Reinforced Clostridial Medium (RCM) | AS-1339

Reinforced Clostridial Medium (RCM) was proposed by Hirsch and Grinsted (1954) for the cultivation and enumeration of Clostridia, other obligate anaerobes, and facultative microorganisms from foodstuffs and environmental materials. The medium complies with harmonised pharmacopoeial specifications of the European Pharmacopoeia (EP 2.6.13), United States Pharmacopoeia (USP <62>), and Japanese Pharmacopoeia (JP Chapter 4.05).

Cross-Reference Compatibility:

• Oxoid: Alternative to CM0149

• BD Difco: Alternative to 218081

• Merck: Alternative to 105411

RCM is free from selective inhibitors. L-Cysteine hydrochloride scavenges dissolved oxygen, maintaining a low Eh suitable for obligate anaerobes. Sodium acetate provides pH buffering and supplementary carbon. Soluble starch absorbs toxic metabolic byproducts. The semi-solid agar (0.5 g/L) retards O₂ diffusion, creating an anaerobic gradient at depth. Polymyxin B (not included) may be added post-sterilisation at 0.02 g/L to inhibit Gram-negative organisms.

• Pharmacopoeial harmonised — complies with EP 2.6.13, USP <62>, JP Chapter 4.05
• Rich nutrient base — meat extract + peptone + yeast extract + glucose supports fastidious anaerobes
• L-Cysteine reducing agent — effective O₂ scavenger without colour interference
• Semi-solid agar (0.5 g/L) — oxygen gradient without solidification
• Starch absorbs toxic anaerobic fermentation byproducts
• No selective inhibitors — broad recovery of strict and facultative anaerobes
• Optional Polymyxin B — selective Gram-negative inhibition

• Pharmaceutical QC: Microbial examination of non-sterile products for Clostridium sporogenes, C. perfringens, C. novyi — EP/USP/JP
• Food microbiology: Anaerobic sporeformer enumeration in cheese, dairy, canned and vacuum-packed foods
• Clinical microbiology: Enrichment of clinical Clostridia from wound, faecal, and blood specimens
• Research: Propagation and maintenance of obligate anaerobe reference strains

1. Dissolve 38.0 g in 1 litre of purified water with gentle heating and agitation.
2. Dispense into test tubes (8–10 mL per tube).
3. Autoclave at 121 °C for 15 minutes.
4. Cool to room temperature. Appearance: clear, yellowish semi-solid.
5. Optional — Polymyxin B: add 0.02 g/L sterile solution aseptically post-autoclave.
6. For strict anaerobes: heat in boiling water bath 15 min; cool rapidly; inoculate immediately; overlay with sterile paraffin or 1.5% agar.

RCM is free from selective inhibitors. L-Cysteine HCl scavenges dissolved O₂, maintaining low Eh for obligate anaerobes. Sodium acetate buffers pH and provides supplementary carbon for acetoclastic organisms. Soluble starch absorbs toxic metabolic byproducts. Semi-solid agar (0.5 g/L) retards O₂ diffusion, creating an anaerobic gradient at depth without gelling.

Inoculum: ≤100 CFU per ATCC strain. Incubation: anaerobic (paraffin overlay or anaerobic jar).

Uninoculated: General laboratory waste per local EPA regulations.

Inoculated Clostridium cultures: Autoclave at 121 °C for ≥60 minutes (spore-forming organisms require extended cycle). Treat as BSL-2/3 biological waste per institutional biosafety policy.

Inoculum: ≤100 CFU per ATCC strain | Incubation: anaerobic (paraffin overlay or anaerobic jar)