CLOSTRIDIUM

By
STAFF MEMBERS of MICROBIOLOGY &
IMMUNOLOGY
FACULTY of MEDICINE , HELWAN UNIVERSITY
Objectives:

- Remember members of the genus Clostridium.

- Recognize serological types of Cl. perfringens .
- Explain methods of diagnosis of gas gangrene.
 CLOSTRIDIUM
Anaerobic, Gram positive, Spore forming bacilli

1- Cl. tetani Tetanus.

2- Cl. botulinum Botulism.
3- Cl. perfringens Gas gangrene &
food poisoning.
4- Cl. difficile pseudomembranous
colitis & antibiotic associated diarrhea.
Clostridium
perfringens
•Serological characters:
- According to the exotoxins produced:
Five types (A-E).
- A&C cause human diseases:
A1: Gas gangrene.
A2: Food poisoning.
C: Pigbel.
 Methods of bacteriological diagnosis

1- Specimen collection.
2- Smear preparation.
3- Cultivation:
A-Colony morphology..
B- Film preparation.
C- Biochemical reactions.
.Laboratory diagnosis:
• Sample: wound exudate aspirated from
deep sites of the wound ?
1. Microscopic examination:
- Gram stained smear.
2. Culture:
- Blood agar incubated anaerobically.
- Anaerobic culture media.
3. Biochemical reactions:
- Sugar fermentation test.
- Stormy clot reaction in litmus milk.
- Nagler's reaction.
4. Animal pathogenicity:
Animal inoculation & animal protection
test.
Sample: wound exudate aspirated from
deep sites of the wound ?
• Morphology:
Gram stain:
-Stain.
-Shape.
-Arrangement.
-Motility.
-Capsule.
-Spores.
(Site, Shape, size)
Gram positive large bacilli, spores are oval, sub terminal
and non projecting. It is capsulated, non-motile.
•Cultural characters:
- Oxygen requirement:

Strict anaerobes.
- Type: …………………….

- Temperature: 35-37°C.

- Time: 48-72 hours.

 Obligate Obligate
aerobes anaerobes

Aerotolerant Micro-
anaerobes aerophilic
Facultative
anaerobes
Anaerobic cultivation

Anaerobic culture methods:

1- Anaerobic GasPak system.
2- Anaerobic culture media :
- Robertson cooked meat medium.
- Thioglycollate broth.
Anaerobic GasPak system
 Special envelope is placed inside the jar, 10 ml distilled
water is added to the envelope immediately before closure,
this will release hydrogen and CO2
 Robertson cooked meat medium

Cooked minced meat

+ broth + reducing
substances (haematin
& glutathione)
 Thioglycollate broth

Sodium thioglycollate
Cl. perfringens is cultured on:
1- Blood agar incubated anaerobically:
- Anaerobic GasPak system.
- Beta haemolysis on blood agar.
- Colonies are large, semi translucent.
2- Neomycin blood agar:
- Selective medium.
3- Anaerobic culture media:
- Robertson cooked meat and thioglycollate broth.
Cl. perfringens on blood agar
Cl. perfringens on Robertson cooked meat &
thioglycollate broth
•Biochemical reactions:
- Sugar fermentation.
- Stormy clot reaction.
- Nagler's reaction.
 - Sugar fermentation:
Cl. perfringens ferment glucose, lactose,
maltose and sucrose producing acid and
large amount of gas.
- Stormy clot reaction :
Litmus milk: contain:
*Lactose: sugar.
*Casein: protein.
*Litmus: PH indicator.
When Cl. perfringens is inoculated on litmus
milk, it ferments lactose producing acid + gas:

*Acid cause coagulation of casein “acid clot”.

*Acid change the PH indicator “litmus” to
pink.
*Gas produced split the clot.
 - Nagler's reaction :
- Opalescence in egg yolk media.
(a visible precipitate around colonies).
- Lecithinase.
- Inhibited by antitoxin.
•Animal pathogenicity:
Animal inoculation &
animal protection test.