ANAEROBIC INFECTIONS
Specimen Collection and
Transportation to the Laboratory
Specimens for the culture of anaerobes
should be taken carefully from the active
site of infection.
Specimen Collection and
Transportation to the Laboratory
The specimen should be placed in a sterile,
tightly closed container.
If one of these is not available!
The entire aspirated specimen should be
transported immediately to the laboratory in the
syringe, with the needle capped or protected by
a rubber stopper.
Types of Specimens
Needle aspiration of pus
Tissue and/or pus specimens
from infected wounds,
empyema, or draining
abscesses.
Specimen Transportation
Stuart’s Transport
Medium
A non-nutrient semi-solid medium
containing sodium thioglycollate to
suppress oxidative changes.
It also maintains the organisms in
a viable state although the
organism do not multiply.
Specimen Transportation
Sterile, tightly closed
universal container for
the transportation of
pus, aspirates, tissues
etc.
Macroscopic Examination
of Specimen
Pus, is observed for yellow
sulfur granules, characteristic of
Actinomyces which can be seen
with the naked eye.
Granules are washed with sterile
water and crushed between two
microscope slides and the
stained using the Gram stain.
Anaerobic Culture Media
Blood Agar
Enriched and Differential
medium
Enriched medium - Contain 5- 10% sheep or
horse blood
Differential medium - Detects different types
of haemolysis produced by different
organisms
Supports the growth of both gram positive
and gram negative organisms
Support the growth of most pathogens
Anaerobic Culture Media
Anaerobic Blood
Agar
Blood agar made selective by the addition
of one or more specific antibiotics
The addition of an aminoglycoside
(neomycin, kanamycin) in a final
concentration of 50 μg/ml inhibits the
majority of aerobic and facultative
bacteria.
Anaerobic Culture Media
Cooked Meat Medium
Supports the growth of organisms that
require anaerobic conditions for
growth.
Fat free minced synthetic cooked
meat in broth (contain hematin &
glutathione that act as reducing
agent)
Also used as a transport medium
Anaerobic System
Gaspak System
Anaerobic jar made of thick glass
or polycarbonate.
Anaerobic atmosphere generated
by introducing a commercially
available disposable Anaerobiosis
generating device,and closing the
lid.
Anaerobic System
Commercially available as
disposable envelope, containing
chemicals which generate H2 ,
CO2 with the addition of water.
Reduced methylene blue is
used as indicator. Remains
colorless anaerobically but
turns blue on exposure to O2
Gram stain
Gram positive Gram negative
cocci
bacilli
aerobic
Anaerobic
catalase
Peptostreptococcus
Peptococcus
+
-
S. aureus - coagulase +
S. saprophyticus – coag - β haemolysis
novo R Strep. Gp A – Bacitracin +
S. epidermidis – coag – Strep, Gp B – CAMP +
novo S Strep. Gp. D – Bile esculin + γ haemolysis
α haemolysis Strept. Gp D
Strep. pneumoniae – optochin +
Viridans group bile solubility +
Strep. Grp D Inulin ferm +
Mouse pathogenicity +
Gram positive
bacilli
aerobic anaerobic
non spore forming
Spore forming C. diphtheriae Spore forming Non Spore forming
Bacillus Listeria C. perfringens Actinomyces
B. anthracis Nocardia C. tetani Propionibacterium
B. cereus C. botulinum
B. subtillis C. difficile
Gram negative
Cocci
Bacilli
Aerobic
N. gonorrhoeae Anaerobic Aerobic Anaerobic
N meningitidis Veillonella Enterobacteriaceae Bacteriodes
E. coli Fusobacterium
Klebsiella
Proteus
Salmonella
Shigella
Fastidious
Haemophilus
Brucella
Cholera Non fermenters
Campylobacter Pseudomonas
Stenotrophonomas
Acinetobacter
Peptostreptococcus
Aerobic plate: no Anaerobic plate:
growth growth
Gram stain:
Gram positive
cocci
Obligate Anaerobe
Susceptible to
metronidazole
Peptococcus
Aerobic plate: no Anaerobic plate:
growth growth
Gram stain:
Gram positive
cocci
Obligate Anaerobe
Susceptible to
metronidazole
Bacteroides sp.
Bacteriodes sp. involved in the following:
Purulent, foul smelling and gangrenous infections
Septic abortions
Peritonitis
Abscesses of brain, liver or lung
Bacteremia or septicemia
Bacteriodes fragilis
Aerobic plate: no Anaerobic plate:
growth growth Gram stain:
Gram negative
bacilli
Obligate Anaerobe
Susceptible to
metronidazole
Clostridium sp.
Clostridium form endospores under adverse
environmental conditions
Spores are a survival mechanism
Spores are characterized on the basis of
position, size and shape
Most Clostridium spp., including C. perfringens
and C. botulinum, C difficile have ovoid
subterminal spores
C. tetani have round terminal (RT) spores
Clostridium perfringens
Microscopy
Gram stain
Organism appear as
thick brick shaped bacilli
Stain irregularly and may
form chains.
Spores are rarely seen
Usually in the absence of
leukocytes.
Clostridium perfringens
Colonies are surrounded by a
double-zone haemolysis on
blood agar.
Note the small area of beta
hemolysis (complete lysis of
red blood cells) surrounded by
a larger zone of alpha
hemolysis (partial hemolysis)
Reverse CAMP Test
Rapid identification of C. perfringens is possible with the
Reverse CAMP test
Used to demonstrate the phenomenon of a synergistic
effect of hemolysis with CAMP factor and the alpha
hemolysin (alpha toxin) of Clostridium perfringens.
Similar to the CAMP test, excepting that the Streptococcus
Group B is plated down the center.
A known Clostridium perfringens at a 45o angle to it.
The test organism is then also plated at a 45o angle to the
Streptococcus Group B.
A positive test shows a C-shaped increase zone of
hemolysis.
Reverse CAMP Test
HANSEN, M. V. & ELLIOT, L. P. New presumptive
identification test for Clostridium perfringens: reverse
CAMP test. Journal of clinical microbiology, 12: 617-619
(1980).
Reverse CAMP Test
C.
Perfringens
Control
Streptococcus
Group B
Control
Test
Nagler’s Reaction
Opacity
Look for opacity around the inoculum in the half of the plate
containing no antitoxin and
no opacity in the half of the plate containing the antitoxin.
Nagler’s Reaction
Positive Nagler’s
Procedure of Nagler’s Reaction
Reaction
Stormy Clot
Clostridium perfringens ferments lactose
producing lactic acid
changing the medium to a pink – red colour
Also hydrolyse the milk protein casein
resulting in the coagulation of the milk (i.e. the formation of a clot)
thus separating the curd from the whey.
The end product of lactose fermentation is the formation of
gas.
Clostridium perfringens produces an abundance of gas
resulting in the breaking up of the clot formed thus referred to as a
stormy clot.
Stormy Clot
“Stormy clot”
clot torn by gas in 24hrs.
Stormy clot positive
Laboratory Diagnosis of
Tetanus
The diagnosis of tetanus depends primarily upon the
clinical manifestation of tetanus including muscle spasm
and rigidity.
Clostridium tetani is recognized by:
Its rhizoidal growth
Drum stick appearance on gram stain
Production of indole
Animal inoculation with control for demonstration of
toxigenicity
Clostridium tetani
Microscopy
Gram stain
Organisms appear as long
thin rods with round
spores at the end (drum
stick spores).
Note: Round terminal spores give cells a “drumstick” or
“tennis racket” appearance.
Clostridium botulinum
The diagnosis of botulism
depends
primarily upon isolation of the
organism or detecting the toxin in
food or the patient’s faeces or
serum.
Clostridium botulinum
Microscopy
Gram stain
Organisms appear as
pleomorphic rods with
oval
sub terminal spores.
Actinomyces israeli
Hard yellow "Sulfur granules"
Characteristically found in pus
from lesions
Diagnostic
Gram positive filamentous
bacilli
"Sulfur granules" actually a
mass of Actinomyces
filaments solidified with
tissue exudates
Actinomyces israeli
Anaerobic, gram-positive bacteria that forms long,
branching filaments resembling the hyphae of fungi.
Aero tolerant
Should be cultured under anaerobic conditions.
Growth is slow
An extended incubation time is usually required (4-
10 days).
Most species are nonhemolytic, nonproteolytic, and
catalase-negative.
THE END
