 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
  58. 58. 58 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
  59. 59. 59 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
  60. 60. Absorption of O2 by Chemical method60  Pyrogallol  •Chromium and sulphuric acid  •Gas-pak  -available commercially Dr.T.V.Rao MD
  61. 61. 61 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
  62. 62. 62 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
  63. 63. A solid or liquid medium maybe used & must provide an anaerobic environment Anaerobic Culture System63 A. 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
  64. 64. Culture of strict anaerobes64  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 O2  Medium usually boiled during preparation and reducing agent added, stored under O2-free atmosphere Dr.T.V.Rao MD
  65. 65. Choosing the Optimal Media65  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
  66. 66. Media chosen according to our needs66  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
  67. 67. A skilled plating the Medium is67 highly essential Dr.T.V.Rao MD Figure 6.10a–b
  68. 68. Anaerobic Glove Chamber68 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
  69. 69. IDENTIFICATION of ANAEROBES69 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
  70. 70. Identification of Anaerobes is70 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
  71. 71. All isolates to the Purified by Sub culturing71  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
  72. 72. Needs several Biochemical Tests for72 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
  73. 73. Antibiotic Sensitivity Testing73  .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
  74. 74. Follow me for More Articles of Interest on Infectious Diseases Dr.T.V.Rao MD 74
  75. 75. 75  The programme is Created by Dr.T.V.Rao for ‘ e ‘ learning resources to Microbiologists in the Developing World.  Email  doctortvrao@gmail.com Dr.T.V.Rao MD
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