Clostridial Agar

Intended use

Recommended for the selective isolation of pathogenic Clostridia from mixed flora.

Composition**

Final pH (at 25°C): 7.0±0.2

**Formula adjusted, standardized to suit performance parameters

Directions

Suspend 46.4 grams in 1000 ml purified/ distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 118°C for 15 minutes. Cool to 45-50°C. Mix well and pour into sterile Petri plates.

Principle And Interpretation

One of the major species of anaerobic bacteria to cause disease in humans is Clostridium. Clostridium species cause tetanus and gas gangrene that ultimately leads to tissue damage. Another Clostridium species produces the lethal botulinum toxin, the causative agent of botulism (1). Clostridial Agar formulated by Vera is recommended for the selective isolation of pathogenic Clostridia form mixed flora (2). The media is well supplemented to support luxuriant growth of Clostridium species.

Tryptone and soya peptone provide the nitrogenous and carbonaceous compounds, long chain amino acids and other essential nutrients, mainly the nitrogen compounds. Dextrose serves as the carbon or fermentable carbohydrate source. L-cystine is an amino acid, which promotes the growth of Clostridia. Sodium thioglycollate and sodium formaldehyde sulphoxylate are the reducing agents that help to create low oxidation-reduction potential enabling the growth of Clostridia. Accompanying enteric bacteria including Proteus, Pseudomonas and Bacillus species are inhibited by neomycin sulphate and sodium azide incorporated in the medium. The ideal method of inoculation of Clostridial Agar is direct inoculation of sterile, cooled medium with the specimen (in tubes). Alternatively agar plates of the medium can also be inoculated by streaking.

Type of specimen

Clinical samples - faeces, wounds, Food and dairy samples.

Specimen Collection and Handling:

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (3,4).

For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (5,6,7).

After use, contaminated materials must be sterilized by autoclaving before discarding.

Warning and Precautions :

In Vitro diagnostic use. For professional use only. 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.

Limitation:

• Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
• Each lot of the medium has been tested for the organisms specified on the COA. It is recommended to users to validate the medium for any specific microorganism other than mentioned in the COA based on the user's unique requirement.

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 beige homogeneous free flowing powder

Gelling
Firm, comparable with 1.45% Agar gel

Colour and Clarity of prepared medium
Yellow coloured, clear to slightly opalescent gel forms in Petri plates

Reaction
Reaction of 4.64% w/v aqueous solution at 25°C. pH : 7.0±0.2

pH
6.80-7.20

Cultural Response
Cultural characteristics observed after an incubation at 35-37°Cfor 18-24 hours.

* - Corresponding WDCM numbers

Storage and Shelf Life

Store between 10-30°C in a tightly closed container and the prepared medium at 2-8°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.

Disposal

User must ensure safe disposal by autoclaving and/or incineration of used or unusable preparations of this product. Follow established laboratory procedures in disposing of infectious materials and material that comes into contact with clinical sample must be decontaminated and disposed of in accordance with current laboratory techniques (3,4).

Reference

1. Alcamo E. I., 2001, Fundamentals of Microbiology, 6th Ed., Jones and Bartlett Publisher
2. Vera, 1962, Presented Pa. Soc. Med. Tech., York, P
3. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.
4. Jorgensen, J.H., Pfaller, M.A., Carroll, K.C., Funke, G., Landry, M.L., Richter, S.S and Warnock.,D.W. (2015) Manual of Clinical Microbiology, 11th Edition. Vol. 1.
5. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C
6. Salfinger Y., and Tortorello M.L., 2015, Compendium of Methods for the Microbiological Examination of Foods, 5th Ed., American Public Health Association, Washington, D.C.
7. Wehr H. M. and Frank J. H., 2004, Standard Methods for the Microbiological Examination of Dairy Products, 17th Ed., APHA Inc., Washington, D.C.