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Laboratory Exercise Instructor Notes
Synthesis and Bioassay of a Volatile Fungistatic Agent 


This exercise can be easily modified to fit particular instructional situations. Methyl salicylate can be purchased and used for a basic demonstration of fungistasis. Students with some background in chemistry may appreciate the synthesis and testing of the active compound.

Advanced students could isolate fungi from natural sources and test them in the assay described here using a various volatile compounds.


Accurate determination of the comparative growth inhibition requires that measurements are made before the hyphae in the control treatment reach the edge of the plate. This may not be possible in all instructional settings. If the time between two lab sessions is too great, one could place the inoculum plugs near the edge of the plate, and measure the colony radius from the edge of the mycelial disk to the edge of the colony; this way, the fungus has farther to grow before reaching the far edge of the plate. Measurements of all treatments should be made at the same time, after the organism has grown substantially. In no case should the control colonies be allowed to reach the edge of the plate and overgrow before measurements are made.

There are a number of ways to express growth or inhibition of treated colonies relative to that of the controls:

100 x (Dt/Dc): relative growth of treated colony as % of control

100 x (Dc-Dt)/Dc: relative inhibition of treated colony, as % of control

100 x ((Dc-Dt)/Dc) - 100: comparative growth inhibition, as used in this publication

Numerical examples:

Dc Dt 100 x (Dt/Dc) 100 x (Dc-Dt)/Dc 100 x ((Dc-Dt)/Dc) - 100
80 80 100 0 -100
80 60 75 25 -75
80 40 50 50 -50
80 20 25 75 -25
80 0 0 100 0



All eight fungal species (Alternaria tenuis, Aspergillus flavus, Aspergillus niger, Fusarium oxysporum, Neurospora crassa, Penicillium chrysogenum, Penicillium notatum, and Rhizopus nigricans) grown on Potato Dextrose Agar were inhibited completely upon exposure to commercially prepared pure methyl salicylate (Sigma-Aldrich Chemical Company); all had a CGI value of 0. When methyl salicylate prepared by the above procedure was used as the experimental treatment, all fungi were inhibited, with CGI values ranging from 0 (Rhizopus nigricans) to -90 (Alternaria tenuis).

The organisms reported here were used because of their availability and have a range of biological properties and significance. Alternaria tenuis causes leaf spot disease on many plant species; Aspergillus flavus is widely distributed in nature and is the source of aflatoxin; Aspergillus niger, a very common soil inhabitant with airborne spores, is commonly called black mold; Fusarium oxysporum causes wilt diseases in many plant species; Neurospora crassa is best known for its use in genetic studies; Penicillium chrysogenum and P. notatum historically were sources of the antibiotic penicillin; Rhizopus nigricans is the common bread mold.