Reinforced Clostridal Agar

Hirsch and Grinstead formulated Reinforced Clostridial Medium (LC-1007) and found that this medium is superior to others in supporting growth and producing high cell counts of Clostridia. When incubated anaerobically, this medium grows various anaerobes and other bacteria. Barnes and Ingram also demonstrated that it can be used to develop vegetative cells in assays of Clostridium perfringens.

Peptone and beef extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is a source of vitamins, particularly of the B-group. Dextrose is the fermentable carbohydrate providing carbon and energy. Sodium chloride supplies essential electrolytes for transport and osmotic balance. Starch in the medium acts as a growth factor, probably functioning like a colloid protector, and neutralizes toxic products that form during the development of the organisms. L-Cysteine hydrochloride is the reducing agent and sodium acetate is the buffer. Bacteriological agar is the solidifying agent.

Since it is a non-selective enrichment medium, it allows the growth of various anaerobic microorganisms and facultative bacteria when incubated anaerobically.

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

Preparation

Suspend 50 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. Cool to 45-50 °C and, if desired, add 0.02 g/L of polymyxin B in a sterile filtered solution.

Instructions for use

Pour plate method:

1. Deposit 1 mL of the initial suspension and/or diluted sample in an empty Petri dish.
2. Add 12-15 mL of agar cooled to 44-47 °C in each Petri dish and mix gently moving the plate.
3. Allow the plates to solidify and incubate in an inverted position at a temperature of 35 ± 2 °C for 40-48 hours in anaerobiosis.

• Barnes, E.M., J.E. Despaul and M. Ingram 1963. The behavior of a food poisoning strain of Clostridium welchii in beef. J. Appl. Bacteriol 26:415.
• MacFaddin J.F. 1985 Media for isolation-cultivation-identification-maintenance of medical bacteria, vol. 1. p. 660-668. Williams & Wilkins, Baltimore MD.