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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
Classification (gram +ve rods)
Non-spore formingSpore forming
• Corynebacterium spp.
• Lactobacillus spp.
• Mycobacterium spp.
• Bacillus spp.
• Clostridium spp.
2
• 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.
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).
3
Clostridium spp.
4
Clostridium spp.
Specimens
• C. perfringens: wound swabs, necrotic tissues, muscle
fragments, exudate from active parts etc..
• C. difficile: stool samples (diarrhoea)
5Diabetic foot
Clostridium spp.
Microscopy (Gram +ve rods)
6
C. perfringens C. tetani
C. difficile C. botulinum
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
7
Double zone of clear beta-hemolysis around
a colony is clearly seen (arrow)
TSCA (C. perfringens)
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
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
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).
10
Advanced techniques
11
• Clostridium difficile rapid test:
• Toxin analysis (PCR & ELIZA): C. perfringens & C. difficile
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
12

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Clostridium spp. (Practical Medical Microbiology, 13)

  • 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. 2 • 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). 3
  • 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) 6 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 7 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). 10
  • 11. Advanced techniques 11 • 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 12