Anaerobic Bacteriology

1. ANAEROBIC
BACTERIOLOGY
Dr.T.V.Rao MD
Dr.T.V.Rao MD 1

2. What Are Anaerobic Microorganisms
2
• Anaerobic
microorganisms
are widespread
and very
important
• Do not require
oxygen for growth
- often extremely
toxic Dr.T.V.Rao MD

3. The Requirements for Growth:
Related to Oxygen
• Oxygen (O2)
Dr.T.V.Rao MD 3
Table 6.1

4. Anaerobes differ from Aerobic
Bacteria
• 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 4

5. DEFINITIONS
• OBLIGAETE ANAEROBE
– Lack superoxide dismutase and/or catalase
– toxic radicals formed by oxidative enzymes kill
organisms
• AERO-TOLERANT ANAEROBES
– survive in presence of oxygen
– Do not use oxygen for energy requirements
• FACULTATIVE ANAEROBES
Dr.T.V.Rao MD 5

6. Anaerobes 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 6

7. Anaerobic and Aerobic Respiration
•Reaction name •Reduc •Oxid. •Reaction •kcal/
t. Stoichiometry mol
•Aerobic •CHO •O2 •C6H12O6 + 6O2 ==> •686
Respiration 6CO2 + 6H2O
•Nitrate •CHO •NO3- •CHO + NO3- + H+ •649
Reduction ==> CO2+ N2+ H2O
•Sulfate •CHO •SO42- •2CHO + SO42-+2H+ •190
Reduction => 2CO2+ H+ 2H2O
•Methanogenesis •CHO •CO2 •4H2 + CO2 ==> CH4 •8.3
or H2 + 2H2O
Dr.T.V.Rao MD 7

8. 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 8

9. Strict Anaerobic Bacteria
9
• 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

10. ROS production during respiration
• 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 10

11. 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 11

12. 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 12

13. Chemical Dynamics in Anaerobic
Bacteria
• 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 13

14. 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 14

15. 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 15

16. FACTORS RESPONSIBLE FOR
THEIR VIRULENCE
1. Lipopolysaccharide
- promotes abscess formation, enhanced coagulation
2. Polysaccharide capsule
- correlated with abscess production
3. Enzymes
a. Collagenase
b. Heparinize
* 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 16

17. Multiplication of the opportunistic
pathogens is facilitated by:
1. Inhibition of phagocytosis & intracellular
killing
by PMN in the presence of Bacteroides by:
a. competition of opsonins
b. inhibition by capsular materials
2. Protection of antibiotic susceptibility
strains in
mixtures thru destruction by the ß-
lactamases
3. Utilization of O2 by facultative species that
aids in producing a suitable environment
for growth of anaerobe
Dr.T.V.Rao MD 17

18. CLINICAL MANIFESTATION
Clinical finding suggestive of
Anaerobic infection
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 18

19. Dr.T.V.Rao MD 19

20. Dr.T.V.Rao MD 20

21. 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 21

22. Pathogenesis of anaerobic
infections
• Contamination of site with spores
• Factors which promote anaerobiasis
– ‘crush’ injuries with interruption of blood supply,
contamination with foreign bodies (dirt), tissue
damage
• Germination of spores
• Toxin release
• Binding of toxin to receptor
• Resulting effect produces symptom(s) of
disease Dr.T.V.Rao MD 22

23. 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 23

24. 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 24

25. 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 25

26. Common Human
Anaerobic Infections
Dr.T.V.Rao MD 26

27. 27
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

28. 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 28

29. 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 neurotransmittors (eg
GABA)
Dr.T.V.Rao MD 29

30. 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 30

31. TETANUS
• Clinical syndromes due
to unregulated
excitatory synaptic
activity resulting in
spastic paralysis
– Generalised
tetanus
– Neonatal tetanus
– localized tetanus
Dr.T.V.Rao MD 31

32. Prevention and treatment
• Active immunization with tetanus toxoid
• Wound toilet and active/passive immunization
of ‘risk’ injuries
– management of wound
– tetanus toxoid
– Anti-tetanus serum (ARS -horse serum) or Human
Tetanus ImmunoGlobulin (HTIG)
– Penicillin or Metronidazole
• Management of patient with tetanus
– reduce stimuli
– respiratory and CVS support
Dr.T.V.Rao MD 32

33. 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 33

34. Clostridium difficle
• 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 34

35. 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 35

36. ANAEROBIC GRAM NEGATIVE BACILLI
• Bacteroides, 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 infctions
– genital infection
Dr.T.V.Rao MD 36

37. 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
• Treatment - surgery and long term penicillin
Dr.T.V.Rao MD 37

38. 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 38

39. Aspiration is ideal
Avoid Swabs
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 39

40. HANDLING
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 40

41. Isolation
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 41

42. A solid or liquid medium maybe used & must provide
an anaerobic environment Anaerobic Culture
System
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 42

43. 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 43

44. IDENTIFICATION
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
chromotography
Species determination is based on fermentation of sugars &
other biochemical determination
Dr.T.V.Rao MD 44

45. 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 45

46. 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 46

47. 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 47

48. 48
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

49. 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 49

50. Acceptable Specimens
50
• 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

51. The accepted specimens for anaerobic
51
processing are as follows:
• Sites • Acceptable
specimen
• CNS • CSF, abscess,
tissue
Abscess,
• Dental/ENT aspirates, tissues
Dr.T.V.Rao MD

52. The accepted specimens for anaerobic
52
processing are as follows:
• Local abscess • Needle aspirates
• Trans tracheal
• Pulmonary aspirates, lung
aspirates, pleural
fluid, tissue,
• Protected
bronchial washing
Dr.T.V.Rao MD

53. The accepted specimens for anaerobic
53
processing are as follows
• Abdominal • Abdominal Abscess
aspirate, fluid and tissues
• Urinary tract
• Suprapubic bladder
• Genital tract aspirate
• Culdocentesis specimen,
• Ulcers/wounds endometrial swabs
• Aspirate/swab pus from deep pockets
or from under skin flaps
• that have been decontaminated
• Others • Deep tissue or bone lesions, blood,
bone marrow, synovial fluid,
• tissues
Dr.T.V.Rao MD

54. Handling other Critical Specimens
54
• 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

55. 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 55

56. 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 56

57. Limitation with Culturing the Specimens
57
• 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

58. 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 58

59. 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 59

60. Testing for anaerobes in Routine
60
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

61. Why Needle Aspiration Preferred
61 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

62. HANDLING
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 62

63. 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 63

64. 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 64

65. Basic Information with Gram
65 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

66. 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 66

67. 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 67

68. 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 68

69. 69 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

70. 70 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

71. 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 71

72. P. aeruginosa Strict aerobe
Enterococcus Facultative
Grows aerobic or anaerobic.
Dr.T.V.Rao MD 72
Bacteriodes fragilis

73. Obligate Anaerobes needs
73 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

74. 74 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

75. Absorption of O2 by Chemical method
• Pyrogallol
• •Chromium and
sulphuric acid
• •Gas-pak
• -available
commercially
Dr.T.V.Rao MD 75

76. 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.
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77. 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
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78. A solid or liquid medium maybe used & must provide an
anaerobic environment Anaerobic Culture System
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
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79. 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 O2
• Medium usually boiled during preparation and
reducing agent added, stored under O2-free
atmosphere
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80. 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.
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81. 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.
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82. 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
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83. TREATMENT
- Susceptibility testing should be done
- surgical drainage & resection of necrotic
tissue
- most are resistant to aminoglycosides
- for Bacteroides group, if resistant to Penicillin
& Cephalosporin, they may use:
a. Clindamycin
b. Metronidazole
c. Chloramphenicol
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Interest on Infectious Diseases
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85. • The programme is Created by
Dr.T.V.Rao MD for ‘ e ‘ learning
resources to Microbiologists
• Email
• doctortvrao@gmail.com
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