3.2.1. Culture Maintenance and Growth
1. Maintain representative isolates of selected fungi (e.g., Fusarium subglutinans) on SAB slants.
2. Prepare working cultures on SAB or PDA plates.
3. Incubate cultures at 20-25°C for 7-14 d (time based on conidiogenesis).
3.2.2. Preparation of Fungal Conidia Inoculum
1. Harvest conidia from 7-14-d cultures with 0.9% NaCl containing 0.1% polysorbate-80 (NaCl-Tween-80).
2. Pour 10 mL of (NaCl-Tween-80) on the surface of fungal growth. Gently scrape the surface of fungal growth with a flame-sterilized loop to loosen conidia from hyphal structures.
3. Filter suspensions of conidia through glass wool. (This step removes most hyphal fragments.)
4. Harvest conidia with centrifugation and suspend pelleted conidia in 0.9% NaCl.
5. Adjust the suspensions for each species to give concentrations of about 107-108 conidia/mL.
3.2.3. Disinfection Test Procedure
1. Product samples to be tested should be representative of the product to be marketed.
2. Aliquots should be taken directly from the final product container immediately prior to testing.
3. Inoculate conidia of the selected fungi into full-strength preservative formulations and incubate at 20-25°C for 96 h. The preservative formulations should contain concentrations of conidia of 105-106/mL.
3.2.4. Viability Determination After Disinfection
1. Harvest the conidia from the preservative formulations with centrifugation and wash the pelleted conidia twice in 0.9% NaCl.
2. Stain conidia suspensions (1.0 mL) with 25 ^L PI for 30 s prior to FCM analysis.
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