Reinforced Clostridial Medium

Intended Use

Reinforced Clostridial Medium is used for cultivating and enumerating clostridia, other anaerobes, and other species of bacteria from foods and clinical specimens. Meets United States Pharmacopeia (USP), European Pharmacopoeia (EP) and Japanese Pharmacopoeia (JP)1-3 performance specifications, where applicable.

Summary and Explanation

Reinforced Clostridial Medium is a semisolid medium formulated by Hirsch and Grinstead.4 Their work demonstrated that the medium outperformed other media in supporting growth
of clostridia from small inocula and produced higher viable cell counts.4 Barnes and Ingram5 used the medium to dilute vegetative cells of Clostridium perfringens. Barnes et al.6 used a solid (agar) version of the medium to enumerate clostridia in food.
Reinforced Clostridial Medium is a nonselective enrichment medium and grows various anaerobic and facultative bacteria when incubated anaerobically.7 This medium has been used to
detect clostridia, bifidobacteria and other anaerobes in food products8-11 and fecal samples.12 Reinforced Clostridial Medium is listed in the USP as the recommended medium for the isolation of Clostridium sp. from nonsterile pharmaceutical products.1

Principles of the Procedure

Reinforced Clostridial Medium contains peptone and beef extract as sources of carbon, nitrogen, vitamins and minerals. Yeast extract supplies B-complex vitamins which stimulate bacterial growth. Dextrose is the carbohydrate source. Sodium chloride maintains the osmotic balance. In low concentrations, soluble starch detoxifies metabolic by-products. Cysteine HCl is the reducing agent. Sodium acetate acts as a buffer. The small amount of agar makes the medium semisolid.

User Quality Control

Identity Specifications
Reinforced Clostridial Medium
Dehydrated Appearance: Light tan, free-flowing, homogeneous.
Solution:                       3.8% solution, soluble in purified water upon
                                    boiling. Solution is medium amber, slightly
                                    opalescent with dark particles and flocculation
                                    when hot.
Prepared Appearance:     Upon cooling, medium amber and becomes
                                    more opalescent.
Reaction of 3.8%
Solution at 25°C:            pH 6.8 ± 0.2
Reinforced Clostridial Medium (prepared)
Appearance:                  Light to medium amber and opalescent with particles.
Reaction at 25°C:           pH 6.8 ± 0.2
Cultural Response
Reinforced Clostridial Medium
Prepare the medium per label directions. Inoculate and incubate tubes with caps tightened at 35 ± 2°C for 18-48 hours. Inoculate 100 mL bottles with C. sporogenes cultures and incubate with caps tightened at 30-35°C for 48 hours.

Reinforced Clostridial Medium (prepared)
Inoculate and incubate bottles with caps tightened at 30-35°C for up to 48 hours.

Formula

Reinforced Clostridial Medium
Approximate Formula* Per Liter
Peptone..................................................................... 10.0 g
Beef Extract................................................................ 10.0 g
Yeast Extract................................................................ 3.0 g
Dextrose...................................................................... 5.0 g
Sodium Chloride.......................................................... 5.0 g
Soluble Starch.............................................................. 1.0 g
Cysteine HCl................................................................ 0.5 g
Sodium Acetate........................................................... 3.0 g
Agar............................................................................ 0.5 g
*Adjusted and/or supplemented as required to meet performance criteria.

Precautions13

1. Biosafety Level 2 practices, containment equipment and facilities are recommended for activities with clinical specimens of human or animal origin containing or potentially containing
C. botulinum or C. tetani or their toxins.
2. Biosafety Level 3 practices, containment equipment and facilities are recommended for all manipulations of cultures of these organisms and for activities with a high potential for aerosol or droplet production, and those involving production quantities of toxin.

Directions for Preparation from Dehydrated Product

1. Suspend 38 g of the powder in 1 L of purified water. Mix thoroughly.
2. Heat with frequent agitation and boil for 1 minute to completely dissolve the powder.
3. Autoclave at 121°C for 15 minutes.
4. Test samples of the finished product for performance using stable, typical control cultures.

Procedure

For pharmaceutical samples, refer to the USP for details on sample collection and preparation for testing of nonsterile products.1 Refer to USP General Chapter <62> for details on the examination of nonsterile products and the isolation of clostridia using Reinforced Clostridial Medium.1

Expected Results

After appropriate incubation time and temperature, subculture each tube or bottle to two Columbia Agar plates. Incubate under both aerobic and anaerobic conditions for 48 hours at 30-35°C to confirm the presence of anaerobic growth. After incubation of these plates, if isolates grow anaerobically only (with or without endospores) and are catalase negative, this indicates the presence of Clostridium sp.1 Perform other confirmatory biochemical testing as necessary.

* Store at 2-8°C.
† QC testing performed according to USP/EP/JP performance specifications.

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