Method for assaying gliotoxin production in Aspergillus fumi

Thursday, November 13, 2003

Description
Method for assaying gliotoxin production in Aspergillus fumigatus

Procedure
Culture and extraction
Culture Aspergillus fumigatus on Sabouraud agar plates for 2 days at 37C and harvest the conidia using sterile 0.5% Tween 20.
Adjust the concentration of conidia 107ml-1 in distilled water using a haemocytometer.
Use 1ml of this conidial suspension to inoculate 100ml of Czapek-dox broth in a 250ml flask.
Incubate culture at 37C in a shaking incubator at 1400rpm for 2 days.
Harvest fungal biomass by filtration through Whatman No1 filter paper in a Buchner funnel.
Extact the filtrate three times by shaking for 10 min with 50 ml chloroform at 25C.
Pool the chloroform fractions and evaporate to dryness on a rotary evaporator at reduced pressure and at 47 C.
Dissolve dried extracts in 200l methanol and store at -70C until use.
Detection of gliotoxin by TLC
Spot 10l of the chloroform extract and a 1mgml-1 gliotoxin standard in methanol onto a Silicagel 60 plate with fluorescence indicator approximately 3cm from the bottom edge.
Develop for 10cm in an inlined tank with a methanol:chloroform (10:90, v:v) solvent system.
Air dry the plates and spray with 25% sulphuric acid in distilled water and heat at 110C for 15min to reveal the gliotoxin spots.

Recipes
To prepare extracts
Haemocytometer
250ml flask
Culture plates containing Sabouraud glucose agar
0.5% Tween 20 (Sigma)
100ml Czapek-Dox medium (30g sucrose, 3g Na2NO3, 0.5g MgSO4.7H2O, 0.5g KCl, 0.01g FSO4 in 1l distilled water - all from Sigma)
100ml glass flask for extraction
Microfuge
Shaking incubator
Buchner funnel and Whatman No1 filter paper to fit
Chloroform & methanol (Hyper Solv, BDH)
Rotary evaporator + suitable tubes to fit
-70C freezer
Deionised-distilled water
For TLC detection
1mgml-1 gliotoxin standard in methanol
Silicagel 60 plate with fluorescence indicator (Merck, Damstadt, Germany)
inlined chromatography tank
Sulphuric Acid (Sigma)
110C oven

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Method for assaying gliotoxin production in Aspergillus fumi