Dehydrated culture media
Reinforced Clostridial Medium EP/USP
For the cultivation and enumeration of Clostridium and other anaerobes.
- Cat. No.
- LC-1007
- Category
- Dehydrated culture media
- Format
- 500 g
- Storage
- 2 to 25 °C
- Minimum guaranteed shelf life
- 15 months
- Standards
- European PharmacopoeiaUSP
Reinforced Clostridial Medium is a semisolid medium. It is recommended for the cultivation and enumeration of anaerobes, particularly Clostridium and other microorganisms, in foods and other samples.
Key features
Applications
Non-selective enumeration of anaerobes and detection of Clostridium.
Industry
Final product quality control, pharmaceutical/veterinary.
Standards
European Pharmacopoeia, USP.
Preparation
38 g in one liter of distilled water; boil for one minute, sterilize in autoclave at 121 °C for 15 minutes.
Principles and uses
It was formulated by Hirsch and Grinstead in 1954. Their work demonstrated that the medium outperformed other media in supporting the growth of Clostridium from small inoculum and produced higher viable cell counts.
Peptone and beef extract provide nitrogen, vitamins, minerals and amino acids essential for growth. Yeast extract is the 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.
Since the medium is a non-selective enrichment one, it allows the growth of various anaerobic microorganisms and facultative bacteria when incubated under anaerobic conditions.
The European Pharmacopoeia, USP, recommends the Reinforced Clostridial Medium in paragraph 2.6.13 "Microbiological examination of non-sterile products: test for specified microorganisms" for the testing of Clostridia in products.
Technical specifications
| Solubility | Without residues |
|---|---|
| Appearance | Fine powder |
| Color of the dehydrated medium | Cream |
| Color of the prepared medium | Clear amber, slightly opalescent |
| Final pH (25 °C) | 6.8 ± 0.2 |
| Amount per litre | 38 g |
| Storage | 2 to 25 °C |
Formula
| Ingredient | g/L |
|---|---|
| Bacteriological agar | 0.5 |
| Cysteine hydrochloride | 0.5 |
| Glucose monohydrate | 5 |
| Beef extract | 10 |
| Peptone | 10 |
| Sodium acetate | 3 |
| Sodium chloride | 5 |
| Soluble starch | 1 |
| Yeast extract | 3 |
Typical formula in g/L, adjusted and/or supplemented as required to meet performance criteria.
Preparation and use
Preparation
Suspend 38 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.
According to the European Pharmacopoeia
Test of specified microorganisms (Clostridia) according to European Pharmacopoeia:
- Used without blood.
- Inoculate and incubate, under anaerobic conditions, the Reinforced Clostridial Medium at a temperature of 30-35 °C for 48 hours.
- Make subcultures on Columbia Agar (LC-1104).
- Add 20 mg/L of gentamicin if necessary.
- Incubate, under anaerobic conditions, at 30-35 °C for 48 hours.
- Colonies grown in anaerobiosis that are catalase-negative indicate presence of clostridia. This result must be confirmed by biochemical tests.
Quality control
According to European Pharmacopoeia
Clostridium sporogenes.
Incubation conditions: 30-35 °C, under anaerobic conditions / 48-72 h.
Inoculation conditions: ≤ 100 CFU.
Rest of strains
Incubation conditions: 30-35 °C, under anaerobic conditions / 48 h.
| Microorganism | Specification |
|---|---|
| Clostridium sporogenesATCC 11437 | Good growth |
| Clostridium perfringensATCC 13124 | Good growth |
| Clostridium sporogenesATCC 19404 | Good growth |
| Clostridium difficileATCC 9689 | Good growth |
Bibliography
- Andrews, W.H. (ed) 1995. Microbial methods p. 1-119. In Official methods of analysis of AOAC International. 16th ed.
- European Pharmacopoeia. 9.0.
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