Differential Reinforced Clostridium Broth

Beef extract and casein peptone provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly of the B-group, essential for bacterial growth. Glucose is the fermentable carbohydrate providing carbon and energy. L-Cysteine hydrochloride is the reducing agent. Starch absorbs any toxic metabolites produced. Ferric ammonium citrate and sodium sulfite are H₂S indicators.

Clostridium reduce sulfite to sulfide, the iron sulfide produced causes the culture medium to turn black. As other bacteria can also produce sulfide, vegetative forms must first be removed from the culture by a relevant treatment (e.g. pasteurization), and the anaerobic spore-forming microorganisms must then be identified. To inhibit the growth of most non-spore-forming microorganisms, add 70 IU/mL polymyxin to the broth.

Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.

Preparation

Suspend 30.1 grams of the medium in one liter of distilled water. Mix well and dissolve by heating with frequent agitation. Boil for one minute until complete dissolution. Dispense into appropriate containers and sterilize in autoclave at 121 °C for 15 minutes.

According to ISO 6461-1

For detection and enumeration of the spores of sulfite-reducing anaerobes (clostridia) according to ISO 6461-1 (UNE-EN 26461-1):

1. Heat the sample in a water bath at 75 ± 5 °C for 15 min.
2. Add 50 mL of sample to a 100 mL bottle containing 50 mL of the double strength medium.
3. Add 10 mL of sample to a series of five 25 mL bottles containing 10 mL of the double strength medium.
4. Add 1 mL of sample to a series of five 25 mL bottles containing 25 mL of the single strength medium.
5. Incubate the inoculated bottles at 36 ± 1 °C for 44 ± 4 h.
6. Bottles in which blackening is observed shall be regarded as positive.
7. Express the results as the most probable number of sulfite-reducing anaerobes (clostridia).

• GIBBS, B.M.: The detection of Clostridium welchii in the Differential Clostridial Medium technique. - J. Appl. Bact., 36; 23-33 (1973).
• HIRSCH, A., a. GRINSTED, E.: Methods for the growth and enumeration of anaerobic spore-formers from cheese, with observations on the effect on nisin. - J. Dairy Res., 21; 101-110 (1954).
• UNE-EN 26461-1 Detección y recuento de los esporos de microorganismos anaerobios sulfito-reductores (clostridia) Parte 1: Método por enriquecimiento en un medio líquido.