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Antifungal Bioassays

Human infections, particularly those involving the skin and mucosal surface as well as plant infection, constitute a serious problem, especially in tropical and subtropical developing countries. Herbal medicines have been important sources of products for the developing countries in treating common infections including fungal diseases. There is a considerable need to discover new fungi toxic compounds for the treatment of many plant and human fungal diseases.

The agar-dilution method is amongst the most convenient method for routine testing.It was used in this study to screen the crude extract of plants collected from various

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Method: Tube dilution Method

In this method Saboured Dextrose Agar was used. Six different species of fungus were inoculated and incubated for 7-days. Measurements were made by calculating the percent growth inhibition.

Names and infections of Fungus species:

• Candida albican

• Candida glabrata
These both are human pathogen and yeast. These cause Candidiases and infect the mucosa and epithelial layer of human.

• Microsporum canis: Plant and human pathogen and are mold. On rice grain it produces deep yellow pigments. In human it causes Tinea capita and Tinea corpis.

• Trichophyton longifusus: Human pathogen and are mold. It infects the nails and hair.

• Fusarium solani: Plant and human pathogen and are mold. In human it causes Keratitis, cutaneus infection, pulmonary infection and many other diseases. It produces mycotoxins which ingestion in body causes allergic and cancer.

• Aspergillus flavus: Plant and human pathogen and are mold. It produces carcinogen aflatoxin.

Nature of Compound Tested
Crude plant extracts and fractions.

Material

• Sabouraud dextrose agar (SDA) pH- 5.5-5.6
• Screw capped test tubes
• Micropipette (100-200 ul)
• Tips and tip box (Sterile)
• DMSO (Dimethyl sulfoxide)
• Glass vials

Methodology

1 Preparation of test sample
24 mg of crude extract dissolved in 1 ml sterile DMSO serving as stock solution.

2 Preparation of media
Sabouraud dextrose agar (SDA) is used for the growth of fungus. Media with acidic (pH 5.5-5.6) containing relatively high concentration of glucose or maltose 2% is prepared by mixing 32.5 gm/500 ml D. water.
It is then steamed to dissolve the contents and dispensed as volumes 4ml into screw caps tubes folword by Autoclaved at 121° C for 15 min.
3 Loading of sample
Tubes were allowed to cool to 50° C and non-solidified SDA is loaded with 66.6 µl of compound pipette from the stock solution. This will give the final concentration of 400 µg/ml (Crude extract) and 200µg/ml of the media for pure compound respectively.Tubes then allowed solidifying in slanting position at room temperature.
- 4 Inoculation of fungus
  Each tube was inoculated with 4mm diameter piece of fungus removed from a seven-day-old culture of fungus.For non-mycelial growth, an agar surface streak is employed.
- 5 Other media supplemented with DMSO and reference antifungal drugs used as negative and positive control respectively. The tubes incubated at 27-29° C for 3-7 days.
- 6 Cultures were examined twice weekly during incubation.

Result Interpretation
Growth in the compound amended media was determined by measuring linear growth (mm) and growth inhibition calculated with reference to the negative control.

Calculating % Inhibition of fungal growth:

% Inhibition = 100 – linear growth in test (mm) X 100
linear growth in control (mm)

The Standard drugs used in the assays were Miconazole and Amphotericin B

CRITERIA
              Percentage Inhibition Activity

             0-39 ————————— Low
             40-59 ————————  Moderate
             60-69 ————————  Good
            Above 70 ——————— Significant activity