Clostridias

Clostridium spp (including C. botulinun, C. perfringene) (Gram Positive)

This is a method with reference to the Thai Pharmacopoeia’s Method, according to Thai Pharmacopoeia, Vol. 1, 1987

Information retrieved from 2nd Revised Draft WHO guidelines on assessing safety and quality of herbal medicine with reference to contaminants and residue, July 2004 , 2004, WHO, Geneva Switzerland

Enrichment
· Boil 2 tubes of 100ml Cook Meat Medium for 100˚C for 10 min
· Cool to 37˚C
· Weigh 10 g of sample to tubes
· Seal up on tube immediately with parafilm
· Another heat to 65˚C for 30min
· Seal the heated tube
· Incubate both tubes for 37˚C for 4X24hrs

· At every 24hrs check for changes

Differentiation between C. perfringnes , C. botulinum , C. tentani

1. All Clostridium spp will not digest any of the meat
2. C. perfringnes will cause the meat to turn pink
3. C. botulinum will cause the tubes to have bubbles and white sediments
4. C. tentani will give off burnt organic smell

Confirmation step

1. Inoculate the broth in duplicates into 5% Defibrinated Sheep’s Blood agar
2. Incubate at 37˚C for 48hrs, one aerobically, another anaerobically.

Differentiation between C. perfringnes , C. botulinum , C. tentani colonies
Colony Morphology
Gram Stain
Spore Stain

Colony morphology
C. botulinum: Irregular
Translucent
Granular surface
Fimbrated spreading edge
Haemolysis

C. perfringnes: Round convex
Translucent
Smooth
Entire
Double zone of haemolysis


C. tentani : Transparent
Long Feathering projection
Haemolysis

Gram stain
All Gram Positive

Spore stain
C. botulinum: Oval, cocci

C. perfringnes: No exosporium observed


C. tentani : Drum Stick Shape