Fluconazole Testing Medium (Twin Pack)

Intended Use

Recommended for fluconazole susceptibility testing by using Candida species.

Composition

**Formula adjusted, standardized to suit performance parameters

Directions

Part A and Part B are sterilized separately as follows:

Part A: - Suspend 2.0 grams of Part A in 100 ml purified / distilled water, add 0.1 ml phosphate buffer to adjust the pH to 7.5. Heat to boiling to dissolve the agar particles completely and then sterilize by autoclaving at 115°C for 10 minutes.

Part B: - Suspend 29.31 grams of Part B in 900 ml purified / distilled water. Mix well, add 2 gram of sodium bicarbonate, after stirring make up the total volume to 1 litre with distilled water. Sterilize by filtration. The medium can be kept at 4°C for two weeks. Complete medium is prepared by aseptically adding equal volume of molten Part A (previously cooled to 50°C) and Part B. Mix thoroughly and dispense.

Principle And Interpretation

To carry out standardization of antifungal drug susceptibility assays as a standard reference method, CLSI had recommended broth macro dilution testing of yeasts. Fluconazole Testing Medium is a chemically defined medium specifically developed for the in-vitro testing of fluconazole by using Candida species. Inhibitory concentration values obtained by using this medium correlate well with the clinical outcome (1,2,3).

The medium contains dextrose and a variety of amino acids, salts and vitamins to support the growth of Candida and other fungi.

Type of specimen

Clinical samples - skin, Palate scrapings, blood sample or scraping from the infected body site.

Specimen Collection and Handling

The inoculum size varies with different fungi. Candida species are grown in Sabouraud Dextrose Broth (M033) at 37°C for 16-18 hours and then diluted with normal saline to give following dilutions:

• Candida albicans .... 10⁵ / ml
• Candida tropicalis .... 10⁵/ml
• Candida krusei .... 10⁵/ml
• Candida guillermondii .... 10⁶/ml
• Candida parapsilosis .... 10⁶/ml
• Candida pseudotropicalis ....10⁶ / ml

Surface inoculate the above diluted cultures and incubate at 28°C for 48 hours to determine MIC value of fluconazole. Dermatophytes are grown in Sabouraud Dextrose Agar (M063) at 28°C for 5-10 days. The mycelial growth is homogenized in 2 ml of 0.85% saline using glass beads. The density of the suspension is adjusted with 0.85% saline to get a 65% light transmission. Inoculate the plates and incubate for about 5-6 days at 28°C.

Check the control plates to ensure that all isolates have grown adequately and determine the Minimum Inhibitory Concentration (MIC).

Warning and Precautions

In Vitro diagnostic Use only. 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. Standard methods should be followed for inoculating the plates.
• 2. Dilutions should be precise to avoid erroreous results.

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

Part A: Cream to light yellow homogeneous coarse powder Part B : White to light yellow homogeneous free flowing powder

Colour and Clarity of prepared medium

Light yellow coloured, opalescent solution may be with fine precipitate

Cultural Response

Cultural characteristics observed after an incubation at 28-30°C for 48 hours .

Storage and Shelf Life

Store dehydrated 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 (4,5).

Reference

1. Cook R. A., McIntyre K. A. and Galgiani J. N., 1990, Antimicrob. Agents and Chemother., 34:1542.
2. Hoeprich P. D. and Finn. P. D., 1972, J. Infect, Dis., 126: 353
3. Pfaller M. A. et al, 1992, Antimicrob. Agents and Chemother.,36:1805.
4. Isenberg, H.D. Clinical Microbiology Procedures Handbook 2nd Edition.
5. 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.