Coagulase Mannitol Broth Base

Intended use

Recommended for simultaneous detection of coagulase production and mannitol fermentation in the differentiation of Staphylococci.

Composition**

Final pH ( at 25°C) 7.4±0.2

**Formula adjusted, standardized to suit performance parameters

# Equivalent to Heart muscle infusion from

Directions

Suspend 35.0 grams in 1000 ml purified/distilled water. Heat if necessary to dissolve the medium completely. Sterilize by autoclaving at 15 lbs pressure (121°C) for 15 minutes. Just before use aseptically add 7-15% v/v sterile, pretested rabbit plasma (coagulase plasma). Mix well and dispense into sterile tubes.

Principle And Interpretation

Coagulase Mannitol Broth Base is used for the isolation of Staphylococcus aureus from clinical specimens and for differentiation of Staphylococcus aureus from other species on the basis of coagulase production and mannitol fermentation. Chapman for the first time introduced medium for selective isolation and differentiation of Staphylococci (1). Tellurite-glycine media were designed by Zebovitz et al (2) and Marwin (3) for selectively isolating coagulase positive Staphylococcal species. Present medium is based on Esber and Faulconer formulation (4).

Coagulase Mannitol Broth Base simultaneously detects mannitol fermentation and coagulase production by Staphylococci (5). Coagulase Mannitol Broth Base is a good substrate for Staphylococci as well as other fastidious bacteria. Coagulase production and mannitol fermentation observed in Coagulase Mannitol Broth Base is presumptive identification of pathogenic Staphylococci (6). Coagulase production is dependent on the presence of a fermentable sugar like mannitol in this case. It is also dependent on the presence of a protein factor in the HMH infusion and blood plasma (4). When mannitol is fermented, the pH of the medium drops. This drop in pH is indicated by the change in colour of the phenol red, which turns yellow, and exhibit yellow medium. An opaque broth due to coagulated plasma forms due to growth of coagulase positive organisms.

Staphylococcus epidermidis a coagulase negative and mannitol non-fermenting species, does not change the colour of the medium. Coagulase negative species may ferment mannitol and produce a yellow colour but opacity will not be formed. Production of gas can be determined by placing a small inverted Durham's tube in the medium tube. Mutant or old cultures of Staphylococcus aureus may be weak coagulase producers. They should be freshly subcultured and rechecked. Escherichia coli ferments mannitol and may be weakly coagulase positive.

For the test, use this medium in 2 to 5 ml amounts after adding about 12-15% plasma. Inoculate by adding about 2 drops of test organism and incubate at 37°C and examine after 2-3 hours and also after 4-5 hours of incubation. Un-inoculated control tubes should also be run in parallel with the fermentation tests.

Type of specimen

Clinical samples - Pus; Food and dairy samples

Specimen Collection and Handling

For food and dairy samples, follow appropriate techniques for sample collection and processing as per guidelines (7,8).

For clinical samples follow appropriate techniques for handling specimens as per established guidelines (9,10).

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

Limitations

1. Individual organisms differ in their growth requirement and may show variable growth patterns on the medium.
2. 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. Other biochemical and serological tests must be performed for confirmation.

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: Light yellow to light pink homogeneous free flowing powder

Colour and Clarity of prepared medium: Red coloured, clear to slightly opalescent solution in tubes

Reaction: Reaction of 3.5% w/v aqueous solution at 25°C. pH : 7.4±0.2

pH: 7.20-7.60

Cultural Response: Cultural characteristics observed with added 7-15% v/v sterile pretested coagulase plasma after an incubation at 35-37°C for 18-48 hours.

Key : *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 (9,10).

Reference

1. Chapman,1946, J. Bact., 51:409.
2. Zebovitz, Evans and Nivens, 1955, J. Bact., 70:686.
3. Marwin, 1958, Am. J. Clin. Pathol., 30:470.
4. Esber and Faulconer, 1959, Am. J. Clin. Pathol., 32:19
5. Schaub and Merrit, 1960, Bull. Johns Hopkins Hosp., 106:25.
6. Mincheu and Cluff, 1961, J. Chron. Dis., 13:354.
7. 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.
8. American Public Health Association, Standard Methods for the Examination of Dairy Products, 1978, 14th Ed., Washington D.C.
9. 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.
10. Isenberg, H.D. Clinical Microbiology Procedures Handbook. 2nd Edition.