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Clostridia Diagnostic

By: Jvo Siegrist, AnalytiX Volume 7 Article 2

Detection, identification and differentiation of Clostridia species

Clostridia are relatively large, gram-positive, rod-shaped bacteria that can undergo only anaerobic metabolism. Most Clostridia cannot grow under aerobic conditions and even can be killed by exposure to O2, but they form endospores that are able to survive long periods of exposure to air and other adverse environmental conditions. The natural sources of Clostridia are anaerobic habitats with organic nutrients, particularly soils, aquatic sediments and the intestinal tracts of animals. Their fermentation of organic compounds, like sugars, produces large amounts of CO2 and H2 as well as volatile organic compounds like acetic and butyric acid, acetone and butanol. Metabolism of substrates like amino acids and fatty acids results in foul-smelling degradation products. Clostridia also have an extended range of extracellular enzymes that degrade large biological molecules in the environment into fermentable compounds. Although there are non-pathogenic Clostridia, this genus produces some of the most potent biological toxins. Three particularly bad actors in this group are C. perfringens, which is responsible for cooked meat-associated food poisoning and wound and surgical infections that lead to gas gangrene, and C. tetani, which is responsible for deadly tetanus infections, and C. botulinum, which causes botulism.

Below are the most well-known pathogenic Clostridia species with their typical properties and occurrence:

Clostridium perfringens

  • produces a huge range of invasins and exotoxins
  • enzymes: hemolysins (ß-hemolysis), lecithinase, extracellular proteases, lipases (phospholipase-C), collagenase, hyaluronidase, saccharolytic enzymes and is able to reduce sulphite to sulphide
  • enterotoxins causes food poisoning
  • found in improperly sterilized canned foods (germination of endospores) and water
  • nonmotile

Clostridium difficile

  • produces two enterotoxins toxin A and toxin B (lethal cytopathic toxin)
  • enzymes: hydrolytic enzymes, p-hydroxyphenylacetate decarboxylase, ferments mannitol
  • formation of p-cresol as the main fermentation product of tyrosin

Clostridium tetani

  • toxin: tetanospasmin (causative tetanus)
  • obligate anaerobe (sensitive to oxygen)
  • sensitive to heat
  • flagella give limited motility
  • terminal spore (resistant to heat and most antiseptics)
  • typical gram-positive, may stain gram-negative or gram-variable, especially in older cells

Clostridium botulinum

  • seven subtypes (A-G) produces different botulinum toxin (types C and D are not pathogenic)
  • grow best in low-oxygen conditions
  • subterminal endospores (resistant to boiling without pressure)
  • occurrence: soil, aquatic sediments, decaying vegetation, found in improperly sterilized canned foods (germination of endospores)
  • acidity, high concentration of sugar, very low levels of moisture or high levels of oxygen inhibits the growth
  • enzyme: lipase production on egg yolk agars

Identification of Clostridia is an important first step toward the control and eradication of this potent pathogen. To aid in the diagnosis, Sigma-Aldrich has developed a broad range of selective media (Table 1), tests (Tables 2 and 3) and anaerobic equipment (Table 4) for the detection, identification and differentiation of Clostridia.

Non-selective 120Media Brand Cat. No.  Description
AC Agar Sigma A3340 Supports the growth of aerobic, anaerobic and microaerophilic microorganisms.
AEA Sporulation Broth (Base), modified Sigma-Aldrich 17170 For early sporulation of C. perfringens from foods.
Alternative Thioglycollate Medium Sigma A0465 Recommended for sterility testing with certain biological products which are turbid or viscous.
Brain Heart Infusion Agar Sigma-Aldrich 70138 A solid medium for the cultivation of fastidious pathogenic bacteria such as Streptococci and Neisseria.
Brewer thioglycollate medium Sigma B2551 Used for testing the sterility of biological products.
Brucella Agar with Hemin and Vitamin K Sigma B2926 Used for the isolation and subculture of anaerobes.
Casein peptone Lecithin Polysorbate Broth Sigma-Aldrich 22089 For the enumeration of samples from pharmaceutical, cosmetic industry.
Columbia Agar Sigma-Aldrich 27688 For the cultivation of fastidious microorganisms.
Cooked Meat Broth Sigma-Aldrich 60865 For the primary cultivation of aerobic, microaerophilic and anaerobic bacteria from clinical specimens.
Reinforced Clostridial Agar Sigma R0898 Used for the cultivation and enumeration of Clostridia.
Skim Milk Agar, modified Sigma-Aldrich 17175 For cultivation and enumeration of microorganisms encountered in dairy industry.
Thioglycolate Broth Sigma-Aldrich 70157 A medium for sterility tests and the cultivation of microaerophilic and anaerobic organisms.
Thioglycolate Broth with Resazurine Sigma-Aldrich 90404 For cultivation of aerobic and anaerobic organisms, and for sterility testing. Any increase in the oxygen content is indicated by a color change of redox indicator resazurin.
Tryptone Soya Broth without Dextrose Sigma T3938 Recommended for the cultivation of anaerobes from root canals and blood.


Differential Media Brand Cat. No.  Description
Blood Agar (Base) Sigma-Aldrich 70133 A non-selective medium for the isolation and cultivation of many pathogenic and nonpathogenic microorganisms.
Gelatin Iron Agar Sigma G0289 Used for detecting gelatin liquefaction and hydrogen sulphide production.
Meat Liver Agar Sigma-Aldrich 46379 For the cultivation of anaerobic microorganisms.
Nutrient Gelatin Sigma-Aldrich 70151 Nutrient gelatin is recommended for the determination of gelatin-liquefying microorganisms and enumeration of proteolytic organisms in water.


Selective Media Brand Cat. No.  Description
Clostridium difficile Agar (Base) Sigma-Aldrich 17145 Used with supplement for cultivation of C. difficile from food and certain pathological specimens.
Wilkins Chalgren Anaerobic Agar Sigma W1761 Used for the isolation of anaerobic bacteria.


Selective Differential Media Brand Cat. No. Description
HiCrome™ M-CP Agar Base Sigma-Aldrich 75605 Selective chromogenic media recommended by the Directive of the Council of the European Union 98/83/EC for isolation and enumeration of C. perfringens from water (see Figure 1).
SPS Agar, modified Sigma-Aldrich 17231 For the selective isolation and enumeration of C. perfringens from foods.

Table 1 ......... Media for Clostridia


Test for Clostridia Diagnostics Brand Cat. No.  Description
Aminopeptidase Test Sigma-Aldrich 75554 For the detection of L-alanine-aminopeptidase which is found primarily in gram-negative microorganisms.
Mannitol Disks Sigma-Aldrich 94438 Used to differentiate bacteria on the basis of mannitol fermentation.
Nitrate Reagent Disks Sigma-Aldrich 08086 Used to detect an organism’s ability to reduce nitrate.
Tributyrin-Strips Sigma-Aldrich 75744 The test principle is hydrolysis of tributyrin. This reaction causes color change of acidobasic indicator.

Table 2 ......... Tests for identification and differentiation of Clostridia


Gram Stain Brands Cat. No.
Gram Staining Kit Sigma-Aldrich 77730
Gram‘s Crystal Violet Solution Sigma-Aldrich 94448
Gram‘s Decolorizer Solution Sigma-Aldrich 75482
Gram’s Fuchsin Solution Sigma-Aldrich 87794
Gram‘s Iodine Solution Sigma-Aldrich 90107
Gram‘s Safranin Solution Sigma-Aldrich 94635

Table 3 ......... Gram staining kit and single solutions


Anaerobic Equipment Brand Cat. No.
Anaerobe Atmosphere Generation Bags Sigma-Aldrich 68061
Anaerobe Indicator Test Sigma-Aldrich 59886
Anaerobe Jar Insert for Petri Disks Sigma-Aldrich 68886
Anaerobic Jar Sigma-Aldrich 28029

Table 4 ......... Anaerobic equipment


Further details about the media, tests and equipment for Clostridia and many other pathogens can be found on our Web site:

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  1. Selmer, T.; Andrei, P. I. p-Hydroxyphenylacetate decarboxylase from Clostridium difficile: A novel glycyl radical enzyme catalysing the formation of p-cresol, Eur. J. Biochem., 268, 1363-1372 (2001).
  2. Wells, C. L..; Wilkins, T. D. Botulism and Clostridium botulinum, In Baron‘s Medical Microbiology, 4th ed., S. Baron Editor, Univ. of Texas Medical Branch, (1996).
  3. Sherris Medical Microbiology, 4th ed., K.J. Ryan and C.G. Ray Editors, McGraw Hill, (2004).
  4. Wells, C. L..; Wilkins, T. D., Clostridia: Sporeforming Anaerobic Bacilli, In Baron‘s Medical Microbiology, 4th ed., S. Baron Editor, Univ. of Texas Medical Branch, (1996).
  5. Elmer Koneman, W. et al Color Atlas and Textbook of Diagnostic Microbiology, 5th ed., Lippincott Williams & Wilkins, (1997).


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