HiMedia Laboratories GmbH, Marie-Curie-Str. 3, 64683, Einhausen, Germany.
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Reinforced Medium for Clostridia, Granulated
For the cultivation and enumeration of Clostridia from pharmaceutical products in accordance with the microbial limit testing by harmonized methodology of USP/EP/BP/JP.
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Granulated™ Media#4fa25a
Intended use
Recommended for the enrichment of Clostridia from pharmaceutical products in accordance with the microbial limit testing by harmonized methodology of USP/EP/BP/JP/IP.
Composition**
| Ingredients | g/L |
|---|---|
| Peptone | 10.000 |
| HM Peptone B# | 10.000 |
| Yeast extract | 3.000 |
| Glucose monohydrate | 5.000 |
| Sodium chloride | 5.000 |
| Soluble starch | 1.000 |
| Cysteine hydrochloride | 0.500 |
| Sodium acetate | 3.000 |
| Agar | 0.500 |
If necessary adjust the pH so that after sterilization it is 6.8±0.2
*pH can also be measured after sterilization at 25°C
**Formula adjusted, standardized to suit performance parameters
# Equivalent to Beef extract
Directions
Suspend 37.54 grams (the equivalent weight of dehydrated medium per litre) in 1000 ml purified/distilled water. Heat to boiling to dissolve the medium completely. Dispense into tubes or flasks as desired. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes or as per validated cycle.
Principle And Interpretation
Reinforced Medium for Clostridia was formulated by Hirsch and Grinsted (1). This media is prepared in accordance with the microbial limit testing by harmonized methodology of USP/EP/BP/JP/IP (2-6). It is recommended for sterility checking of non-sterile products, nutritional and dietary supplements. It can be used to initiate growth from small inocula and to obtain the highest viable count of clostridia. Barnes and Ingram used the broth medium for diluting an inoculum of vegetative cells of Clostridium perfringens (7,8). It can be used in studies of spore forming anaerobes, especially Clostridium butyricum in cheese, for enumeration of Clostridia in tube dilution counts or for preparation of plates for isolation (8). Other spore forming anaerobes, Streptococci and Lactobacilli also grow in these media. These are enriched but non-selective media.
Peptone, yeast extract and HM peptone B carbonaceous and nitrogenous substances, long chain amino acids, vitamins and other necessary nutrients for the growth of clostridia. Glucose monohydrate is a fermentable carbohydrate in the medium while sodium chloride maintains osmotic equilibrium. Cysteine hydrochloride acts as reducing agent. Small amount of soluble starch removes toxic metabolites from the medium. Sodium acetate also acts as a good buffering agent. Small quantity of agar keeps the medium semi solid and helps in maintaining anaerobic conditions.
Type of specimen
Pharmaceutical samples
Specimen Collection and Handling
For pharmaceutical samples, follow appropriate techniques for sample collection, processing as per pharmaceutical guidelines (10,2,3,8,5). After use, contaminated materials must be sterilized by autoclaving before discarding.
Warning and Precautions
Read the label before opening the container. Wear protective gloves/protective clothing/eye protection/face protection. Follow good microbiological lab practices while handling specimens and culture. Standard precautions as per established guidelines should be followed while handling clinical specimens. Safety guidelines may be referred in individual safety data sheets.
Limitations
- Some Clostridium species may show poor growth due to nutritional variations.
- Further biochemical tests must be carried out for complete identification.
Performance and Evaluation
Performance of the medium is expected when used as per the direction on the label within the expiry period when stored at recommended temperature.
Quality Control
Appearance
Cream to yellow coloured granular medium
Colour and Clarity of prepared medium
Light yellow coloured clear solution in tubes.
pH
6.60-7.00
Growth Promotion Test
Growth promotion was carried out in accordance with the harmonized method of USP/EP/BP/JP/IP, and growth was observed under anaerobic conditions after an incubation at 30-35°C for <=48 hours
Growth promoting properties
Clearly visible growth of microorganism comparable to that previously obtained with previously tested and approved lot of medium occurs at the specified temperature for not more than the shortest period of time specified inoculating not more than 100 cfu under anaerobic conditions (at 30-35°C for <=48 hours).
Cultural Response
Cultural characteristics observed in an anaerobic atmosphere, after an incubation at 30-35°C for 24-48 hours.
| Organism | Inoculum (CFU) | Growth | Incubation temperature | Incubation period |
|---|---|---|---|---|
| Growth Promoting | ||||
| Clostridium sporogenes ATCC 11437 | 50-100 | good - luxuriant | 30-35 °C | <=48 hrs |
| Clostridium sporogenes ATCC 19404 (00008*) * | 50-100 | good - luxuriant | 30-35 °C | <=48 hrs |
| **Phocaeicola vulgatus ATCC 8482 | 50-100 | good - luxuriant | 30-35 °C | <=48 hrs |
| Additional Microbiological testing | ||||
| Bacteroides fragilis ATCC 23745 | 50-100 | good - luxuriant | 30-35 °C | 24-48 hrs |
| Clostridium perfringens ATCC 13124 (00007*) | 50-100 | good - luxuriant | 30-35 °C | 24-48 hrs |
Key: (*) Corresponding WDCM numbers * Formerly known as Bacteroides vulgatus
Storage and Shelf Life
Store between 10-30°C in a tightly closed container and the prepared medium at 20-30°C. Use before expiry date on the label. On opening, product should be properly stored dry, after tightly capping the bottle in order to prevent lump formation due to the hygroscopic nature of the product. Improper storage of the product may lead to lump formation. Store in dry ventilated area protected from extremes of temperature and sources of ignition Seal the container tightly after use. Product performance is best if used within stated expiry period.
| Product Name | Reinforced Medium for Clostridia, Granulated |
|---|---|
| SKU | GMH443 |
| Product Type | Granulated |
| Physical Form | Granular |
| Origin | Animal |
| Packaging type | HDPE |
| References | 1.Hirsch and Grinsted, 1954, J. Dairy Res., 21:101. 2.Barnes and Ingram, 1956, J. Appl. Bact., 19:117. 3.Lewis and Angelotti (Eds.), 1964, Examination of Foods for Enteropathogenic and Indicator Bacteria, Dept. of HEW, PHSPublication, 1142, Washington. |
| Customized Product Available | No |
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