Clostridia Diagnostic
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 | Product. 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 | Product 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 Differential Media | Brand | Product 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. |
Test for Clostridia Diagnostics | Brand | Product 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 3. Gram staining kit and single solutions
Table 4. Anaerobic equipment
References
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