ANTIFUNGAL ASSAY

DR. KHAJISTA JABEEN

Antifungals are used to kill or prevent further
growth of fungi.

In medicine, they are used as a treatment for

infections such as athlete's foot, ringworm and
thrush and work by exploiting differences between
mammalian and fungal cells.

They kill off the fungal organism without

dangerous effects on the host.
Unlike bacteria, both fungi and humans are eukaryotes.
Thus, fungal and human cells are similar at the
molecular level, making it more difficult to find a target
for an antifungal drug to attack that does not also exist
in the infected organism.

Consequently, there are often side effects to some of

these drugs. Some of these side effects.

Antifungal activity of natural extracts and pure

compounds can be detected by inhibition of various
fungi, yeast or filamentous, by samples that are placed
in contact with them. Several methods for detecting
activity are available, but since they are not equally
sensitive or not based upon the same principle, results
will be profoundly influenced by the method
There is considerable need to discover new
fungitoxic compounds in view of the many plant
and human fungal diseases. Some of the common
plant fungal diseases are potato late blight, tobacco
blue mould, downy mildew, ergot of rye, cereal
rusts, corn blight and grape downy mildew.

Some fungi can be beneficial to man since they

attack harmful insects (Blank et al, 1965; Brass et al,
1979).
 Agar tube dilution assay

Materials
1. Test fungi Epidermophyton floccosum, Trichophyton
mentogrophytes, T.rubrum, T.simii, T. schoenleinii,
Microsporum canis, Pseudallescheria boydii, Candida
albicans, etc
2. Sabouraud dextrose agar (composition in gm/l, pepto
complex 10, glucose 40, agar 15).
3. Dimethyl sulfoxide (DMSO)
4. Screw test tubes
5. Incubator
6. Micropipettes
7. Magnetic stirrer
8. Autoclave
9. Standard antifungal drugs such as
amphotericin-B, miconazole, ketoconozole,
flueytopsine etc.
10. Test sample (crude extract, pure natural
product or synthetic compound)
The following steps are involved in agar tube dilution
assay:
1. Test sample is dissolved in sterile DMSO to serve
as stock solution.

2. Sabouraud dextrose agar is prepared by mixing

Sabouraud 4% glucose agar and agar agar in distilled
water.

3. It is then stirred with a magnetic stirrer to dissolve

it and a known amount is dispensed into screw
capped test tubes.

4. Test tubes containing media are autoclaved at

121°C for 15 minutes.
5. Tubes are allowed to cool to 50°C and the test sample of desired
concentrations pipetted from the stock solution into the non-
solidified Sabouraud agar media.
6. Tubes are then allowed to solidify in a slanting position at room
temperature.
7. Each tube is inoculated with a 4 mm diameter piece of
inoculum removed from a seven day old culture of fungi.
8. All culture containing tubes are inoculated at optimum
temperature of 28–30°C for growth for 7–10 days. Humidity
(40% to 50%) is controlled by placing an open pan of water in the
incubator.
9. Cultures are examined atleast twice weekly during the
incubation.
10. After the incubation for 7–10 days, the test tubes with no
visible growth of the microorganism is taken to represent the
minimum inhibitory concentration (MIC) of the test sample
which is expressed in μg/ml.
 DIRECT BIOAUTOBIOGRAPHY METHOD

This is an elegant method was designed for the

detection of fungitoxic substances. This method is
particularly suitable for rapid separation of antifungal
substances from natural sources (Homans et al., 1970).
Materials

1. TLC plates (silica gel)

2. Inorganic stock solution. The following inorganic salts
are dissolved in 1 liter of dist. H2O:-

KH2PO4 7g
Na2HPO4.2H2O 3g
KNO3 1g
MgSO4.7H2O 1g
NaCl l g

3. UV lamp
 4. Test fungi such as Aspergillus niger, Ascochyta
pisi, Botrytis cinerea, Colletrichum lindemuthianun,
Fusarium culmorum, Penicillium expansum,
Glomerella cingulata, Cladosporium cucumerinum
5. Organic solvents
6. TLC tanks
7. Autoclave
8. Glucose
9. Incubator
10. Hot air blower
11. Test sample (crude extract, pure natural product
or synthetic compound)
Direct bioautography method:
1. The TLC chromatogram is developed in a TLC
tank by using a suitable solvent
system. Volatile organic solvents are generally used
and the chromatograms are dried by using a hot air
blower.
2. UV absorbing spots are located and marked
under a UV lamp.
3. After locating the UV absorbing spots, the
chromatograms are usually sprayed with a conidial
suspension (caution). During the spraying, care
should be taken to avoid the plates becoming too
wet.
4. The conidial suspension containing Cladosporium
cucumerinun (or any other fungus against which
activity is needed to be determined) is taken up in the
inorganic medium. The solution is autoclaved at
120°C for 20 min. Just before making the conidial
suspension, 10 ml of a 30% aqueous solution of
glucose is added per 60 ml of the autoclaved solution.
5. After spraying, the chromatograms are incubated in
a moist atmosphere for 2–3 days at 25°C.
6. Inhibition zones indicate the presence of a
fungitoxic product (or its conversion or decomposition
products which are fungitoxic).
https://
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-activity

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Bank, U., Reinhold, D. and Ansorge, S.Allerg.ImmunoL,

37, 119 (1991).

Brass, G., Shainhouse, J.Z. and Stevens, D.A.,

Antimicrob. Agents. Chemother., 15, 763 (1979).

Homans, A.L. and Fuchs, A., J.Chromatog., 51, 327

(1970).