After growing aflatoxigenic Aspergillus flavus NRRL 3357 on a potato dextrose auger slant for six days at 30 degrees Celsius, elute the fungal spores from the test tube with 10 milliliters of water containing Tween 20. Vortex the test tube for 20 seconds. Filter the fungal spore suspension through glass wool placed in a funnel, and use the vortexed filtrate for dilution.
Dilute a 100 microliter aliquot of the filtered suspension with 9.9 milliliters of water. After vortexing, count the spores with a hemocytometer. Using the onscreen formula, calculate the volume of water needed to dilute one volume of the original suspension from the slant to 1000 spores per microliter.
Add one volume of the original suspension to the calculated amount of water. Next, gently crack the peanut pods and remove the hulls. Place the seeds in a sterile beaker.
Add 0.05%hydrogen peroxide solution until the beaker is 70%full, allowing seeds to imbibe water for three hours. Decant the hydrogen peroxide solution from the seeds and add approximately three times the amount of 80%ethanol water mixture to cover the seeds. Incubate for one minute.
Then rinse the seeds twice with equal volumes of sterile distilled water. Add the fivefold volume of 3%hydrogen peroxide solution to the beaker and allow it to stand for five minutes. After decanting the hydrogen peroxide solution, rinse the seeds twice with equal volumes of sterile water.
Place a 90 millimeter diameter filter paper into the lid and moisten the paper with one milliliter of sterile water. Place the lid on the dish. Place the seeds on a sterile paper towel.
Using forceps, remove the seed's skin. Place the de-skinned seeds promptly in the Petri dish to avoid dehydration. Cut about one third of the seed containing the embryonic axis.
Split the seed into cotyledons, and using a sharp drill bit, drill approximately 1.5 to two millimeter deep into the middle of the outer side of each cotyledon. Place four to six drilled pieces of the seeds on a 1.5%auger dish. Using a 10 microliter pipette, add two microliters of the spore suspension into the drilled cavity of each half cotyledon piece.
After covering the dish with a lid, incubate at 30 degrees Celsius without light for 72 hours.
