Team:NCTU Formosa/Fungal Experiment
Fungi Experiment
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Dual Cultures on Potato Dextrose Agar
In order to know whether the peptides are effective in inhibiting hyphae and the accurate concentration of peptides for inhibition, we measured the zone of inhibition.
Putting the mycelium on the Petri plates of Potato dextrose agar, we cultivated plates in 20℃ for 2 days. After making sure that the mycelial colony has developed, we put the sterile blank paper which the different concentrations of peptides and the negative control were added respectively.
Observation
The white part is mycelium, the holes are added different concentrations of peptides and negative control HEPES respectively.
If the peptides can inhibit the mycelium, then there will be a range around the holes that the mycelium can not occupied. Otherwise, the mycelium will grow to ignore the holes, like the hole containing HEPES.
The whole scene will look like a white circle, while the edges of effective peptides concave.
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Spore Germination: inhibition measurement for spores
In order to know whether the antifungal peptides can inhibit the germination of spores or not and the accurate concentration of antifungal peptides for inhibition, we conducted the spore germination experiment.
Preparation of Spore Suspension Solution
The concentration of the spore suspension solution (1.5*10^5 spores/ml) was calculated and adjusted by using a hemacytometer and a microscope. After adjusting the concentration of the solution, we mixed it with 2% glucose solution and each added different doses of peptides together into PCR tubes to pipet. Then we drew 15μl solution from the PCR tubes to the Petri dishes. After 20℃, 6 hours of culture, we calculated the percentage of spore germination afterward.
Percentage of Spore Germination
We directly observed Petri dishes under the microscope. By calculating the amount of the germinated spores and ungerminated spores, we calculated the rate of spore germination under dose response of peptides. Then, we compared all the solution with the control, and we knew the effectiveness of every peptide we predicted for the spore germination. Finally, the results were made and shown the MIC and IC50.
Results
Sample
HEPES buffer, which has no toxic on cells, is applied to be the negative control.
Reference
Antifungal Mechanism of a Novel Antifungal Protein from Pumpkin Rinds against Various Fungal Pathogens.J. Agric. Food Chem. 2009, 57, 9299–9304. DOI:10.1021/jf902005g
