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Dr.T.V.

Rao MD 1

ANAEROBIC BACTERIA
AND
BASIC CULTURE METHODS

Dr.T.V.Rao MD
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
Defining Anaerobes
3

 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
Strict Anaerobic Bacteria
4

 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

 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
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

•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 2H 2O MD
Dr.T.V.Rao 7
ROS production during respiration
8

 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
Chemical Dynamics in Anaerobic Bacteria
9

 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
Anaerobic environments exist in
10
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
Anaerobic Environments
11

Dr.T.V.Rao MD
Anaerobes and Oxygen
12

 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
FACTORS THAT INHIBIT THE
GROWTH OF ANAEROBES BY OXYGEN
13

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
ANAEROBES OF CLINICAL IMPORTANCE
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 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

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16

Dr.T.V.Rao MD
Anaerobic Bacteria of Medical Interest
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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
Gram-positive anaerobes
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 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
Gram-negative anaerobes
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 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
ANAEROBIC GRAM NEGATIVE BACILLI

20

 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
FACTORS RESPONSIBLE FOR
THEIR VIRULENCE
21

* develop thrombophlebitis & septic emboli

Dr.T.V.Rao MD
CLINICAL MANIFESTATION
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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

 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
Clostridium perfringens
25

 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
Clostridium tetani
26

 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
Clostridium difficile
27

 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
Clostridium botulinum
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 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
ANAEROBIC GRAM NEGATIVE BACILLI

29

 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
ACTINOMYCES
30

 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
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

 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
Culture methods
33

 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
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

 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
The accepted specimens for anaerobic
36
processing are as follows:

 Sites  Acceptable
specimen

 CNS  CSF, abscess, tissue


 Dental/ENT Abscess,
aspirates, tissues
Dr.T.V.Rao MD
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
The accepted specimens for anaerobic
processing are as follows
38

 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
Handling other Critical Specimens
39

 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
Unacceptable Specimens
40

 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
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
Dr.T.V.Rao MD
 pus cells.
Limitation with Culturing the Specimens
42

 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
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

 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
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

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
HANDING AND TRANSPORT OF
49
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
Transporting
50

 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
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

 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
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

 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
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
P. aeruginosa Strict aerobe

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

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
Culture of strict anaerobes
64

 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
Choosing the Optimal Media
65

 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
Media chosen according to our needs
66

 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
A skilled plating the Medium is
67
highly essential

Dr.T.V.Rao MD
Figure 6.10a–b
Anaerobic Glove Chamber
68

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
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

 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
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
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Developing World.
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Dr.T.V.Rao MD