Lecture notes about medically important species in genus Clostridium (Hussein Al-tameemi, Iraq)

1. GRAM POSITIVE BACILLI
Hussein A. Abid
B. Sc. M. Clin. L. T.
Middle Technical University
Ba’aquba Medical Technical Institute
Medical Laboratory Technology Department
Practical Week: 13

2. Classification (gram +ve rods)
Non-spore formingSpore forming
• Corynebacterium spp.
• Lactobacillus spp.
• Mycobacterium spp.
• Bacillus spp.
• Clostridium spp.
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• Bacterial spores are highly resistant, dormant structures
(i.e. no metabolic activity) formed in response to adverse
environmental conditions.
• They help in the survival of the organisms during adverse
environmental conditions; they do not have a role in
reproduction.

3. Clostridium spp.
• Gram-positive rods (at least early in growth), in singles,
pairs, or chains.
• Most are obligate anaerobes, but some are
microaerophiles.
• Produces endospores, but not aerobically; spore shape
and position are variable, but usually distend the cell
• Most are catalase-negative
• Most are isolated from soil, sewage, or marine sediments
• Key pathogens are C. tetani (tetanus), C. botulinum
(botulism), C. perfringens (food poisoning and gas
gangrene), and C. difficile (pseudomembranous colitis).
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4. Clostridium spp.
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5. Clostridium spp.
Specimens
• C. perfringens: wound swabs, necrotic tissues, muscle
fragments, exudate from active parts etc..
• C. difficile: stool samples (diarrhoea)
5Diabetic foot

6. Clostridium spp.
Microscopy (Gram +ve rods)
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C. perfringens C. tetani
C. difficile C. botulinum

7. C. perfringens (Culture)
• Samples are cultured on BAP anaerobically (i.e. using jar)
• Tryptose sulphite cycloserine agar (TSCA)
• Robertson’s Cooked Meat broth & Thioglycolate broth media
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Double zone of clear beta-hemolysis around
a colony is clearly seen (arrow)
TSCA (C. perfringens)

8. C. perfringens (Culture)
• On egg yolk medium it shows white precipitate due to
production of Lecithinase or phospholipase (Nagler’s
reaction)
• Reverse CAMP test (CAMP positive)
Positive Nagler reactionProcedure 8

9. C. difficile (Culture)
• Blood agar (anaerobically): large, flat colonies with barnyard
smell.
• Cycloserine Cefoxitin Fructose Agar (CCFA)/ selective: 4 mm
colonies appear as yellow and ground class-like and have
filamentous edge, the odor associated with colonies is very
distinct and typically like elephant or horse manure.
9CCFABlood agar

10. C. tetani (Culture)
• On blood agar it produces a
fine film of growth.
• Swarming due to its motility.
• On fresh BAP it is hemolytic
(alpha first followed by beta
hemolysis).
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11. Advanced techniques
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• Clostridium difficile rapid test:
• Toxin analysis (PCR & ELIZA): C. perfringens & C. difficile

12. Simple differentiation chart
C. tetaniC. botulinumC. difficileC. perfringensCharacter
MotileMotileMotileNonmotileMotility
–––+Lecinthase
Eggyolk
agar
–+––Lipase
––++Glucose
Acidformation
–++–Lactose
–++–Sucrose
––+–Maltose
++++Hydrolysis of gelatin
–+–+Digestion of casein
V–––Indole production
Terminal,
spherical
endospore
ToxinsToxins
Stormy-clot in
Litmus milk
Additional features
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