This document describes Clostridium perfringens, including its morphology, cultural characteristics, pathogenicity, and laboratory diagnosis. It notes that C. perfringens is a gram-positive, spore-forming bacterium that can cause gas gangrene, food poisoning, and necrotizing enteritis. Laboratory diagnosis involves microscopic examination of samples, culturing in anaerobic conditions to observe hemolysis, and biochemical tests of fermentation and toxin production. Definitive identification requires animal testing using antitoxins. Treatment involves antibiotics like penicillin along with antitoxins.

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.

15. Anaerobic or clostridial cellulitis

17.  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.

18.  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:

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

20.  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.

22. FOOD POISONING

23. 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.

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

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

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

28. 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.

29. 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.

30. 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

31. 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

32.  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

33. 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.

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