Clostridium perfringens, their Properties and their Detection

By:, AnalytiX Volume 10 Article 4

Role of Clostridium perfringens and their detection, identification, and differentiation
Product Manager Microbiology

Clostridia are relatively large, Gram-positive, rod-shaped bacteria that can undergo only anaerobic metabolism. Most Clostridia cannot grow under aerobic conditions and can even be killed by exposure to O2; however, 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, such as 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. The three particularly lethal members of this group are C. perfringens, which is responsible for cooked meat-associated food poisoning and wound and surgical infections that lead to gas gangrene, C. tetani, which is responsible for deadly tetanus infections, and C. botulinum, which causes botulism.

Clostridium perfringens is found in undercooked or improperly sterillized canned foods (germination of endospores) and in water (surface water). The natural contamination source is human and animal feces mainly transmitted into food by water. C. perfringens produces an extensive range of invasins and exotoxins. The enterotoxins cause the undesirable, mostly meat-associated, food poisoning and wound and surgical infections that lead to gas gangrene.

C. perfringens plays a subsidiary role in water examination4. Clostridia are spore builders and are resistant to heating, chlorination and other stress factors. In contrast to vegetative cells like coliforms (E. coli, enterococci), which are less resistant, C. perfringens has the advantage of surviving longer6. Therefore, while a fecal contamination is detected mostly by coliforms as an indicator, which could disappear after a processing step, C. perfringens remains present. The organism is not a hazard in water; rather, it is problematic when the water comes in contact with food.

In consideration of the aforementioned facts, it is obvious that detection and identification of C. perfringens is an important step towards the control and eradication of this potent pathogen. Some characteristic enzymes of C. perfringens are hemolysins (ß-hemolysis), lecithinase, extracellular proteases, lipases (phospholipase-C), collagenase, hyaluronidase, saccharolytic enzymes and enzymes to reduce sulphite to sulphide. These enzymes are also used as detection and differentiation targets. It is also notable that C. perfringens is a non-motile bacterium and it is the most important of the sulphite-reducing clostridia. Also, C. perfringens normally grows at 44 °C, whereas some other clostridia are inhibited at this temperature. This property is used in ISO methods to give the medium more selectivity4.

For detection of C. Perfringens, mCP and TSC agar have been recommended4, 5. However, there are problems associated with each of these media. The mCP method is very onerous for routine screening and bacterial colonies cannot be used for further biochemical testing. In addition, the recovery of C. perfringens was rejected by ISO in favor of methods based on TSC agar6. TSC detects all sulphite-reducing clostridia, however, and not only C. perfringens; in some cases, exces-sive blackening of the agar frustrated counting of the lower dilutions. On the other hand, if the contamination was too high, TSC did not consistently produce black colonies and, as a consequence, the colonies’ white color provided false negative results.

To prevent this problem, Sigma-Aldrich has introduced a new medium called CP ChromoSelect Agar, a chromogenic media from the new generation. Studies have shown that this medium produces counts that are not significantly different from those obtained from other media. The identification of typical and atypical colonies isolated from all media has demonstrated that CP ChromoSelect Agar (see Figure 1) was more specific to C. perfringens in water samples than mCP (see Figure 2) and TSCF agar.


Figure 1. Scanning electron micrograph of C. perfringens grown on a silicon wafer

Figure 1 Scanning electron micrograph of C. perfringens grown on a silicon wafer (source: S. Melville, Department of Biological Sciences, Virginia Tech University)

Figure 2. CP ChromoSelect Agar (C. perfringens appears as green colonies)

Figure 2 CP ChromoSelect Agar (C. perfringens appears as green colonies)


To keep Clostridium perfringens under control, 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.

Cat. No. Description
17170 AEA Sporulation Broth (Base), modified
15997 Brewer Agar
27544 Clostridial Differential Broth
27546 Clostridial Nutrient Medium
12398 CP ChromoSelect Agar
G0289 Gelatin Iron Agar
61348 Lactose Gelatin Medium (Base)
75605 m-CP Agar Base
14305 Motility Nitrate Medium
70151 Nutrient Gelatin
39727 Perfringens Agar Base
91365 Reinforced Clostridial Agar
85627 SPS Agar
17231 SPS Agar, modified
86128 Sulphite Iron Agar
T3938 Tryptone Soya Broth without Dextrose
93745 TSC Agar
93735 TSN Agar

Table 1 Common and new media for Colostridium perfringens (a complete list of clostridia media can be found on


Test for Clostridia Diagnostics Cat. No. Description
Aminopeptidase Test 75554 For the detection of L-alanine-aminopeptidase which is found primarily in Gram-negative microorganisms.
Kovac’s Reagent for indoles (isoamylic alcohol as solvent) 67309 For the differentiation of clostridia. In the presence of oxygen, some bacteria are able to split tryptophan into indole and
Kovac’s Reagent for indoles (butanol as solvent) 60983 a-aminopropionic acid. Clostridium perfringens is, for example, an indole-negative bacterium.
Mannitol Disks 94438 Used to differentiate bacteria on the basis of mannitol fermentation.
Nitrate Reagent Disks 08086 Used to detect an organism’s ability to reduce nitrate.
Tributyrin-Strips 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 Cat. No.
Gram Staining Kit 77730
Gram’s Crystal Violet Solution 94448
Gram’s Decolorizer Solution 75482
Gram’s Fuchsin Solution 87794
Gram’s Iodine Solution 90107
Gram’s Safranin Solution 94635

Table 3 Gram staining kit and single solutions


Anaerobic Equipment Cat. No.
Anaerobe atmosphere generation bags 68061
Anaerobe indicator test 59886
Anaerobic jar 28029

Table 4 Anaerobic equipment


Further details about the media, tests, and equipment for clostridia and many other pathogens can be found on our website

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  1. Sherris Medical Microbiology, 4th ed., K.J. Ryan and C.G. Ray Editors, McGraw Hill, 2004.
  2. 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.
  3. Elmer Koneman, W. et al Color Atlas and Textbook of Diagnostic Microbiology, 5th ed., Lippincott Williams & Wilkins, 1997.
  4. ISO 6461 Part 2. Water Qualily – detection and enumeration of Clostridium perfringens. (Revision of ISO 6461-2: 1986).
  5. Council Directive 98/83/EC, Relating to the Quality of Water Intended for Human Consumption, Official Journal of the European Communities, p.L330-32-L330/53 (1998).
  6. National Standard Method, Enumeration of Clostridium perfringens by membrane filtration, issue no 3.1, reference no W5i3.1 (2005).


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