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ANAEROBIC BACTERIA
AND
BASIC CULTURE METHODS
Dr.T.V.Rao MD
What Are Anaerobic Microorganisms
2
Anaerobic
microorganisms are
widespread and very
important
Dr.T.V.Rao MD
Defining Anaerobes
3
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
Oxygen Toxicity
5
Dr.T.V.Rao MD
The Requirements for Growth:
Related to Oxygen
6
Oxygen (O2)
Dr.T.V.Rao MD
Table 6.1
Anaerobic and Aerobic Respiration
•Reaction name •Reduct. •Oxid. •Reaction •kcal/
Stoichiometry mol
•Aerobic •CHO •O2 •C6H12O6 + 6O2 ==> •686
Respiration 6CO2 + 6H2O
Dr.T.V.Rao MD
Anaerobic environments exist in
10
Nature too
Anaerobic environments (low reduction potential)
include:
Dr.T.V.Rao MD
Anaerobic Environments
11
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Anaerobes and Oxygen
12
Dr.T.V.Rao MD
FACTORS THAT INHIBIT THE
GROWTH OF ANAEROBES BY OXYGEN
13
Dr.T.V.Rao MD
ANAEROBES OF CLINICAL IMPORTANCE
14
CLOSTRIDIA
C tetani; C perfringens; C difficile; C botulinum
BACTEROIDES
B fragilis;
Prevotella
Porphyromonas
ACTINOMYCES
FUSOBACTERIUM
ANAEROBIC STREPTOCOCCI
Dr.T.V.Rao MD
Sites and Infection produced by
Anaerobes
15
Dr.T.V.Rao MD
16
Dr.T.V.Rao MD
Anaerobic Bacteria of Medical Interest
17
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
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Gram-positive anaerobes
18
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Gram-negative anaerobes
19
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ANAEROBIC GRAM NEGATIVE BACILLI
20
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FACTORS RESPONSIBLE FOR
THEIR VIRULENCE
21
Dr.T.V.Rao MD
CLINICAL MANIFESTATION
22
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
Dr.T.V.Rao MD 23
COMMON HUMAN
ANAEROBIC INFECTIONS
CLOSTRIDIA
24
Several species
associated with human
disease
Dr.T.V.Rao MD
Clostridium perfringens
25
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Clostridium tetani
26
Dr.T.V.Rao MD
ANAEROBIC GRAM NEGATIVE BACILLI
29
Dr.T.V.Rao MD
ACTINOMYCES
30
Dr.T.V.Rao MD
Dr.T.V.Rao MD 31
CULTURING OF
ANAEROBES
Culturing of anaerobes need
32
special skills
Culture of anaerobes is extremely difficult due to the
need to exclude oxygen, slow growth and complex
growth requirements
Dr.T.V.Rao MD
Culture methods
33
Dr.T.V.Rao MD
Appropriate Specimens for Anaerobic
34
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
Acceptable Specimens
35
Sites Acceptable
specimen
Dental/ENT Abscess,
aspirates, tissues
Dr.T.V.Rao MD
The accepted specimens for anaerobic
processing are as follows:
37
Dr.T.V.Rao MD
Handling other Critical Specimens
39
Dr.T.V.Rao MD
Interpretation by Physicians and
41
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
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pus cells.
Limitation with Culturing the Specimens
42
Dr.T.V.Rao MD
Diagnosis
43
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
Basic needs in Anaerobic Medium
44
Dr.T.V.Rao MD
Testing for anaerobes in Routine
45
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
LABORATORY DIAGNOSIS
46
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
Why Needle Aspiration Preferred
47
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
B. HANDLING
48
Dr.T.V.Rao MD
Basic Information with Gram
51
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
Gram stain can be Guiding factor
52
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
Interpretation of Gram Staining
53
Dr.T.V.Rao MD
Anaerobic culturing Needs Define
54
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
Enterococcus Facultative
Grows aerobic or anaerobic.
56 Dr.T.V.Rao MD
Bacteriodes fragilis
Obligate Anaerobes needs Optimal
57
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
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
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
Absorption of O2 by Chemical method
60
Pyrogallol
•Chromium and
sulphuric acid
•Gas-pak
-available
commercially
Dr.T.V.Rao MD
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
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
A solid or liquid medium maybe used & must provide an
anaerobic environment Anaerobic Culture System
63
A. ANAEROBIC JAR
1. Candle Jar
- reduces O2 environment
- only ↑ CO2 tension
Dr.T.V.Rao MD
Media chosen according to our needs
66
Dr.T.V.Rao MD
A skilled plating the Medium is
67
highly essential
Dr.T.V.Rao MD
Figure 6.10a–b
Anaerobic Glove Chamber
68
Dr.T.V.Rao MD
IDENTIFICATION of ANAEROBES
69
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
Identification of Anaerobes is
70
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
All isolates to the Purified by Sub culturing
71
Dr.T.V.Rao MD
Needs several Biochemical Tests for
72
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
Antibiotic Sensitivity Testing
73
.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
Follow me for More Articles of Interest on
Infectious Diseases
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75
doctortvrao@gmail.com
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