**3.4 Isolation, purification, identification and preservation of mycotoxigenic fungi**

Isolation & purification of *Aspergillus* spp*.* and *Fusarium* spp. were collected from stored maize grain samples which was conducted by blotter method [64, 65]. In this

*Aflatoxins and Fumonisins Contamination of Maize in Bangladesh: An Emerging Threat… DOI: http://dx.doi.org/10.5772/intechopen.101647*

method, 400 maize grains were tested for the identification of toxigenic *Aspergillus* spp. and *Fusarium* spp. for each sample collected from different locations and 40 plastic pestridishes were used for each sample. Then 10 maize grains were placed in the sterile plastic petridish containing three layers of wet blotter papers. The petridish was incubated at 25 ± 1° C under 12/12 h light and darkness cycle for 7 days. Each seed was observed under stereo microscope (Stemi 508, Germany) in order to record the presence of fungal colonies and temporary slides were prepared from the fungal colonies for morphological identification under compound microscope (Primo Star, Germany). Or one of the quarter kilo samples from each trader milled into fine floor using a Laboratory Milling machine. Ten grams of the ground sample was mixed with 100 ml sterile water to make a stock solution and serially diluted up to dilution 103 . The suspension was plated in Potato Dextrose Agar Medium (PDA) [66, 67] by mixing 1 ml suspension in molten PDA cooled to 40° C. Isolation media was prepared by weighing 39 g of PDA into 1 L of water. The mixture was autoclaved for 15 min at 121° C and 15 PSI pressure. The media was allowed to cool to about 50° C and then amended with 25 ng/L of streptomycin and tetracycline [68, 69]. Petri dishes were labeled appropriately and a milliliter of the diluted sample was poured into a sterile petri dish aseptically and then 18 ml of PDA media at 40° C will was poured on the same plate and the mixture swirled gently to mix. The mixture was allowed to cool and solidify in the laminar flow hood and then sealed using parafilm for incubation. The plates were incubated at room temperature for 5–7 days.
