1.
RATHEESH R.L
CLOSTRIDIUM

2.
 There are three medically important species are
there,
 1. Cl. Perfringens or Cl. welchii
 2. Cl. Tetani
 3. Cl. Botulinum

3.
Clostridium Perfringens
MORPHOLOGY
 They are large, gram positive, capsulated and
non-motile.
 The size varies from 4-6mu m to 1 mu m
 They occurs either in single form or as chains

4.
CLOSTRIDIUM PERFRINGENS

5.
CULTURAL CHARACTERISTICS
 Mostly they are anaerobic,
 which is growing in 37 degree C and in the
ph of 5.5 to 8.

6.
NUTRIENT AGAR MEDIUM
 Two types of colonies will be formed
 One is round, smooth, regular, convex,
grayish yellow which measures 2-4mm in
diameter.
 Second, umbonate (having a projection) with
opaque brownish centre and a lighter,
translucent periphery with a curved edge.

7.
BLOOD AGAR MEDIUM
 Clostridium perfringens usually produces a
double zone of hemolysis
 The inner zone shows complete hemolysis
(beta-hemolysis), whereas the outer zone
may display partial hemolysis (alpha-
hemolysis).

8.
Robertson’s cooked meat media
 After 24-48 hrs of incubation, medium becomes
turbid with production of gas.
 The meat pieces become red but not digested.

9.
Marshal’s medium
 This medium contains polymyxin – B, iron citrate
and neomycin.
 The colonies formed will be black in colour.

10.
PATHOGENESIS
 CLOSTRIDIAL GAS GANGRENE
 FOOD POISONING
 NECROTIZING ENTERITIS

11.
CLOSTRIDIAL GAS GANGRENE
 Clostridial gas gangrene is a highly lethal necrotizing
soft tissue infection of skeletal muscle caused by toxin-
and gas producing Clostridium species.
 The organism lives in the intestinal tract of human and
animals.
 The spores of organism are widely distributed in
nature, present in dust,soil,human skin and feces

12.
 When the wound gets contaminated by soil, dust,
or feces three types of clinical conditions may be
produced
 Simple wound infection
 Anaerobic or clostridial cellulitis
 Anaerobic myositis

13.
SIMPLE WOUND INFECTION
 Clostridiae bacilli will be present over the
wounded tissue with out producing any wound
infection.
 Clostridiae bacilli can’t be multiply because of
lack of anaerobic conditions in open wound
 So that they will delay in the process of wound
healing.

14.
Anaerobic or clostridial cellulitis

15.
 These organisms are present in soil and dust.
 Clostridia have been isolated from the mucous
membranes of humans, including the GI tract and
the female genital tract.
 Clostridia may also colonize the skin, especially
around the perineum.
 Bacterial multiplication and the production of
soluble proteins called exotoxins require a low
oxygen tension.

16.
 Other bacteria are also capable of producing gas,
and nonclostridial organisms have been isolated
in 60-85% cases of gas gangrene.
 C perfringens produces at least 20 exotoxins. The
most important exotoxins and their biologic
effects are as follows:

17.
 Alpha toxin - necrotizing, hemolytic, cardiotoxic
 Beta toxin - necrotizing
 Iota toxin - necrotizing
 Delta toxin - hemolysin
 Phi toxin - Hemolysin, cytolysin

18.
 The alpha toxin produced by the organism
destroys cell membrane of muscle fibers and
leading to massive necrosis of muscle tissue.
 Hemolytic toxins cause lysis of RBC’s and
resulting to hemolytic anemia and
hemoglobinuria.

19.
FOOD POISONING

20.
NECROTIZING ENTERITIS
 Necrotizing enterocolitis (NEC) or necrotizing
enteritis is the death of tissue in the intestine.
 It occurs most often in premature or sick babies.

21.
LABORATORY DIAGNOSIS
 It includes,
• Hematological investigations
• Bacteriological investigations

22.
Hematological investigations
 Usually it does not cause any changes in the total
count or differentiated count

23.
Bacteriological investigations
 1. microscopic studies
 2. culture studies
 3. biochemical studies
 4. Nagler’s reaction test
 5. animal pathogenecity test

24.
microscopic studies
 Usually the specimens are collected as wound
swabs, exudates or discharges, edges of affected
muscles, necrotized tissue and muscle fragments.
 Gram staining of the smear made from the above
specimen shows gram positive, spore bearing
,chain shaped bacilli.

25.
culture studies
 Specimen is inoculated in the blood agar medium
or cooked meat medium and incubated
anaerobically for 48-72 hours.
 In that beta hemolysis will occur around the
colonies.

26.
BIOCHEMICAL STUDIES
 1. sugar fermentation test: fermentation of
glucose, maltose and lactose will occur with the
production of acid and gas.
 2. indole test: negative
 3. H2S Production test: positive
 4. litmus milk test: production of acid and gas
occurs

27.
NAGLER’S REACTION TEST
 Label and dry a egg yolk media plate an mark the
plate into two halves.
 Inoculate 2-3 drops of Clostridium perfringes type A
antitoxin in half of the plate, spread over the surface
of agar using a spreader and allow to absorb and dry.
Mark the side of the plate in which the antitoxin is
inoculated.
 Place the inoculated organism over the medium

28.
 Incubate anaerobically at 35-37 degree C for 24-
48 hrs.
 Examine the plate for an opalescent halo around
the inoculum and inhibition by antitoxin.Observance Inference
A zone of opacity in the antitoxin-free half only
but not on other half due to nutralization of the
alpha toxin.
positive study
A zone of opacity on both sides of the plate or
no reaction on the agar.
negative study

29.
ANIMAL PATHOGENECITY TEST
 In this test 0.1 ml of organism containing broth
will administer intra muscularly to a lower limb of
guinea pig
 Same amount is injected in control animal which
is protected with 300units of antitoxin before test.
 On autopsy, the injected limb shows swelling with
sub cutaneous crunching of tissue due to the
accumulation of gas bubbles.

30.
TREATMENT
 The common drugs are penicillin, sulphonamides
and metronidazole
 Anti toxins are administered with antibiotics